Geraint P. Williams

Endogenous Cortisol and TGF-β in Human Aqueous Humor Contribute to Ocular Immune Privilege by Regulating Dendritic Cell Function

Aqueous humor (AqH) has been shown to have significant immunosuppressive effects on APCs in animal models. We wanted to establish whether, in humans, AqH can regulate dendritic cell (DC) function and to identify the dominant mechanism involved. Human AqH inhibited the capacity of human peripheral blood monocyte-derived DC to induce naive CD4+ T cell proliferation and cytokine production in vitro, associated with a reduction in DC expression of the costimulatory molecule CD86. This was seen both for DC cultured under noninflammatory conditions (immature DC) and for DC stimulated by proinflammatory cytokines (mature DC). DC expression of MHC classes I/II and CD83 was reduced (mature DC only). Myeloid DC from peripheral blood were similarly sensitive to the effects of human AqH, but only under inflammatory conditions. The addition of α-melanocyte stimulating hormone and vasoactive intestinal peptide did not cause significant inhibition at physiological levels. However, the addition of exogenous cortisol at physiological levels recapitulated the AqH-induced reduction in CD86 and inhibition of DC-induced T cell proliferation, and blockade of cortisol in AqH partially reversed its suppressive effects. TGF-β2 had an additional effect with cortisol, and although simultaneous blockade of cortisol and TGF-β2 in AqH reduced its effectiveness, there was still a cortisol- and TGF-β–independent component. In humans, AqH regulates DC maturation and function by the combined actions of cortisol and TGF-β2, a pathway that is likely to contribute to the maintenance of immune privilege in the eye.

Incidence, presenting features, and diagnosis of cicatrising conjunctivitis in the United Kingdom.

PurposeCicatrising conjunctival disorders are uncommon, and are difficult to diagnose and manage. This study was designed to assess the annual incidence and underlying diagnosis of patients with cicatrising conjunctivitis (CC) within the United Kingdom.MethodsClinical data of newly diagnosed cases of CC were reported via the British Ophthalmological Surveillance Unit at diagnosis and at 12 months follow-up.ResultsA total of 50 (61%) ocular mucous membrane pemphigoid (OcMMP), 16 (20%) Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS-TEN) and 16 (20%) other causes of CC, equating to an incidence of 0.8, 0.2, and 0.2 per million, respectively, were reported. Although diagnosis of SJS-TEN was usually within a median of 7 days of symptom-onset, that for OcMMP and other CC was a median 225 days for both. At diagnosis, 64/163 (39%) eyes had moderate/severe conjunctival inflammation, and 102/164 (62%) had symblepharon formation. Although 43/82 (52%) patients were commenced on immunosuppression or had this therapy modified, at follow-up there was an increase in the number of symblepharon, despite control of inflammation (P<0.001). Mortality only occurred in the SJS-TEN group (4/16 (25%)).ConclusionCC has a substantial morbidity and for non-SJS-TEN causes, diagnosis is frequently delayed. The proportion of patients given immunosuppressive therapy to prevent disease progression may be less than optimal. These data highlight the need for developing patient access to specialist-designated centres with expertise in CC.

Evaluation of early and late presentation of patients with ocular mucous membrane pemphigoid to two major tertiary referral hospitals in the United Kingdom

PurposeOcular mucous membrane pemphigoid (OcMMP) is a sight-threatening autoimmune disease in which referral to specialists units for further management is a common practise. This study aims to describe referral patterns, disease phenotype and management strategies in patients who present with either early or established disease to two large tertiary care hospitals in the United Kingdom.Patients and MethodsIn all, 54 consecutive patients with a documented history of OcMMP were followed for 24 months. Two groups were defined: (i) early-onset disease (EOD:<3years, n=26, 51 eyes) and (ii) established disease (EstD:>5years, n=24, 48 eyes). Data were captured at first clinic visit, and at 12 and 24 months follow-up. Information regarding duration, activity and stage of disease, visual acuity (VA), therapeutic strategies and clinical outcome were analysed.ResultsPatients with EOD were younger and had more severe conjunctival inflammation (76% of inflamed eyes) than the EstD group, who had poorer VA (26.7%=VA<3/60, P<0.01) and more advanced disease. Although 40% of patients were on existing immunosuppression, 48% required initiation or switch to more potent immunotherapy. In all, 28% (14) were referred back to the originating hospitals for continued care. Although inflammation had resolved in 78% (60/77) at 12 months, persistence of inflammation and progression did not differ between the two phenotypes. Importantly, 42% demonstrated disease progression in the absence of clinically detectable inflammation.ConclusionsThese data highlight that irrespective of OcMMP phenotype, initiation or escalation of potent immunosuppression is required at tertiary hospitals. Moreover, the conjunctival scarring progresses even when the eye remains clinically quiescent. Early referral to tertiary centres is recommended to optimise immunosuppression and limit long-term ocular damage.

Validation of a fornix depth measurer: a putative tool for the assessment of progressive cicatrising conjunctivitis

Background/aims Documentation of conjunctival forniceal foreshortening in cases of progressive cicatrising conjunctivitis (PCC) is important in ascertaining disease stage and progression. Lower fornix shortening is often documented subjectively or semi-objectively, whereas upper forniceal obliteration is seldom quantified. Although tools such as fornix depth measurers (FDMs) have been described, their designs limit upper fornix measurement. The purpose of this study was to custom-design a FDM to evaluate the upper fornix and to assess variability in gauging fornix depth. Methods A polymethylmethacrylate FDM was constructed using industry-standard jewellery computer software and machinery. Two observers undertook a prospective independent evaluation of central lower fornix depth in a heterogeneous cohort of patients with clinically normal and abnormal conjunctival fornices both subjectively and by using the FDM (in mm). Upper central fornix depth was also measured. Agreement was assessed using Bland–Altman plots. Results Fifty-one eyes were evaluated. There was 100% intraobserver agreement to within 1 mm for each observer for lower fornix measurement. The mean difference in fornix depth loss using the FDM between observer 1 and 2 was 1.19%, with 95% confidence of agreement (±2SD) of −15% to +20%. In total, 86% (44/51) of measurements taken by the two observers agreed to within 10% of total lower fornix depth (ie, ±1 mm) versus only 63% (32/51) of the subjective measurements. Mean upper fornix difference was 0.57 mm, with 95% confidence of agreement of between −2 and +3 mm. Conclusions This custom-designed FDM is well tolerated by patients and shows low intraobserver and interobserver variability. This enables repeatable and reproducible measurement of upper and lower fornix depths, facilitating improved rates of detection and better monitoring of progression of conjunctival scarring.

Elevation of conjunctival epithelial CD45INTCD11b⁺CD16⁺CD14⁻ neutrophils in ocular Stevens-Johnson syndrome and toxic epidermal necrolysis.

PURPOSE Ocular complications related to Stevens-Johnson Syndrome (SJS)-Toxic Epidermal Necrolysis (TEN) may persist and progress after resolution of systemic disease. This is thought to be related in part to persistent ocular innate-immune signaling. In this study, our aim was to characterize infiltrative conjunctival cellular profiles during acute (<12 months) and chronic (>12 months) disease. METHODS Consecutive patients presenting with SJS-TEN over a 12-month period were followed for 1 year. Detailed clinical examination and conjunctival impression cell recovery was analyzed by flow cytometry for the presence of intraepithelial leukocytes and compared with healthy controls (n = 21). RESULTS Ten patients were recruited of whom six had acute disease and five were classified as TEN (SCORTEN = 1, n = 4). Conjunctival inflammation was graded as absent/mild in a total of nine patients; but despite this, evidence of fornix shrinkage was observed in nine subjects. This inversely correlated with disease duration (P < 0.05). A reduction in percentage of CD8αβ(+) T cells compared with controls (80% vs. 57%; P < 0.01) was associated with a corresponding increase in the number/percentage of CD45(INT)CD11b(+)CD16(+)CD14(-) neutrophils (186 vs. 3.4, P < 0.01, 31% vs. 0.8%, P < 0.001). Neutrophils inversely correlated with disease duration (r = -0.71, P = 0.03), yet there was no absolute change in the CD8αβ(+) or neutrophil populations during the study period (P = 1.0). CONCLUSIONS These data highlight that a neutrophilic infiltrate is present in mildly inflamed or clinically quiescent conjunctival mucosa in patients with ocular SJS-TEN, where neutrophil numbers inversely correlate with disease duration. Neutrophil persistence endorses the hypothesis of an unresolved innate-inflammatory process that might account for disease progression.

Rheumatoid corneal melt: autoimmunity or infection?

The effect of immunosuppression may mask the typical signs of infective keratitis, potentially resulting in a devastating delay in diagnosis.

Aqueous Humor Suppression of Dendritic Cell Function Helps Maintain Immune Regulation in the Eye during Human Uveitis

PURPOSE Noninfectious uveitis is characterized by a dysregulated inflammatory or immune response in the eye. It is unclear whether this represents a failure of immune privilege or an overwhelming inflammatory drive that has exceeded the capacity of regulatory mechanisms that are still functioning. The authors investigated immune regulation in the human eye during intraocular inflammation (uveitis) and its impact on dendritic cell (DC) function and subsequent T-cell responses. METHODS Myeloid DCs were isolated from the aqueous humor (AqH) and peripheral blood of patients with active uveitis and characterized by flow cytometry. The effect of uveitis AqH was interrogated in an in vitro model of peripheral blood monocyte-derived DCs from healthy controls. RESULTS Myeloid DCs isolated from uveitic AqH were characterized by elevated major histocompatibility complex classes I and II (MHC I/II), but reduced CD86 compared with matched peripheral blood DCs. Exposure of peripheral blood monocyte-derived DCs from healthy controls to the inflammatory AqH supernatant recapitulated this phenotype. Despite interferon gamma (IFNγ)-dependent upregulation of MHC I, inflammatory AqH was overall suppressive to DC function, with reduced CD86 expression and diminished T-cell responses. This suppressive effect was equal to or greater than that induced by noninflammatory AqH, but was glucocorticoid independent (in contrast to noninflammatory AqH). CONCLUSIONS These data indicate that the ocular microenvironment continues to regulate DC function during uveitis, despite IFNγ-driven upregulation of MHC expression, supporting the hypothesis that immune regulation within the eye is maintained during inflammation.

Cortisol Biosynthesis in the Human Ocular Surface Innate Immune Response

Innate immune responses have a critical role in regulating sight-threatening ocular surface (OcS) inflammation. While glucocorticoids (GCs) are frequently used to limit tissue damage, the role of intracrine GC (cortisol) bioavailability via 11-beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in OcS defense, remains unresolved. We found that primary human corneal epithelial cells (PHCEC), fibroblasts (PHKF) and allogeneic macrophages (M1, GM-CSF; M2, M-CSF) were capable of generating cortisol (M1>PHKF>M2>PHCEC) but in corneal cells, this was independent of Toll-like receptor (TLR) activation. While PolyI∶C induced maximal cytokine and chemokine production from both PHCEC (IFNγ, CCL2, CCL3, and (CCL4), IL6, CXCL10, CCL5, TNFα) and PHKF (CCL2, IL-6, CXCL10, CCL5), only PHKF cytokines were inhibited by GCs. Both Poly I∶C and LPS challenged-corneal cells induced M1 chemotaxis (greatest LPS-PHKF (250%), but down-regulated M1 11β-HSD1 activity (30 and 40% respectively). These data were supported by clinical studies demonstrating reduced human tear film cortisol∶cortisone ratios (a biomarker of local 11β-HSD1 activity) in pseudomonas keratitis (1∶2.9) versus healthy controls (1∶1.3; p<0.05). This contrasted with putative TLR3-mediated OcS disease (Stevens-Johnson Syndrome, Mucous membrane pemphigoid) where an increase in cortisol∶cortisone ratio was observed (113.8∶1; p<0.05). In summary, cortisol biosynthesis in human corneal cells is independent of TLR activation and is likely to afford immunoprotection under physiological conditions. Contribution to ocular mucosal innate responses is dependent on the aetiology of immunological challenge.

Defining the Limits of Normal Conjunctival Fornix Anatomy in a Healthy South Asian Population

Purpose Quantifying the extent of conjunctival fibrosis for documentation of progression in conjunctival scarring disease is a clinical challenge. Measurement of forniceal foreshortening facilitates monitoring of these disorders. This study aims (1) to define the limits of the normal human conjunctival fornices and how these alter with age and (2) to provide normative data for upper and lower fornix depths (FDs) and fornix intercanthal distance (FICD) within a healthy South Asian, racially distinct population. Design Epidemiologic, cross-sectional study. Participants A total of 240 subjects with national origins from South Asia, with no known ocular history and normal adnexal and conjunctival examination, aged 20 to 80 years. Methods An FICD modification of a custom-designed fornix depth measurer (FDM) was validated and used for measurement of both lower and upper FDs together with FICDs in 480 healthy eyes with no ocular comorbidities. Data were analyzed using repeated-measures analysis of variance and presented as means with 95% confidence intervals (CIs). Main Outcome Measures Mean lower and upper FDs and FICD for the entire cohort, stratified according to age decade and sex. Results For this South Asian population, the overall upper and lower FDs were 15.3 mm (95% CI, 14.9–15.6) and 10.9 mm (95% CI, 10.7–11.1), respectively, with FICD defined as 32.9 mm (95% CI, 32.5–33.4) (upper) and 31.7 mm (95% CI, 31.3–32.1) (lower). With increasing age, a progressive reduction of all measured parameters (P < 0.001) was noted, with female subjects having significantly shallower fornices (upper FD, P < 0.001; lower FD, P < 0.001; upper FICD, P = 0.081; and lower FICD, P = 0.015). Conclusions This is the first study to define the limits of normal upper FD and FICDs in any population group. Our study demonstrates sex variations and progressive conjunctival shrinkage with age. Although it provides important, objective data for normal forniceal anatomy, further study is recommended in other populations to confirm the generalizability of these data or to enable normal comparative datasets for the assessment of conjunctival scarring disorders among all anthropological groups.

Neuron J is a rapid and reliable open source tool for evaluating corneal nerve density in herpes simplex keratitis.

PURPOSE In vivo confocal microscopy (IVCM) demonstrates reduction in corneal sub-basal nerve density in herpes simplex keratitis (HSK). Image J is an open source image-analysis platform that can be combined with a nerve tracer, Neuron J. We sought to compare the reliability and speed of corneal nerve density quantification between these modalities and their relation to clinical damage. METHODS A total of 16 eyes (14 patients) with chronic HSK was assessed clinically and by IVCM. Randomly ordered triplicate, representative images from the central cornea were presented to two masked observers and corneal sub-basal nerve density was measured using Image J/Neuron J. Agreement was quantified using intraclass correlation coefficients (ICC), Bland-Altman plots together with mean difference, and level of agreement (LoA). RESULTS The median nerve density was measured at 7.1 mm/mm(2) (quartiles, 3.3-11.2), with Neuron-J demonstrating good intra-/interobserver agreement (ICC, 0.96-0.99; P < 0.001; mean difference, 0.1-1.4; LoA, <±3.3). Intraeye reliability was less consistent (mean difference, 1.7-2.3; LoA, ±8.8-9.8). Neuron J was highly comparable to Image J for both observers (ICC, 1.0; P < 0.001; mean difference, <0.2; LoA, ±<1.2) and significantly faster than Image J (median, 49 vs. 102 seconds, P < 0.001). Diminished nerve density was associated with corneal opacification and reduction in visual acuity (both P = 0.03). CONCLUSIONS The IVCM combined with Neuron J affords objective, user-friendly, and fast quantification of corneal nerve damage in HSK. It provides semiobjective phenotyping of the sequelae of neurotrophic corneal damage and offers a potential tool for measuring vulnerability to relapse or additional infections. Further exploration in a larger longitudinal cohort is warranted.