Purpose.: The present study was designed to understand the role of inflammatory cytokines secreted by corneal epithelial cells in keratoconus (KC) and the response to treatment with cyclosporine A (CyA). Methods.: The study involved 129 Indian KC patients clinically graded according to Amsler-Krumeich classification and 20 healthy, nonectatic subjects as controls. Tear levels of matrix metalloproteinase-9 (MMP9), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) were measured using ELISA kits. Gene expression was measured by qPCR in corneal epithelial cells obtained by debridement from subjects undergoing ocular surface surgeries. In addition, epithelial cells were stimulated with TNFα and treated with CyA to study its role on MMP9 expression. Finally, 20 KC patients (27 eyes) with inflammatory symptoms were treated with topical CyA application. Results.: We observed that MMP9, TNFα, and IL6 levels were strongly upregulated at the mRNA level in KC patient epithelia. Similarly, tears collected from KC patients exhibited high levels of MMP9 and IL6 protein. Cyclosporine A treatment significantly reduced the mRNA expression levels of IL6 and TNFα in both short- and long-term treatments; however, it reduced MMP9 levels only in long-term treatment in cultured corneal epithelial cells. Subsequent treatment of KC patients with CyA for approximately 6 months reduced tear MMP9 levels and led to local reduction in corneal curvatures as determined by corneal topography maps. Conclusions.: The data indicate that corneal epithelium contributes to elevated MMP9 and inflammatory cytokine expression in tears of KC patients. Cyclosporine A treatment reduced MMP9 and inflammatory cytokine levels in an in vitro inflammation model system. In KC patients, CyA treatment reduced MMP9 levels measured in tears with concomitant arrest of disease progression. Therefore, CyA might be a novel treatment strategy in KC patients but requires additional evaluation in larger cohorts. (ClinicalTrials.gov number, NCT01746823.)

The functionality of stem cells is tightly regulated by cues from the niche, comprising both intrinsic and extrinsic cell signals. Besides chemical and growth factors, biophysical signals are important components of extrinsic signals that dictate the stem cell properties. The materials used in the fabrication of scaffolds provide the chemical cues whereas the shape of the scaffolds provides the biophysical cues. The effect of the chemical composition of the scaffolds on stem cell fate is well researched. Biophysical signals such as nanotopography, mechanical forces, stiffness of the matrix, and roughness of the biomaterial influence the fate of stem cells. However, not much is known about their role in signaling crosstalk, stem cell maintenance, and directed differentiation. Among the various techniques for scaffold design, nanotechnology has special significance. The role of nanoscale topography in scaffold design for the regulation of stem cell behavior has gained importance in regenerative medicine. Nanotechnology allows manipulation of highly advanced surfaces/scaffolds for optimal regulation of cellular behavior. Techniques such as electrospinning, soft lithography, microfluidics, carbon nanotubes, and nanostructured hydrogel are described in this review, along with their potential usage in regenerative medicine. We have also provided a brief insight into the potential signaling crosstalk that is triggered by nanomaterials that dictate a specific outcome of stem cells. This concise review compiles recent developments in nanoscale architecture and its importance in directing stem cell differentiation for prospective therapeutic applications.

Purpose: The purpose of this study was to study the status and association among tear-soluble factors, corneal dendritic cell density, vitamin D, and signs and symptoms in dry eye disease (DED). Methods: A total of 33 control subjects and 47 evaporative dry eye patients were included in the study. DED diagnosis and classification was based on the 2017 Report of the Tear Film & Ocular Surface Society International Dry Eye Workshop (TFOS DEWS II). DED workup, including tear film break-up time (TBUT), Schirmer's test I (STI), corneal and conjunctival staining, ocular surface disease index (OSDI) scoring, and in vivo confocal microscopy (to assess corneal dendritic cell density [cDCD] and subbasal nerve plexus [SBNP] features) was performed in the study subjects. Tear fluid using Schirmer's strip and serum were collected from the subjects. Multiplex ELISA or single analyte ELISA was performed to measure 34 tear-soluble factors levels including vitamin D. Results: Significantly higher OSDI discomfort score, lower TBUT, and lower STI were observed in DED patients. cDCD was significantly higher in DED patients. No significant difference was observed in SBNP features. Tear fluid IL-1β, IL-17A, MMP9, MMP10, MMP9/TIMP ratio, and VEGF-B were significantly higher in DED patients. Significantly lower tear fluid IL-2, IP-10, NPY, VEGF-A, and vitamin D was observed in DED patients. These dysregulated tear factors showed significant associations with DED signs and symptoms. Conclusions: Altered tear fluid soluble factors with potential to modulate nociception exhibited a distinct association with ocular surface discomfort status, TBUT, STI, and cDCD. This implies a functional relationship between the various tear-soluble factors and dry eye pathogenesis, indicating new molecular targets for designing targeted therapies.

In Notch signaling, cell-bound ligands activate Notch receptors on juxtaposed cells, but the relationship between ligand endocytosis, ubiquitylation and ligand-receptor interaction remains poorly understood. To study the specific role of ligand-receptor interaction, we identified a missense mutant of the Notch ligand Jagged1 (Nodder, Ndr) that failed to interact with Notch receptors, but retained a cellular distribution that was similar to wild-type Jagged1 (Jagged1^WT) in the absence of active Notch signaling. Both Jagged1^WT and Jagged1^Ndr interacted with the E3 ubiquitin ligase Mind bomb, but only Jagged1^WT showed enhanced ubiquitylation after co-culture with cells expressing Notch receptor. Cells expressing Jagged1^WT, but not Jagged1^Ndr, trans-endocytosed the Notch extracellular domain (NECD) into the ligand-expressing cell, and NECD colocalized with Jagged1^WT in early endosomes, multivesicular bodies and lysosomes, suggesting that NECD is routed through the endocytic degradation pathway. When coexpressed in the same cell, Jagged1^Ndr did not exert a dominant-negative effect over Jagged1^WT in terms of receptor activation. Finally, in Jag1^Ndr/Ndr mice, the ligand was largely accumulated at the cell surface, indicating that engagement of the Notch receptor is important for ligand internalization in vivo. In conclusion, the interaction-dead Jagged1^Ndr ligand provides new insights into the specific role of receptor-ligand interaction in the intracellular trafficking of Notch ligands.