Inflammation may play a significant role in Keratoconus (KC), but the implication of immunity on this inflammatory response is unknown. Therefore, our aim was to determine the expression levels of Toll-like receptors 2 (TLR2) and 4 (TLR4) in monocytes and neutrophils from patients with KC and control subjects for demonstrating the role of innate immunity in KC. We also study the correlation between TLR2/TLR4 expression and serum levels of proinflammatory markers (IL-1β, IL-6, TNF-α, MMP-9 and NF-κB). Forty patients with bilateral KC (55% males; mean age; 33.1 ± 10.9 years) and 20 control subjects (55% males; mean age; 30.4 ± 7.6 years) were included in the study. Our results showed that mean expression of TLR2 and TLR4 in both neutrophils and monocytes was significantly higher in patients with KC compared to control subjects (all p < 0.0001). Furthermore, KC patients also showed higher serum levels of IL-1B, IL-6, TNF-α, MMP-9 (all p < 0.0001) and NF-κB (p = 0.036). In addition, we found a strong correlation between TLR2 expression in both monocytes and neutrophils (all p < 0.0001), and TLR4 in monocytes (all p < 0.05) with serum levels of IL-1B, IL-6, TNF-α and MMP-9. In conclusion, these findings suggest that TLRs may play an important role in the pathophysiology of KC.

Purpose To elucidate dysregulated proteins in keratoconus (KC) to provide a better understanding of the molecular mechanisms that lead to the development of the disease using sequential window acquisition of all theoretical mass spectra (SWATH-MS) as a protein quantification tool of the tear proteomic profile. Methods Prospective cross-sectional study that includes 25 keratoconic eyes and 25 healthy eyes. All participants underwent a clinical, tomographic, and aberrometric exam. Tear sample was collected using Schirmer strips and analyzed by liquid chromatography with tandem mass spectrometry. SWATH-MS was used as a quantification tool of the tear proteomic profile. The expression of the quantified proteins was compared between groups, and the biological and molecular functions of the dysregulated proteins as well as their functional relationships were studied by in silico analysis. Results A total of 203 proteins were quantified in tear samples of patients with KC and control participants, of which 18 showed differential expression between groups ( P < 0.05). An increase in the expression of 7 proteins and a decrease in the expression of 11 proteins were observed. Protein–protein interactions and gene ontology analysis showed the involvement of these dysregulated proteins in structural, inflammatory-immune, iron homeostasis, oxidative stress, and extracellular matrix proteolysis processes. Conclusions Tear protein quantification has revealed the dysregulation of proteins involved in biological processes previously associated with KC. Among them, iron homeostasis should be highlighted as a relevant pathway in the KC pathophysiology, and it should be taken into account in the development of therapeutic targets to cope with tissue damage derived from iron accumulation and toxicity.

This research study describes the design, optimization, and characterization of two different types of chitosan-based nanoparticles as novel drug delivery systems of a protein drug, lactoferrin. A preclinical consistent base was obtained for both nanosystems, being considered as the first pharmacological treatment for keratoconus as an alternative to current invasive clinical methods. Both types of nanoparticles were obtained via the ionotropic gelation technique. The size and morphology of the nanoparticles were studied as a function of the preparation conditions. A mean size of 180.73 ± 40.67 nm, a size distribution [polydispersity index (PDI)] of 0.170 ± 0.067, and positive ζ-potential values, ranging from 17.13 to 19.89 mV, were achieved. Lactoferrin was successfully incorporated into both types of nanocarriers. In vitro release profiles showed a lactoferrin enhanced, prolonged, and controlled delivery from the polymeric matrix. These formulations also demonstrated no stability or cytotoxicity problems, as well as appropriate mucoadhesive properties, with a high permanence time in the ocular surface. Thus, both types of nanoparticles may be considered as nanocarriers for the controlled release of lactoferrin as novel topical ophthalmic drug delivery systems.

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TLR2 and TLR4 expression in corneal and conjunctival epithelial cells may constitute predictive biomarkers for the early detection of KC.

Alzheimer’s Disease (AD) is one of the main neurodegenerative diseases worldwide. Unfortunately, AD shares many similarities with other dementias at early stages, which impedes an accurate premortem diagnosis. Therefore, it is urgent to find biomarkers to allow for early diagnosis of the disease. There is increasing scientific evidence highlighting the similarities between the eye and other structures of the CNS, suggesting that knowledge acquired in eye research could be useful for research and diagnosis of AD. For example, the retina and optic nerve are considered part of the central nervous system, and their damage can result in retrograde and anterograde axon degeneration, as well as abnormal protein aggregation. In the anterior eye segment, the aqueous humor and tear film may be comparable to the cerebrospinal fluid. Both fluids are enriched with molecules that can be potential neurodegenerative biomarkers. Indeed, the pathophysiology of AD, characterized by cerebral deposits of amyloid-beta (Aβ) and tau protein, is also present in the eyes of AD patients, besides numerous structural and functional changes observed in the structure of the eyes. Therefore, all this evidence suggests that ocular changes have the potential to be used as either predictive values for AD assessment or as diagnostic tools.

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Keratoconus (KC) is a corneal disorder whose etiology shares a close relationship with Lactoferrin (LTF) dysregulation and Toll-like Receptors 2 (TLR2) overexpression. This study shows how these two important biomarkers are clinically and molecularly interrelated, increasing knowledge about KC pathophysiology, and opening the door to future therapies. In this prospective clinical study, serum and tear LTF concentrations were quantified in 90 KC patients and 60 controls. A correlation analysis with multiple blood and tear immunoinflammatory mediators, and KC-associated tomographic parameters, was performed. An in vitro study using HEK-BlueTMhTLR2 cell cultures was also conducted to determine the expression and functionality of TLR2 under the influence of LTF treatment. As a result, a LTF decreased was observed in KC patients compared to controls (p < 0.0001), evidencing the strong correlation with TLR2 overexpression at systemic and ocular surface level, with inflammatory mediator upregulation and with KC severity. In stimulated cell cultures, TLR2 expression was decreased using 2 mg/mL of LTF. The levels of secreted embryonic alkaline phosphatase (SEAP) and interleukin-8 (IL-8) were also reduced in supernatants after LTF treatment. As conclusions, the dysregulation of LTF and TLR2 in the ocular surface of KC patients contributes to KC severity by maintaining a detrimental chronic immune–inflammatory state. The immunomodulatory properties of LTF on TLR2 expression suggest its potential as a therapeutic approach for KC.