Serum Differentially Modulates the Clonal Growth and Differentiation of Cultured Limbal and Corneal Epithelium

Purpose. The stem cell-containing limbal epithelium is in proximity with highly vascularized tissue, as opposed to the transient amplifying cell-containing corneal basal epithelium, which resides on top of avascular corneal stroma. We therefore speculate that limbal stem cells are preferentially under the modulation of serum-derived factors. Methods. Using a previously reported serum-free, chemically defined culture system for ocular surface epithelium, a culture condition primarily supporting transient amplifying cells of both corneal and limbal epithelia, we compared the clonal growth measured by colony-forming efficiency (CFE), colony size, and BrdU labeling, as well as colony differentiation measured by colony morphology and immunofluorescence staining, with the monoclonal antibody AE-5 against keratin K3 when fetal bovine serum (FBS) was added at different concentrations. Results. The addition of 1% FBS decreased CFE and colony size in peripheral corneal cultures but had no effect in limbal cultures. Both cultures showed no obvious difference in colony morphology or BrdU labeling and AE-5 staining. In contrast, at 10% or 20% FBS, CFE and colony size increased in limbal cultures, but dose dependently decreased in peripheral corneal cultures. The presence of a unique subpopulation of progenitor cells in limbal cultures different from transient amplifying cells in corneal cultures was further supported by the emergence of a higher proportion of a unique type (B) colonies in limbal cultures that had high BrdU labeling and heterogeneous or negative AE-5 staining, indicative of their being in a proliferating, undifferentiated state. These colonies showed continuous growth in late cultures and could be passaged into serum-free medium. Conclusio

Correlation between Ocular Demodex Infestation and Serum Immunoreactivity to Bacillus Proteins in Patients with Facial Rosacea

Purpose—To investigate correlation between ocular Demodex infestation and serum. Design—A prospective study to correlate clinical findings with laboratory data. Participants—We consecutively enrolled 59 patients: 34 men and 25 women with a mean age of 60.4±17.6 years (range, 17–93). Methods—Demodex counting was performed based on lash sampling. Serum immunoreactivity to two 62-kDa and 83-kDa proteins derived from B oleronius was determined by Western blot analysis. Facial rosacea, lid margin, and ocular surface inflammation were documented by photography and graded in a masked fashion. Main Outcome Measures—Statistical significance based on correlative analyses of clinical and laboratory data. Results—These 59 patients were age matched, but not gender matched, regarding serum immunoreactivity, ocular Demodex infestation, or facial rosacea. There was a significant correlation between serum immunoreactivity and facial rosacea (P = 0.009), lid margin inflammation (P = 0.040), and ocular Demodex infestation (P = 0.048), but not inferior bulbar conjunctival inflammation (P = 0.573). The Demodex count was significantly higher in patients with positive facial rosacea (6.6±9.0 vs. 1.9±2.2; P = 0.014). There was a significant correlation of facial rosacea with lid margin inflammation (P = 0.016), but not with inferior bulbar conjunctival inflammation (P = 0.728). Ocular Demodex infestation was less prevalent in patients with aqueous tear-deficiency dry eye than those without (7/38 vs. 12/21; P = 0.002). Conclusions—The strong correlation provides a better understanding of comorbidity between Demodex mites and their symbiotic B oleronius in facial rosacea and blepharitis. Treatments directed to both warrant future investigation

In Vivo Downregulation of Innate and Adaptive Immune Responses in Corneal Allograft Rejection by HC-HA/PTX3 Complex Purified From Amniotic Membrane

Citation: He H, Tan Y, Duffort S, Perez VL, Tseng SCG. In vivo downregulation of innate and adaptive immune responses in corneal allograft rejection by HC-HA/PTX3 complex purified from amniotic membrane. Invest Ophthalmol Vis Sci. 2014;55:164755: -165655: . DOI:10.1167 PURPOSE. Heavy chain-hyaluronic acid (HC-HA)/PTX3 purified from human amniotic membrane (AM) was previously observed to suppress inflammatory responses in vitro. We now examine whether HC-HA/PTX3 is able to exert a similar effect in vivo, using murine models for keratitis and corneal allograft rejection. METHODS. The in vitro effect of HC-HA/PTX3 was tested using OTII ovalbumin (OVA) transgenic, purified CD4 þ T cells, or IFN-c/lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Cytokine production was measured by ELISA, while cell surface markers and cell proliferation were determined by flow cytometry. In vivo effects of HC-HA/PTX3 were analyzed by quantifying the recruitment of enhanced green fluorescence-labeled macrophages and by measuring the expression of arginase 1 (Arg-1), IL-10, and IL-12 in LPS-induced keratitis in the macrophage Fas-induced apoptosis (Mafia) mouse. The effect of corneal allograft survival in a complete major histocompatibility complex (MHC) mismatched mouse model was assessed by grading corneal opacification. RESULTS. In vitro studies demonstrated that HC-HA/PTX3 significantly enhanced the expansion of FOXP3 T cells and suppressed cell proliferation and protein expression of IFN-c, IL-2, CD25, and CD69 in activated CD4 þ T cells. Furthermore, immobilized HC-HA/PTX3 significantly upregulated IL-10 gene expression but downregulated that of IL-12 and IL-23 in activated RAW264.7 cells. Finally, in vivo subconjunctival injection of HC-HA/PTX3 significantly prolonged corneal allograft survival, suppressed macrophage infiltration, and promoted M2 polarization by upregulating Arg-1 and IL-10 but downregulating IL-12. CONCLUSIONS. HC-HA/PTX3 can suppress inflammatory responses in vivo by modulating both innate and adaptive immunity of macrophages and CD4 þ T cells

Air exposure-induced squamous metaplasia of human limbal epithelium

PURPOSE. Squamous metaplasia is a pathologic process that frequently occurs in nonkeratinized stratified ocular surface epithelia. The mechanism for this occurrence is largely unknown except for vitamin A deficiency. METHODS. Human limbal explants were cultured under airlift with or without p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 or in a submerged manner for different durations up to 2 weeks. Epithelial cell proliferation, differentiation, limbal stem cell maintenance, and expansion were studied using certain markers such as Ki67, p63, K10 and K12 keratins, filaggrin, Pax6, ABCG-2, and Musashi-1. Expression of phospho-p38 MAPK and its downstream transcription factors, C/EBP alpha and C/EBP beta, were studied by immunohistochemistry. Epithelial cells harvested from explants after 2 weeks of culturing under different conditions were seeded onto 3T3 feeder layers and cultured for 12 days. The differentiation of clonal epithelial cells was investigated by double staining to K12 and K10 keratins. RESULTS. The squamous metaplasia model was successfully created by culturing human limbal explants at an air-liquid interface (airlift) for 2 weeks. Increased stratification and hyperproliferation only happened in the limbal, but not the corneal, epithelium in airlift, but not submerged, cultures. Epithelial proliferation was associated with a transient increase of limbal epithelial stem cells. Abnormal epidermal differentiation-evidenced by positive expression of K10 keratin in suprabasal cells and filaggrin in superficial cells-ensued. Clones generated from epithelial cells harvested from airlift culture only expressed K12 keratin without K10. As early as 2 days in airlift cultures, p38 expression emerged in limbal basal epithelial cells and gradually extended to the cytoplasm and nuclei. Furthermore, addition of the p38 inhibitor SB203580 abolished abnormal epidermal differentiation without affecting limbal epithelial proliferation. Expression of C/EBP alpha and C/EBP beta, downstream of the p38 MAPK signaling pathway, was strongly induced by airlift culture and partially was inhibited by SB203580. CONCLUSIONS. Dryness resulting from exposure activates p38 MAPK signaling coupled with abnormal epidermal differentiation without intrinsic alteration of stem cells in the limbus. On the ocular surface, p38 inhibitors may have the potential to revert the pathologic process of squamous metaplasia induced by dryness

Selective Activation of p120ctn-Kaiso Signaling to Unlock Contact Inhibition of ARPE-19 Cells without Epithelial-Mesenchymal Transition

Contact-inhibition ubiquitously exists in non-transformed cells and explains the poor regenerative capacity of in vivo human retinal pigment epithelial cells (RPE) during aging, injury and diseases. RPE injury or degeneration may unlock mitotic block mediated by contact inhibition but may also promote epithelial-mesenchymal transition (EMT) contributing to retinal blindness. Herein, we confirmed that EMT ensued in post-confluent ARPE-19 cells when contact inhibition was disrupted with EGTA followed by addition of EGF and FGF-2 because of activation of canonical Wnt and Smad/ZEB signaling. In contrast, knockdown of p120-catenin (p120) unlocked such mitotic block by activating p120/Kaiso, but not activating canonical Wnt and Smad/ZEB signaling, thus avoiding EMT. Nuclear BrdU labeling was correlated with nuclear release of Kaiso through p120 nuclear translocation, which was associated with activation of RhoA-ROCK signaling, destabilization of microtubules. Prolonged p120 siRNA knockdown followed by withdrawal further expanded RPE into more compact monolayers with a normal phenotype and a higher density. This new strategy based on selective activation of p120/Kaiso but not Wnt/β-catenin signaling obviates the need of using single cells and the risk of EMT, and may be deployed to engineer surgical grafts containing RPE and other tissues