Long-Term Effects of Adipose-Derived Stem Cells for the Treatment of Bilateral Limbal Stem Cell Deficiency.

PURPOSE: To determine the safety and feasibility of human autologous adipose tissue-derived adult mesenchymal stem cells (ASCs) for ocular surface regeneration in patients with bilateral limbal stem-cell deficiency (LSCD). METHODS: A phase IIa clinical trial was designed (https://Clinicaltrials.gov, NCT01808378) with 8 patients, 3 of whom had aniridia, 2 meibomian glands diseases, 2 multiple surgeries and 1 chronic chemical injury. The therapeutic protocol was as follows: 6-mm of central corneal epithelium was removed, 400,000 ASCs were injected into each limboconjunctival quadrant, 400,000 ASCs were suspended over the cornea for 20 min, and finally the cornea was covered with an amniotic membrane patch. RESULTS: No adverse events were detected after a mean of 86,5 months of follow-up. One year after surgery, 6 of the 8 transplants were scored as successful, five patients had improved uncorrected visual acuity (mean of 12 letters), two patients presented epithelial defects (also present at baseline) and the mean percentage of corneal neovascularization was of 28.75% (36.98%, at baseline). Re-examination 24 months after treatment disclosed preserved efficacy in 4 patients. At the last visit (after a mean of 86,5 months of follow up) epithelial defects were absent in all patients although improvement in all of the variables was only maintained in patient 3 (meibomian glands agenesia). CONCLUSION: ASCs are a feasible and conservative therapy for treating bilateral LSCD. The therapeutic effect differs between etiologies and diminishes over time.

A Pilot Study of a Panel of Ocular Inflammation Biomarkers in Patients with Primary Sjögren's Syndrome.

Ocular diseases have a strong impact on individuals, the effects of which extend from milder visual impairment to blindness. Due to this and to their prevalence, these conditions constitute important health, social and economic challenges. Thus, improvements in their early detection and diagnosis will help dampen the impact of these conditions, both on patients and on healthcare systems alike. In this sense, identifying tear biomarkers could establish better non-invasive approaches to diagnose these diseases and to monitor responses to therapy. With this in mind, we developed a solid phase capture assay, based on antibody microarrays, to quantify S100A6, MMP-9 and CST4 in human tear samples, and we used these arrays to study tear samples from healthy controls and patients with Sjögren's Syndrome, at times concomitant with rheumatoid arthritis. Our results point out that the detection of S100A6 in tear samples seems to be positively correlated to rheumatoid arthritis, consistent with the systemic nature of this autoinflammatory pathology. Thus, we provide evidence that antibody microarrays may potentially help diagnose certain pathologies, possibly paving the way for significant improvements in the future care of these patients.

The analysis of human conjunctival epithelium proteome in ocular surface diseases using impression cytology and 2D-DIGE.

Conjunctival impression cytology samples from patients with meibomian gland dysfunction (MGD), dry eye (DE), and healthy subjects (CT) were collected for determination of the degree of squamous metaplasia (SM) by PAS-hematoxylin staining and for comparative proteomic analyses by 2D-DIGE. The protein spots with discriminant expression were identified by MALDI-TOF/TOF mass spectrometry. Three independent statistical studies were conducted: i). Analysis of differential protein expression between study groups: We observed increased expression of proteins S100A4, S100A8, retinal dehydrogenase-1, peroxiredoxin-1, annexin-A1, annexin-A2, α-enolase, and glutathione S-transferase-P in DE, whereas the highest expression of peroxiredoxin-6, actin cytoplasmic-1, peroxiredoxin-2, and heat shock protein HSP-90-α was observed in MGD; ii). Correlation between changes in the proteome profile and the grade of SM: The expression of 5 different cytokeratins (KRT1, KRT4, KRT8, KRT10, and KRT13) correlated with the degree of SM; iii). Proteome profile differences between pathological and CT groups: An overall proteome analysis revealed upregulation of 9 proteins in the pathological groups (Annexin-A1, α-enolase, Annexin-A2, S100A8, cytokeratin-1, Peroxiredoxin-2 and Leukocyte elastase inhibitor) and downregulation of 2 proteins (Galectin-3 and Lipocalin-1). In conclusion, a sensitive proteomic approach to study conjunctival tissue collected from minimally invasive impression cytology was implemented. Differential proteomics analyses showed that in comparison with the MGD, the DE patients presented higher overexpression of proteins related to antimicrobial defense, tissue-damage response, and regulation of body fluid secretions. Changes in MGD proteome were associated with oxidative stress and anti-apoptotic processes. We found a correlation between the grade of SM and expression of proteins associated with cytoskeleton and keratinization. The studied pathological groups shared elements related to the defense and inflammatory responses. Dot blot assays of proteins ANXA1, S100A8, and S100A4 validated the proteomic results obtained from 2D-DIGE experiments and confirmed the correlation between the expression of these proteins and the clinical parameters.

Clinical and microbiological profile of infectious keratitis in an area of Madrid, Spain.

INTRODUCTION: To study antibiotic susceptibility in bacterial keratitis (BK), its profile over 10 years and its influence on ophthalmological practice. METHODS: Retrospective review of BK with positive corneal scraping over a 10-year period. Risk factors for keratitis, visual acuity (VA), empirical topical treatment, corneal infection characteristics and outcomes were analyzed for BK due to Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Pseudomonas aeruginosa and Propionibacterium acnes. RESULTS: 389 positive corneal scrapings were collected. All Gram-positive bacteria were susceptible to vancomycin. P. aeruginosa demonstrated >90% sensitivity to the most-commonly-used topical antibiotics. Susceptibility to methicillin was 90.2% for S. aureus and 66.3% for S. epidermidis. The results of 215 patients were available. 1.9% required enucleation and 2.8% required surgical treatments. Final VA improved after treatment in keratitis due to S. aureus (p=0.026) and S. epidermidis (p=0.005). There was a correlation between S. aureus resistance to methicillin (p=0.002) and levofloxacin (p=0.043) and enucleation (20% and 10%, respectively) compared with a 0% rate of enucleation in S. aureus-susceptible keratitis. CONCLUSIONS: BK due to S. pneumoniae is very aggressive irrespective of antibiotic sensitivity. S. aureus was frequently isolated in patients with systemic diseases. It causes severe keratitis and remains moderately resistant to methicillin and levofloxacin. For this reason, keeping vancomycin in empirical regimens is believed to be necessary.

Intraocular minocycline for the treatment of ocular pythiosis.

PURPOSE: A case of ocular pythiosis successfully treated with surgery and intraocular and oral minocycline is reported. SUMMARY: A 30-year-old man who wore corrective contact lenses traveled to Brazil and Colombia where he swam in salt and fresh waters while wearing contact lenses. He sought treatment at an emergency department after 2 weeks of suffering with a painful corneal ulcer, redness, and loss of vision in his right eye that had been treated at other centers with ophthalmic moxifloxacin for 10 days and with fortified topical antibiotics (amikacin and vancomycin) for 2 days. Examination using a slit lamp revealed a deep central corneal ulcer with surrounding white infiltrate, endothelial plaque, and hypopyon. Due to infection severity, the patient was admitted and received empirical antibiotic therapy and i.v. and topical antifungals. During the first corneal transplantation, the patient's original infection relapsed and was treated with voriconazole and liposomal amphotericin B intraocular injections. A subsequent infection developed, and a second keratoplasty was performed. One month after hospital admission, the patient was diagnosed with ocular pythiosis and therapy with oral minocycline was initiated. After severe infection relapse in the anterior chamber, the patient underwent a third penetrating keratoplasty, where minocycline intraocular injection was administered. After this intervention, complete infection control was achieved, and the patient was discharged 45 days after admission with oral minocycline and 1% cyclosporine and 0.3% ofloxacin eye drops. CONCLUSION: A patient with ocular pythiosis was successfully treated with penetrating keratoplasty and 2 months of treatment with intracameral and oral minocycline.

Herpes Simplex Keratitis in Rheumatoid Arthritis Patients.

PURPOSE: To describe a series of 5 patients with herpes simplex virus keratitis (HSK) and rheumatoid arthritis (RA) under immunosuppressive treatment. METHODS: Retrospective study. Detailed data were obtained regarding symptoms and signs at the initial evaluation, treatment, microbiological diagnostic tests, evolution, and outcomes. RESULTS: Five patients with HSK and RA were identified. Bilateral involvement occurred in 2 patients (40%). Epithelial keratitis was diagnosed in 5 eyes. Three eyes showed severe melting with eye perforation. Gram-positive bacterial co-infections were common in the group with stromal keratitis. We did not find differences in the evolution of the disease based on anti-rheumatoid treatment. CONCLUSIONS: The characteristics of HSK in patients with RA differed from HSK in immunocompetent patients. The stromal keratitis cases were very aggressive and difficult to manage, with perforation and gram-positive bacterial co-infection as frequently associated conditions. Prophylactic therapy at standard doses was unsuccessful to avoid recurrences.

Recurrent ocular surface inflammation associated with human herpesvirus 6 infection.

OBJECTIVES: To report a case of atypical herpes keratitis and bilateral conjunctivitis associated with human herpesvirus 6 (HHV-6). METHODS: An immunocompetent 34-year-old man was referred for herpetic epithelial keratitis in his left eye, which was non-responsive to topical acyclovir. Biomicroscopy revealed a central dendritic ulcer with a white stromal infiltrate beneath the ulcer. RESULTS: The corneal scraping multiplex polymerase chain reaction (CLART ENTHERPEX, Genomica, Spain) was positive for HHV-6 and negative for herpes simplex virus, varicella zoster virus, cytomegalovirus, and Epstein-Barr virus. An improvement of the keratitis and visual acuity was achieved with topical fluorometholone and systemic valacyclovir. One year later, the patient complained of redness of the eyes. A slit-lamp examination disclosed bilateral follicular conjunctivitis, and HHV-6 DNA was once again detected in a conjunctival scraping of both eyes. CONCLUSIONS: Human herpesvirus 6 may be another causative agent for corneal ulcers and conjunctivitis in isolation. Stromal necrosis is a rare manifestation of herpetic dendritic keratitis. In these cases, we should consider the presence of HHV-6 in the differential diagnosis, even in immunocompetent patients.

Staphylococcus aureus Blepharitis Associated with Multiple Corneal Stromal Microabscess, Stromal Edema, and Uveitis.

We report a case of an immunocompetent woman with atypical marginal keratitis. She presented with recurrent episodes of multiples microabscess distributed in a triangular pattern associated with stromal oedema and anterior chamber uveitis, affecting both eyes, but not simultaneously. The episodes responded to steroid drops, corneal inflammation was coincidental with a worsening of her blepharitis in the affected eye and S. aureus was isolated from the lids.

Comparative study of limbal stem cell deficiency diagnosis methods: detection of MUC5AC mRNA and goblet cells in corneal epithelium.

PURPOSE: To evaluate a limbal stem cell deficiency (LSCD) diagnosis method based on the detection of the MUC5AC transcript by reverse transcription-polymerase chain reaction (RT-PCR) in comparison with the standard diagnostic method based on goblet cell detection by periodic acid-Schiff (PAS)-hematoxylin staining, using samples obtained from corneal epithelium impression cytology (IC). DESIGN: Transversal, comparative case series. PARTICIPANTS: We studied 59 eyes from 43 patients clinically diagnosed with LSCD. METHODS: Impression cytology was used to gather cells from corneal and conjunctival epithelium from the same eye. The presence of goblet cells in the cornea was determined by PAS-hematoxylin staining, whereas the presence of the MUC5AC transcript was detected by RT-PCR using a custom-designed primer pair. MAIN OUTCOME MEASURES: Goblet cells in the corneal epithelium were detected by light microscopy, and the MUC5AC transcript was detected as the corresponding PCR amplicon in agarose gels. RESULTS: Our study included 59 corneal samples, together with their respective conjunctival samples for RT-PCR assays. Of these, 47 samples were also available for comparative PAS-hematoxylin staining. The MUC5AC amplicon was detected in 56 of 59 (94.9%) corneal epithelium samples. In contrast, conventional IC staining detected goblet cells in only 17 of 47 (36.2%) samples; these were not found in 27 of 47 (57.4%) samples (negative results), and 3 of 47 (6.4%) showed inconclusive results. CONCLUSIONS: The detection of the MUC5AC transcript in corneal epithelium is a more sensitive method to diagnose LSCD than the conventional PAS-hematoxylin method, although a minimum RNA concentration of 1.2 ng/µl is required for negative results to be reliable. Moreover, RT-PCR is a highly specific and more objective technique. Overall, these findings indicate that molecular analysis facilitates a more precise clinical diagnosis of LSCD, thereby reducing the risk of surgical failure.