Identification and Analysis of Primary Cilia in the Corneal Endothelial Cells of Patients with Bullous Keratopathy.

PURPOSE: To identify primary cilia in human corneal endothelial cells (CECs) obtained from patients with bullous keratopathy (BK). METHODS: This study involved CEC specimens obtained from 10 eyes of 10 consecutive patients (three males and seven females; mean age: 74.5 years, range: 68-90 years) with BK who underwent Descemet's stripping automated endothelial keratoplasty at Baptist Eye Institute, Kyoto, Japan between August 2019 and September 2020. Three corneal buttons obtained from 3 patients who underwent penetrating keratoplasty for keratoconus were used as 'non-BK' controls. All specimens were evaluated with immunofluorescence staining using an antibody against acetylated α-tubulin. RESULTS: Ciliary expression was observed in six of the 10 CEC specimens; i.e. in two specimens obtained from BK patients after glaucoma surgery (trabeculectomy), in two specimens obtained from patients with Fuchs endothelial corneal dystrophy, and in two specimens obtained from a patient with BK after laser iridotomy for primary angle closure. There was acetylated α-tubulin staining but no hair-like structures in two specimens, and ciliary expression was unknown in two specimens due to the absence of cells. The length of the primary cilia varied between all specimens. In contrast, no primary cilia were observed in the corneal buttons obtained from the three keratoconus patients. CONCLUSION: The findings in this study clearly demonstrate the expression of primary cilia in the CECs of patients afflicted with BK.

Intracellular pH affects mitochondrial homeostasis in cultured human corneal endothelial cells prepared for cell injection therapy.

This study aimed to uncover the mechanism responsible for the clinical efficacy of cell injection therapy with fully differentiated cultured cells. Analysis of polarized expression of ion transporters on cultured human corneal endothelial cells (CECs) subpopulations (SPs) was performed. The intracellular pH (pHi) between two CEC SPs, distinct in the proportion of differentiated cells, was measured, and the association with mitochondrial respiration homeostasis was investigated. The effects of the ion transporter inhibition by their selective inhibitors or siRNA transfection were also explored. Na+/K+-ATPase, Aquaporin 1, SLC4A11, NBCe1, NHE1 as transporters, and ZO-1, were all selectively expressed in differentiated SPs, but were almost null in the cell-state-transitioned SPs. We also confirmed that the pHi of CEC SPs affected their mitochondrial respiration by modulating the expression of these ion transporters via inhibitors or siRNA transfection. Ion and water transporters might participate in the maintenance of pHi and mitochondria homeostasis in differentiated SPs, which may contribute, combined with integral barrier functions, to efficient water efflux. The differences in intracellular pH between the two SPs is attributed to variations in the expression profile of specific ion transporters and mitochondrial functions, which may associate with the efficacy of the SPs in cell injection therapy.

Investigation of the relationship between ocular sarcoidosis and dry eye.

A relationship between ocular sarcoidosis (OcSar) and dry eye (DE) has previously been reported. Here we investigated the tear function in OcSar, and the other uveitis, Vogt-Koyanagi-Harada disease (VKH), to elucidate the association between OcSar and DE. This study involved 27 eyes of 27 female OcSar patients [mean age: 67.3 ± 13.5 (SD) years], 18 eyes of 18 female VKH patients (53.1 ± 14.1 years), and 17 eyes of 17 female healthy control subjects (59.7 ± 9.6 years). In all examined eyes, tear meniscus height (TMH, mm), fluorescein breakup time (FBUT, s), scoring of keratoconjunctival epithelial damage (ED), and the Schirmer 1 test (ST1, mm/5 min) were analyzed. In the OcSar patients, VKH patients, and control subjects, the respective TMH, FBUT, ED, and ST1 values (mean ± SD) were 0.26 ± 0.10, 0.24 ± 0.09, and 0.24 ± 0.08, 4.3 ± 2.5, 7.3 ± 2.9, and 6.3 ± 3.1, 1.1 ± 1.1, 0.7 ± 0.8, and 0.1 ± 0.3, and 12.9 ± 10.5, 21.5 ± 12.1, and 24.3 ± 10.5. Statistically, the ST1 values were significantly lower in the OcSar patients than in the VKH patients (P = 0.004) and control subjects (P = 0.001). ST1 findings revealed that the neural reflex arc and lacrimal gland system, which attenuate the vicious cycle between the tear film and ocular surface epithelium in DE, are significantly impaired in OcSar cases, thus indicating a possible association between OcSar and DE.

The relationship between nasal and conjunctival cultures of antimicrobial-resistant isolates of methicillin-resistant Staphylococcus aureus.

PURPOSE: Nasal screening is performed to avoid the complications of postoperative surgical site infections (SSI), especially those due to antimicrobial-resistant species such as methicillin-resistant Staphylococcus aureus (MRSA). This study examined the relationship between bacterial isolates from the conjunctiva and the nasal cavity. METHODS: All patients were diagnosed with ocular surface infections, and the organisms in the conjunctiva and the nasal cavity were isolated. We investigated the relationship of the following antimicrobial-resistant bacteria between the conjunctiva and the nose: MRSA, methicillin-resistant CNS (MRCNS), levofloxacin-resistant (LVFX-R) Corynebacterium spp. Data were analyzed using Fisher's exact test, and the odds ratio was examined. RESULTS: This study included 188 eyes of 188 subjects (87 males and 101 females; mean age 58.5 years, range 11-97 years). MRSA (4 eyes), MRCNS (29 eyes), and LVFX-R Corynebacterium spp. (41 eyes) were identified from the conjunctiva, and MRSA (6 eyes), MRCNS (38 eyes), and LVFX-R Corynebacterium spp. (41 eyes) were identified from the nasal cavity. There was a significant relationship detected between the conjunctiva and the nose for MRSA, MRCNS, and LVFX-R Corynebacterium spp. MRSA displayed high sensitivity (0.750, 95% confidence interval [CI]; 0.301 to 0.987) and specificity (0.984, 95% CI; 0.953 to 0.996) in nasal cavity cultures, and the odds ratio was 181.00 times (95% CI; 18.41 to 2320). CONCLUSION: This study showed a significant relationship between conjunctival and nasal cultures of MRSA, MRCNS, and LVFX-R Corynebacterium spp., suggesting that nasal cavity culture is a potentially useful screening method for detecting resistant bacteria, especially MRSA, in the conjunctiva.

Current Evidence for Corynebacterium on the Ocular Surface.

Corynebacterium species are commonly found in the conjunctiva of healthy adults and are recognized as non-pathogenic bacteria. In recent years, however, Corynebacterium species have been reported to be potentially pathogenic in various tissues. We investigated Corynebacterium species on the ocular surface and reviewed various species of Corynebacterium in terms of their antimicrobial susceptibility and the underlying molecular resistance mechanisms. We identified a risk for Corynebacterium-related ocular infections in patients with poor immunity, such as patients with diabetes or long-term users of topical steroids, and in those with corneal epithelial damage due to trauma, contact lens wear, lagophthalmos, and trichiasis. The predominant strain in the conjunctiva was C. macginleyi, and the species associated with keratitis and conjunctivitis were C. macginleyi, C. propinquum, C. mastitidis, C. pseudodiphtheriticum, C. accolens, C. striatum, C. xerosis, and C. bovis. Overall, Corynebacterium species present on the ocular surface were resistant to quinolones, whereas those in the nasal cavity were more susceptible. The prevalence of fluoroquinolone-resistant Corynebacterium has not changed in the past 10 years; however, Corynebacterium species remain susceptible to third-generation cephems. In conclusion, the use of third-generation cephems should be a reasonable and pragmatic approach for treatment of ocular infections caused by Corynebacterium species.

Polarized Expression of Ion Channels and Solute Carrier Family Transporters on Heterogeneous Cultured Human Corneal Endothelial Cells.

Purpose: To clarify the expression profiles of ion channels and transporters of metabolic substrates among heterogeneous cultured human corneal endothelial cells (cHCECs) distinct in their effectiveness in reconstituting the corneal endothelium. Methods: Integrated proteomics for cell lysates by liquid chromatography-tandem mass spectrometry was carried out from three aliquots of cHCECs enriched in either cluster of definition (CD)44-/+ (mature) cHCECs or CD44++/+++ cell-state transition (CST) cHCECs. The expression profiles of cations/anions, monocarboxylic acid transporters (MCTs), and solute carrier (SLC) family proteins, as well as carbonic anhydrases (CAs), were investigated. Results: The polarized expression of cations/anions, MCTs, and SLC family proteins, as well as CAs, was clarified for mature and CST cHCECs. Most SLC4 family members, including SLC4A11 and SLC4A4 (NBCe1), were upregulated in the CST cHCECs, whereas SLC9A1 (Na+/H+ exchanger isoform one [NHE1]) and CA5B were detected only in the mature cHCECs. In addition, SLC25A42, catalyzing the entry of coenzyme A into the mitochondria, and SLC25A18, functioning as a mitochondrial glutamate carrier 2 (both relevant for providing the substrates for mitochondrial bioenergetics), were selectively expressed in the mature cHCECs. Conclusions: Our findings may suggest the relevance of qualifying the polarized expression of these ion channels and transporter-like proteins to ensure not only the suitability but also the in vivo biological functionality of cHCECs selected for use in a cell-injection therapy.

The trend of resistance to antibiotics for ocular infection of Staphylococcus aureus, coagulase-negative staphylococci, and Corynebacterium compared with 10-years previous: A retrospective observational study.

OBJECTIVE: To retrospectively identify epidemiological trends of infection on the ocular surface and investigate trends of resistance to bacterial antibiotics compared with 10-years previous for Staphylococcus aureus, coagulase-negative staphylococci (CNS), and Corynebacterium in Japan. MATERIALS AND METHODS: Bacterial isolate samples were collected from the conjunctival sacs of eyes afflicted with conjunctivitis, keratitis, dacryocystitis, and hordeolum from September 2004 through November 2005 (n = 145 isolates) and September 2014 through November 2015 (n = 195 isolates) at the Baptist Eye Institute, Kyoto, Japan. The prevalence of methicillin-resistant S. aureus (MRSA), methicillin-resistant CNS (MR-CNS), and fluoroquinolone-resistant Corynebacterium were examined, and susceptibility of isolated bacteria to levofloxacin (LVFX), cefmenoxime (CMX), chloramphenicol (CP), erythromycin (EM), vancomycin (VCM), and arbekacin (ABK) were compared between both time periods using the disc susceptibility method. RESULTS: Over the 10-year period from initial to final examination, the prevalence of MRSA and MR-CNS significantly decreased from 52% to 22% (P < 0.05) and from 47% to 25% (P < 0.05), respectively, yet there was no change in the prevalence of fluoroquinolone-resistant Corynebacterium (60% and 54%; P = 0.38). Antibiotic-resistance trend analysis revealed that susceptibility to antibiotics in 2014-2015 was similar to that in 2004-2005. MRSA and MR-CNS were susceptible to CP (88%), VCM (100%), and ABK (100%), while fluoroquinolone-resistant Corynebacterium was susceptible to CMX (100%), VCM (100%), and ABK (96%). CONCLUSION: The prevalence of MRSA and MR-CNS significantly decreased between the two time periods, yet more than 50% of the Corynebacterium isolates were still resistant to LVFX. Although no increase in bacterial resistance to antibiotics was found, a cautionary use of fluoroquinolone eye drops should be considered.