Cornea Publications

Olsen MV, Lyngstadaas AV, Bair JA, Hodges RR, Utheim TP, Serhan CN, Dartt DA. Maresin 1, a specialized proresolving mediator, stimulates intracellular [Ca ] and secretion in conjunctival goblet cells. J Cell Physiol 2020;Abstract
Mucin secretion from conjunctival goblet cells forms the tear film mucin layer and requires regulation to function properly. Maresin 1 (MaR1) is a specialized proresolving mediator produced during the resolution of inflammation. We determined if MaR1 stimulates mucin secretion and signaling pathways used. Cultured rat conjunctival goblet cells were used to measure the increase in intracellular Ca ([Ca ] ) concentration and mucin secretion. MaR1-increased [Ca ] and secretion were blocked by inhibitors of phospholipase C, protein kinase C, Ca /calmodulin-dependent protein kinase II, and extracellular-regulated kinase 1/2. MaR1 added before addition of histamine counterregulated histamine-stimulated increase in [Ca ] and secretion. We conclude that MaR1 likely has two actions in conjunctival goblet cells: first, maintaining optimal tear film mucin levels by increasing [Ca ] and stimulating mucin secretion in health and, second, attenuating the increase in [Ca ] and overproduction of mucin secretion by counterregulating the effect of histamine as occurs in ocular allergy.
Erickson S, Sullivan AG, Barabino S, Begovic E, Benitez-Del-Castillo JM, Bonini S, Borges JS, Brzheskiy V, Bulat N, Cerim A, Craig P, Cușnir V, Cușnir V, Cușnir V, Doan S, Dülger E, Farrant S, Geerling G, Goldblum D, Golubev S, Gomes JAP, González-Méijome JM, Grupcheva CN, Gündüz UÖmür, Horwath-Winter J, Källmark F, Karanadze N, Karcic HH, Karcic S, Kontadakis G, Messmer EM, Mrugacz M, Murphy C, O'Leary OE, Procopciuc V, Pult H, Raus P, Şahin A, Setälä N, Stanila A, Stanila DM, Utheim TP, Vehof J, Versura P, Villani E, Willcox MDP, Wolffsohn JS, Zagórski Z, Zoega GMár, Sullivan DA, Sullivan DA, Gomes JAP, Versura P, Willcox MDP. TFOS European ambassador meeting: Unmet needs and future scientific and clinical solutions for ocular surface diseases. Ocul Surf 2020;Abstract
The mission of the Tear Film & Ocular Surface Society (TFOS) is to advance the research, literacy, and educational aspects of the scientific field of the tear film and ocular surface. Fundamental to fulfilling this mission is the TFOS Global Ambassador program. TFOS Ambassadors are dynamic and proactive experts, who help promote TFOS initiatives, such as presenting the conclusions and recommendations of the recent TFOS DEWS II™, throughout the world. They also identify unmet needs, and propose future clinical and scientific solutions, for management of ocular surface diseases in their countries. This meeting report addresses such needs and solutions for 25 European countries, as detailed in the TFOS European Ambassador meeting in Rome, Italy, in September 2019.
Inomata T, Nakamura M, Iwagami M, Midorikawa-Inomata A, Sung J, Fujimoto K, Okumura Y, Eguchi A, Iwata N, Miura M, Fujio K, Nagino K, Hori S, Tsubota K, Dana R, Murakami A. Stratification of Individual Symptoms of Contact Lens-Associated Dry Eye Using the iPhone App DryEyeRhythm: Crowdsourced Cross-Sectional Study. J Med Internet Res 2020;22(6):e18996.Abstract
BACKGROUND: Discontinuation of contact lens use is mainly caused by contact lens-associated dry eye. It is crucial to delineate contact lens-associated dry eye's multifaceted nature to tailor treatment to each patient's individual needs for future personalized medicine. OBJECTIVE: This paper aims to quantify and stratify individual subjective symptoms of contact lens-associated dry eye and clarify its risk factors for future personalized medicine using the smartphone app DryEyeRhythm (Juntendo University). METHODS: This cross-sectional study included iPhone (Apple Inc) users in Japan who downloaded DryEyeRhythm. DryEyeRhythm was used to collect medical big data related to contact lens-associated dry eye between November 2016 and January 2018. The main outcome measure was the incidence of contact lens-associated dry eye. Univariate and multivariate adjusted odds ratios of risk factors for contact lens-associated dry eye were determined by logistic regression analyses. The t-distributed Stochastic Neighbor Embedding algorithm was used to depict the stratification of subjective symptoms of contact lens-associated dry eye. RESULTS: The records of 4454 individuals (median age 27.9 years, SD 12.6), including 2972 female participants (66.73%), who completed all surveys were included in this study. Among the included participants, 1844 (41.40%) were using contact lenses, and among those who used contact lenses, 1447 (78.47%) had contact lens-associated dry eye. Multivariate adjusted odds ratios of risk factors for contact lens-associated dry eye were as follows: younger age, 0.98 (95% CI 0.96-0.99); female sex, 1.53 (95% CI 1.05-2.24); hay fever, 1.38 (95% CI 1.10-1.74); mental illness other than depression or schizophrenia, 2.51 (95% CI 1.13-5.57); past diagnosis of dry eye, 2.21 (95% CI 1.63-2.99); extended screen exposure time >8 hours, 1.61 (95% CI 1.13-2.28); and smoking, 2.07 (95% CI 1.49-2.88). The t-distributed Stochastic Neighbor Embedding analysis visualized and stratified 14 groups based on the subjective symptoms of contact lens-associated dry eye. CONCLUSIONS: This study identified and stratified individuals with contact lens-associated dry eye and its risk factors. Data on subjective symptoms of contact lens-associated dry eye could be used for prospective prevention of contact lens-associated dry eye progression.
Aggarwal S, Kheirkhah A, Cavalcanti BM, Cruzat A, Jamali A, Hamrah P. Correlation of corneal immune cell changes with clinical severity in dry eye disease: An in vivo confocal microscopy study. Ocul Surf 2020;Abstract
PURPOSE: To evaluate corneal immune dendritiform cell (DC) changes in dry eye disease (DED) using in vivo confocal microscopy (IVCM) and to correlate IVCM parameters with clinical severity. METHODS: This was a retrospective, cross-sectional study including 300 eyes of 150 DED patients and 49 eyes of 49 age-matched controls. Severity of DED was based on the Dry Eye Workshop (DEWS) classification. IVCM images of subbasal layer of the central cornea were analyzed for DC density and morphology (including number of dendrites per DC, DC size and DC field). RESULTS: DC density was significantly higher in DED compared to controls (93.4 ± 6.3 vs. 25.9 ± 3.9 cells/mm; P < 0.001). Morphologically, number of dendrites, DC size and field were significantly larger in DED (3.3 ± 0.1, 106.9 ± 4.7 μm, 403.8 ± 20.1 μm than controls (2.3 ± 0.1, 62.5 ± 5.7 μm, 241.4 ± 24.4 μm, P < 0.001). Significantly higher DC density compared to controls was observed as early as Level 1 DED severity (87 ± 10 cells/mm, p < 0.001. Significant morphological changes in DC were detected for Levels 2 to 4 (p=<0.001, and p =< 0.05) for dendrites and DC field, respectively. Similarly, DC size showed significant increase at DED level 3-4. (p < 0.05). Linear regression analysis showed that both conjunctival and corneal staining were independently associated with DC density, while corneal staining was independently associated with DC morphology. CONCLUSION: DC density and morphology correlated with clinical severity of DED. While, DC density is increased in mild DED, morphological changes are seen only in severe cases. IVCM may be a powerful tool to detect early immune changes and may complement clinical examination in DED.
McKay TB, Serjersen H, Hjortdal J, Zieske JD, Karamichos D. Characterization of Tear Immunoglobulins in a Small-Cohort of Keratoconus Patients. Sci Rep 2020;10(1):9426.Abstract
Keratoconus (KC) is classically considered a non-inflammatory condition caused by central corneal thinning that leads to astigmatism and reduced visual acuity. Previous studies have identified increased systemic levels of pro-inflammatory factors, including interleukin-6, tumor necrosis factor-α, and matrix metalloproteinase-9, suggesting that KC may have an inflammatory component in at least a subset of patients. In this study, we evaluated the levels of different immunoglobulins (light and heavy chains) based on Ig α, Ig λ, Ig κ, Ig µ, and Ig heavy chain subunits in non-KC tears (n = 7 control individuals) and KC tears (n = 7 KC patients) using tandem-liquid chromatography mass spectrometry. The most abundant Ig heavy chains detected in both control individuals and KC patients were Ig α-1 and Ig α-2 likely correlating to the higher IgA levels reported in human tears. We identified significant differences in immunoglobulin κ-chain V-II levels in KC patients compared to control individuals with no significant difference in Ig κ/Ig λ ratios or heavy chain levels. Our study supports previous findings suggesting that KC possesses a systemic component that may contribute to the KC pathology. Further studies are required to define causality and establish a role for systemic immune system-dependent factors and pro-inflammatory processes in KC development or progression.
Ting DSJ, Foo VH, Yang LWY, Sia JT, Ang M, Lin H, Chodosh J, Mehta JS, Ting DSW. Artificial intelligence for anterior segment diseases: Emerging applications in ophthalmology. Br J Ophthalmol 2020;Abstract
With the advancement of computational power, refinement of learning algorithms and architectures, and availability of big data, artificial intelligence (AI) technology, particularly with machine learning and deep learning, is paving the way for 'intelligent' healthcare systems. AI-related research in ophthalmology previously focused on the screening and diagnosis of posterior segment diseases, particularly diabetic retinopathy, age-related macular degeneration and glaucoma. There is now emerging evidence demonstrating the application of AI to the diagnosis and management of a variety of anterior segment conditions. In this review, we provide an overview of AI applications to the anterior segment addressing keratoconus, infectious keratitis, refractive surgery, corneal transplant, adult and paediatric cataracts, angle-closure glaucoma and iris tumour, and highlight important clinical considerations for adoption of AI technologies, potential integration with telemedicine and future directions.
Yang M, Bair JA, Hodges RR, Serhan CN, Dartt DA. Resolvin E1 Reduces Leukotriene B-Induced Intracellular Calcium Increase and Mucin Secretion in Rat Conjunctival Goblet Cells. Am J Pathol 2020;Abstract
Leukotriene B (LTB) is a major proinflammatory mediator important in host defense whereas resolvins (Rvs) are produced during the resolution phase of inflammation. We determined the actions of both RvE1 and RvD1 on LTB-induced responses of goblet cells cultured from rat conjunctiva. The responses measured were an increase in the intracellular [Ca] ([Ca]) and high molecular weight glycoprotein secretion. Treatment with RvE1 or RvD1 for 30 min significantly blocked the LTB-induced [Ca] increase. The actions of RvE1 on LTB-induced [Ca] increase were reversed by siRNA for the RvE1 receptor and of RvD1 were reversed by an inhibitor to the RvD1 receptor. The RvE1 and RvD1 block of LTB-stimulated increase in [Ca] was also reversed by an inhibitory peptide to β-adrenergic receptor kinase (BARK1). LTB- and block of the LTB-stimulated increase in [Ca] by RvE1 and RvD1 were partially mediated by the depletion of intracellular Ca stores. RvE1, but not RvD1, counter-regulated the LTB-induced high molecular weight glycoprotein secretion. Thus, both RvE1 and RvD1 receptors directly inhibit LTB by phosphorylating the LTB receptor (BLT1) using BARK1. RvE1 receptor counter-regulates the LTB-induced increase in [Ca] and secretion while RvD1 receptor only counter-regulates LTB-induced [Ca] increase.
Golan S, Vingopoulos F, Olson LC, Patel HH, Pinchover S, Magro CM, Levine B, Lelli GJ. Lacrimal tissue resection in Fasanella Servat operation and the correlation to dry eye. Orbit 2020;39(3):171-174.Abstract
: Fasanella-Servat operation (FSO) was previously reported to be associated with post-operative dry eyes due to accessory lacrimal gland resection during the surgery.We performed a retrospective, cohort study to determine the frequency of lacrimal tissue resection during FSO and its correlation with post-operative eye dryness and keratopathy.: Review of all patients who underwent FSO at New York-Presbyterian Weill Cornell Hospital over a two-year period (2013-2015). Patients were included only if they had adequate histopathological specimens of the resected tissue obtained during surgery. Outcomes included the study of the pathological specimen for the presence of lacrimal tissue; Post-operative dry eye symptoms and pre- and post-operative corneal epitheliopathy.: 46 patients with a total of 58 eyelid resections were studied.Eight eyelids (13.7%) were found to have lacrimal tissue present in the pathology specimens.Postoperatively, nine patients reported some symptoms of dry eye and new-onset keratopathy was noted in four eyes (6.8%), only one of which had lacrimal tissue present in histopathology specimen obtained from surgery.: Previous studies found lacrimal tissue present in up to 43% of specimens resected during FSO. Our data found a lower rate of lacrimal tissue resection during FSO, and did not find an association between lacrimal tissue resection and post-operative dryness or epitheliopathy.: Our study is one of few to examine histopathological resections from the FSO.We found that lacrimal tissue is not frequently resected during FSO, and when it is resected, there is no increased incidence of post-operative dryness or keratopathy.
Vasilikos I, Haas J, Teixeira GQ, Nothelfer J, Neidlinger-Wilke C, Wilke H-J, Seitz A, Vavvas DG, Zentner J, Beck J, Hubbe U, Mizaikoff B. Infrared attenuated total reflection spectroscopic surface analysis of bovine-tail intervertebral discs after UV-light-activated riboflavin-induced collagen crosslinking. J Biophotonics 2020;:e202000110.Abstract
The tensile strength of the intervertebral disc (IVD) is mainly maintained by collagen cross-links. Loss of collagen cross-linking combined with other age-related degenerative processes contributes to tissue weakening, biomechanical failure, disc herniation and pain. Exogenous collagen cross-linking has been identified as an effective therapeutic approach for restoring IVD tensile strength. The current state-of-the-art method to assess the extent of collagen cross-linking in tissues requires destructive procedures and high-performance liquid chromatography (HPLC). In this study, we investigated the utility of infrared attenuated total reflection (IR-ATR) spectroscopy as a non-destructive analytical strategy to rapidly evaluate the extent of UV-light-activated riboflavin (B2)-induced collagen crosslinking (UVA-CXL) in bovine IVD samples. Thirty five fresh bovine-tail IVD samples were equally divided into five treatment groups: (i) untreated, (ii) cell culture medium DMEM only, (iii) B2 only, (iv) UV-light only, and (v) UV-light-B2. A total of 674 measurements have been acquired, and were analyzed via partial least squares discriminant analysis. This classification scheme unambiguously identified individual classes with a sensitivity >91% and specificity >92%. The obtained results demonstrate that IR-ATR spectroscopy reliably differentiates between different treatment categories, and promises an excellent tool for potential in vivo, non-destructive, and real-time assessment of exogenous IVD crosslinking. This article is protected by copyright. All rights reserved.
Soh YQ, Kocaba V, Weiss JS, Jurkunas UV, Kinoshita S, Aldave AJ, Mehta JS. Corneal dystrophies. Nat Rev Dis Primers 2020;6(1):46.Abstract
Corneal dystrophies are broadly defined as inherited disorders that affect any layer of the cornea and are usually progressive, bilateral conditions that do not have systemic effects. The 2015 International Classification of Corneal Dystrophies classifies corneal dystrophies into four classes: epithelial and subepithelial dystrophies, epithelial-stromal TGFBI dystrophies, stromal dystrophies and endothelial dystrophies. Whereas some corneal dystrophies may result in few or mild symptoms and morbidity throughout a patient's lifetime, others may progress and eventually result in substantial visual and ocular disturbances that require medical or surgical intervention. Corneal transplantation, either with full-thickness or partial-thickness donor tissue, may be indicated for patients with advanced corneal dystrophies. Although corneal transplantation techniques have improved considerably over the past two decades, these surgeries are still associated with postoperative risks of disease recurrence, graft failure and other complications that may result in blindness. In addition, a global shortage of cadaveric corneal graft tissue critically limits accessibility to corneal transplantation in some parts of the world. Ongoing advances in gene therapy, regenerative therapy and cell augmentation therapy may eventually result in the development of alternative, novel treatments for corneal dystrophies, which may substantially improve the quality of life of patients with these disorders.
Koulouri I, Hellwinkel O, Altenähr S, Spitzer M, Fritz S, Feuerstacke J, Filev F. A new storage solution for the hypothermic preservation of corneal grafts: an experimental study. Cell Tissue Bank 2020;Abstract
In this experimental study we used for the first time Tiprotec as a solution for corneal preservation and cold storage. We compared the resultant endothelial cell morphology and viability with this obtained after preservation of the ex-vivo corneas with both usual standard techniques: conventional cold storage (using Eusol-C) and organ culture. This prospective, in vitro, 3-armed parallel study was performed with the use of 90 porcine corneas (examined for their endothelial quality and transparency) randomly selected for preservation in three storage methods (each 30 corneas): organ culture, standard cold storage (Eusol-C) and experimental cold storage (Tiprotec) Endothelium cell quantity and quality as well as corneal opacification were assessed. The degree of endothelial transparency was significantly reduced over time with all preservation media, without any significant difference among the three groups at any point of time. A reduction in endothelial cell density was also observed with all three preservation media after 30 days of storage without statistically significant differences between groups. The number of hexagonal and pentagonal endothelium cells was significantly reduced overtime in all media with significantly more hexagonal and pentagonal in the organ culture group compared to the cold storage groups. We could show that the cryopreservation medium Tiprotec, used until now for the preservation of vascular grafts, was of similar quality compared to the medium Eusol-C for the hypothermic storage of corneal tissue for an extended period of time up to 30 days. In comparison to organic culture with culture medium KII, both Tiprotec and Eusol-C were found less effective in preserving endothelial cell quality, as assessed by the morphometric analysis, and viability, as assessed by the degree of vacuolization at least up to the 30th day of storage. However, both, Tiprotec- and Eusol-C-preserved corneas demonstrated a certain capacity to recover after their submission in organ culture.
Han X, Yang S, Kam WR, Sullivan DA, Liu Y. The carbonic anhydrase inhibitor dorzolamide stimulates the differentiation of human meibomian gland epithelial cells. Curr Eye Res 2020;Abstract
PURPOSE: Clinical studies have indicated that the long-term use of topical antiglaucoma drugs, such as carbonic anhydrase inhibitors (CAIs), may lead to meibomian gland dysfunction (MGD). We hypothesize that these adverse effects involve a direct influence on human MG epithelial cells (HMGECs). The purpose our present investigation was to test our hypothesis and determine whether exposure to dorzolamide, a CAI, impacts the proliferation, intracellular signaling and differentiation of HMGECs. : We cultured immortalized (i) HMGECs with vehicle or various concentrations of dorzolamide for 6 days. Cells were enumerated with a hemocytometer, and examined for their morphology, Akt signaling activity, accumulation of neutral lipids, phospholipids and lysosomes, and the expression of protein biomarkers for lipogenesis regulation, lysosomes and autophagosomes. : Our results show that a high, 500 µg/ml concentration of dorzolamide causes a significant decrease in Akt signaling and the proliferation of iHMGECs. However, the high dose of dorzolamide also promotes the differentiation of iHMGECs. This response features increases in the number of lysosomes, the accumulation of phospholipids, and the expression of the light chain 3A biomarker for autophagosomes. In contrast, the therapeutic amount (50 µg/ml) of dorzolamide has no impact on the proliferative or differentiative abilities of iHMGECs. : Our results support our hypothesis and demonstrate that the CAI dorzolamide does exert a direct influence on the proliferation and differentiation of iHMGECs. However, this effect is elicited only by a high, and not a therapeutic, amount of dorzolamide.
Yang J, LeBlanc ME, Cano I, Saez-Torres KL, Saint-Geniez M, Ng Y-S, D'Amore PA. ADAM10 and ADAM17 proteases mediate proinflammatory cytokine-induced and constitutive cleavage of endomucin from the endothelial surface. J Biol Chem 2020;295(19):6641-6651.Abstract
Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily -glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.
Wang Y, Carreno-Galeano JT, Singh RB, Dana R, Yin J. Long-term Outcome of Punctal Cauterization in the Management of Ocular Surface Diseases. Cornea 2020;Abstract
PURPOSE: To evaluate the long-term outcomes of surgical occlusion of lacrimal puncta using thermal cautery in the management of ocular surface diseases. METHODS: We reviewed medical records of 80 consecutive patients from a single academic center who underwent punctal cauterization. Patient demographics, ocular history, symptoms, and signs of ocular surface diseases pre- and post-cauterization were recorded. RESULTS: A total of 80 patients (171 puncta) were included, with an average age of 59 years and a follow-up duration of 27 months. The most common ocular morbidity was ocular graft-versus-host disease (n = 36), followed by primary keratoconjunctivitis sicca (n = 15). Indications for punctal cauterization included plug loss (n = 51), difficulty in plug fitting (n = 11), plug-related complications (n = 6), recanalization of previous cauterization (n = 7), and severe ocular surface disease requiring permanent punctal closure (n = 4). After punctal cauterization, the percentage of eyes with severe (21%) and moderate (25%) dry eye decreased significantly (8% and 19% at 3 months and 6% and 17% at 12 months, P = 0.0006). Fifty-four percent of patients reported improvement in their symptoms. The rate of recanalization was 21% during the follow-up period. The use of topical corticosteroids was associated with higher recanalization rate. Associated complications were limited to temporary pain and swelling. CONCLUSIONS: Punctal cauterization is an effective modality in treating severe ocular surface diseases in patients who repeatedly lose punctal plugs, and it can be easily performed in a clinic setting without major complications. However, cauterization may need to be repeated in up to a quarter of cases because of recanalization.
Wang J, Liu Y, Kam WR, Li Y, Sullivan DA. Toxicity of the cosmetic preservatives parabens, phenoxyethanol and chlorphenesin on human meibomian gland epithelial cells. Exp Eye Res 2020;196:108057.Abstract
Recently, we discovered that the cosmetic preservatives, benzalkonium chloride and formaldehyde, are especially toxic to human meibomian gland epithelial cells (HMGECs). Exposure to these agents, at concentrations approved for human use, leads within hours to cellular atrophy and death. We hypothesize that these effects are not unique, and that other cosmetic preservatives also exert adverse effects on HMGECs. Such compounds include parabens, phenoxyethanol and chlorphenesin, which have been reported to be toxic to corneal and conjunctival epithelial cells, the liver and kidney, as well as to irritate the eye. To test our hypothesis, we examined the influence of parabens, phenoxyethanol and chlorphenesin on the morphology, signaling, survival, proliferation and lipid expression of immortalized (I) HMGECs. These cells were cultured under proliferating or differentiating conditions with varying concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin for up to 5 days. We monitored the signaling ability, appearance, number and neutral lipid content of the IHMGECs, as well as their lysosome accumulation. Our findings show that a 30-min exposure of IHMGECs to these preservatives results in a significant reduction in the activity of the Akt pathway. This effect is dose-dependent and occurs at concentrations equal to (chlorphenesin) and less than (all others) those dosages approved for human use. Further, a 24-h treatment of the IHMGECs with concentrations of methylparaben, ethylparaben, phenoxyethanol and chlorphenesin close to, or at, the approved human dose induces cellular atrophy and death. At all concentrations tested, no preservative stimulated IHMGEC proliferation. Of particular interest, it was not possible to evaluate the influence of these preservatives, at close to human approved dosages, on IHMGEC differentiation, because the cells did not survive the treatment. In summary, our results support our hypothesis and show that methylparaben, ethylparaben, phenoxyethanol and chlorphenesin are toxic to IHMGECs.