Velika Deligianni

Disinfection of Ocular Cells and Tissues by Atmospheric-Pressure Cold Plasma

Background Low temperature plasmas have been proposed in medicine as agents for tissue disinfection and have received increasing attention due to the frequency of bacterial resistance to antibiotics. This study explored whether atmospheric-pressure cold plasma (APCP) generated by a new portable device that ionizes a flow of helium gas can inactivate ocular pathogens without causing significant tissue damage. Methodology/Principal Findings We tested the APCP effects on cultured Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Candida albicans, Aspergillus fumigatus and Herpes simplex virus-1, ocular cells (conjunctival fibroblasts and keratocytes) and ex-vivo corneas. Exposure to APCP for 0.5 to 5 minutes significantly reduced microbial viability (colony-forming units) but not human cell viability (MTT assay, FACS and Tunel analysis) or the number of HSV-1 plaque-forming units. Increased levels of intracellular reactive oxygen species (ROS) in exposed microorganisms and cells were found using a FACS-activated 2′,7′-dichlorofluorescein diacetate probe. Immunoassays demonstrated no induction of thymine dimers in cell cultures and corneal tissues. A transient increased expression of 8-OHdG, genes and proteins related to oxidative stress (OGG1, GPX, NFE2L2), was determined in ocular cells and corneas by HPLC, qRT-PCR and Western blot analysis. Conclusions A short application of APCP appears to be an efficient and rapid ocular disinfectant for bacteria and fungi without significant damage on ocular cells and tissues, although the treatment of conjunctival fibroblasts and keratocytes caused a time-restricted generation of intracellular ROS and oxidative stress-related responses.

A novel plasma source for sterilization of living tissues

A source for the production of low-power plasmas at atmospheric pressure, to be used for the nondamaging sterilization of living tissues, is presented. The source, powered by radiofrequency and working with a helium flow, has a specific configuration, studied to prevent the formation of electric arcs dangerous to living matter. It is capable of killing different types of bacteria with a decimal reduction time of 1?2?min; on the contrary, human cells such as conjunctival fibroblasts were found to be almost unharmed by the plasma. A high concentration of OH radicals, likely to be the origin of the sterilizing effect, is detected through their UV emission lines. The effect of the UV and the OH radicals on the fibroblasts was analysed and no significant effects were detected.

Tailored Approach to the Treatment of Vernal Keratoconjunctivitis

PURPOSE To develop a standardized clinical grading system for the management of patients with vernal keratoconjunctivitis (VKC) and to identify the risk factors associated with a worsened outcome of the disease, including decrease of visual acuity and ocular complications development. DESIGN Retrospective cohort study. PARTICIPANTS A total of 207 consecutive patients with VKC, referred to our Cornea and External Diseases Center from 1997 to 2007, were included in the study. A total of 110 of those patients were included in the follow-up study (range 1-10 years). METHODS Classification and regression tree (CART) analysis was performed to separate the patients into 5 subgroups by therapeutic approach. Regression tree and multivariate logistic regression analyses were performed during follow-up to identify predictors of worse visual outcome. MAIN OUTCOME MEASURES Age, gender, duration and course of disease, signs, symptoms, overall symptoms score, history of atopy, markers of allergy, best-corrected visual acuity, and therapy were collected at baseline and during follow-up. The number of relapses and number of patients with decreased visual acuity were also evaluated in the follow-up. RESULTS A decision tree for VKC treatment was developed by CART analysis, and a new clinical grading system was proposed accordingly. Sixteen patients were classified as grade 0 (absence of symptoms and no therapy); 59 patients were classified as grade 1 (presence of symptoms without photophobia, occasional use of anti-allergic eye drop); 74 patients were classified as grade 2 (presence of symptoms including photophobia, daily anti-allergic treatment); 22 patients with superficial punctuate keratopathy (SPK) were classified as grade 3 (daily anti-allergic treatment associated with occasional topical steroid); and 36 patients were classified as grade 4 (diffuse SPK or corneal ulcer; pulsed high-dose topical steroid). A higher number of relapses and a higher baseline grade of VKC were the main predictor factors for worse visual outcome. CONCLUSIONS This grading system allows for identifying the more severe forms of VKC that are at higher risk of recurrences, corneal ulceration, and worse final visual outcome. FINANCIAL DISCLOSURE(S) The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Vernal keratoconjunctivitis-like disease in adults.

PURPOSE To identify clinical, demographic, immunologic, and health-related quality-of-life data from a cohort of vernal keratoconjunctivitis (VKC) patients with the onset of the disease after puberty (VKC-like disease). DESIGN Retrospective, observational case series. METHODS Forty-nine patients with late-onset VKC-like disease from among 600 consecutive VKC patients. History of disease, test results for allergen sensitivity, signs and symptoms, impact of disease on work productivity, health-related quality of life, and treatment satisfaction were assessed. In addition, multiplex bead analysis for Th1/Th2 cytokines were carried out in tear samples from 20 VKC patients (10 adults and 10 children) and from 10 normal subjects. RESULTS A family history of allergy was positive in only 28% and positive prick test results were present in 55% of the 49 VKC-like adult patients. Based on typical signs and symptoms, 48% were affected by the limbal form, 33% were affected by the tarsal form, and 19% were affected by the mixed form. Corneal ulcer complicated the disease in only 2 adult patients. Although the disease was not considered a limiting factor for work, productivity was reduced by 26% and social activities were reduced by 31% during active flare-ups. No significant differences were found in tear cytokine pattern production between VKC in children and VKC in adults. CONCLUSIONS A late onset VKC-like disease can appear in young adults with signs and symptoms similar to those in pediatric disease, but with less corneal involvement.

Ocular toxicology : the Draize eye test

Purpose of review Superficial ocular tissues are frequently exposed to damage produced by chemical compounds applied on or around the ocular surface for cosmetic, therapeutic or accidental reasons. An experimental test measuring objectively and in a reproducible way any potential damaging effect would certainly help in prospectively minimizing unwanted effects. The Draize eye test, although commonly employed to date, does not seem to be quite satisfactory in this respect. Recent findings The limits of the Draize eye test and the results of some modified versions of the test are analysed. In particular, the good predictivity of the so-called low-volume Draize eye test and the recent findings of studies on the in-vitro and ex-vivo alternatives to the Draize eye test are presented. Summary The Draize eye test, despite criticisms, has been used in the clinical setting for a long time and still remains the reference protocol. To date, only a combination of alternative methods, none of which is devoid of serious criticisms, seems to be able to exhaustively recognize potential irritants and avoiding for that purpose, in some cases, the use of living animals.

Atmospheric-Pressure Cold Plasma Induces Transcriptional Changes in Ex Vivo Human Corneas

Background Atmospheric pressure cold plasma (APCP) might be considered a novel tool for tissue disinfection in medicine since the active chemical species produced by low plasma doses, generated by ionizing helium gas in air, induces reactive oxygen species (ROS) that kill microorganisms without substantially affecting human cells. Objectives In this study, we evaluated morphological and functional changes in human corneas exposed for 2 minutes (min) to APCP and tested if the antioxidant n-acetyl l-cysteine (NAC) was able to inhibit or prevent damage and cell death. Results Immunohistochemistry and western blotting analyses of corneal tissues collected at 6 hours (h) post-APCP treatment demonstrated no morphological tissue changes, but a transient increased expression of OGG1 glycosylase that returned to control levels in 24 h. Transcriptome sequencing and quantitative real time PCR performed on different corneas revealed in the treated corneas many differentially expressed genes: namely, 256 and 304 genes showing expression changes greater than ± 2 folds in the absence and presence of NAC, respectively. At 6 h post-treatment, the most over-expressed gene categories suggested an active or enhanced cell functioning, with only a minority of genes specifically concerning oxidative DNA damage and repair showing slight over-expression values (<2 folds). Moreover, time-related expression analysis of eight genes up-regulated in the APCP-treated corneas overall demonstrated the return to control expression levels after 24 h. Conclusions These findings of transient oxidative stress accompanied by wide-range transcriptome adjustments support the further development of APCP as an ocular disinfectant.