The Pathological Mechanism of Neuronal Autophagy-Lysosome Dysfunction After Ischemic Stroke.

The abnormal initiation of autophagy flux in neurons after ischemic stroke caused dysfunction of autophagy-lysosome, which not only led to autophagy flux blockage, but also resulted in autophagic death of neurons. However, the pathological mechanism of neuronal autophagy-lysosome dysfunction did not form a unified viewpoint until now. In this review, taking the autophagy lysosomal dysfunction of neurons as a starting point, we summarized the molecular mechanisms that led to neuronal autophagy lysosomal dysfunction after ischemic stroke, which would provide theoretical basis for the clinical treatment of ischemic stroke.

Bioactive PLGA/tricalcium phosphate scaffolds incorporating phytomolecule icaritin developed for calvarial defect repair in rat model.

BACKGROUND/OBJECTIVES: For treatment of large bone defects challenging in orthopaedic clinics, bone graft substitutes are commonly used for the majority of surgeons. It would be proposed in the current study that our bioactive scaffolds could additionally serve as a local delivery system for therapeutic small molecule agents capable of providing support to enhance biological bone repair. METHODS: In this study, composite scaffolds made of poly (lactic-co-glycolic acid) (PLGA) and tricalcium phosphate (TCP) named by P/T was fabricated by a low-temperature rapid prototyping technique. For optimizing the scaffolds, the phytomolecule icaritin (ICT) was incorporated into P/T scaffolds called P/T/ICT. The osteogenic efficacies of the two groups of scaffolds were compared in a successfully established calvarial defect model in rats. Bone regeneration was evaluated by X-ray, micro-computerised tomography (micro-CT), and histology at weeks 4 and/or 8 post-implantation. In vitro induction of osteogenesis and osteoclastogenesis was established for identification of differentiation potentials evoked by icaritin in primary cultured precursor cells. RESULTS: The results of radiographies and decalcified histology demonstrated more area and volume fractions of newly formed bone within bone defect sites implanted with P/T/ICT scaffold than that with P/T scaffold. Undecalcified histological results presented more osteoid and mineralized bone tissues, and also more active bone remodeling in P/T/ICT group than that in P/T group. The results of histological staining in osteoclast-like cells and newly formed vessels indicated favorable biocompatibility, rapid bioresorption and more new vessel growth in P/T/ICT scaffolds in contrast to P/T scaffolds. Based on in vitro induction, the results presented that icaritin could significantly facilitate osteogenic differentiation, while suppressed adipogenic differentiation. Meanwhile, icaritin demonstrated remarkable inhibition of osteoclastogenic differentiation. CONCLUSION: The finding that P/T/ICT composite scaffold can enhance bone regeneration in calvarial bone defects through facilitating effective bone formation and restraining excessive bone resorption. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: The osteogenic bioactivity of icaritin facilitated PLGA/TCP/icartin composite scaffold to exert significant bone regeneration in calvarial defects in rat model. It might form an optimized foundation for potential clinical validation in bone defects application.

Microbial Contamination of Periorbital Tissues and Accessories of Children.

PURPOSE: To investigate normal flora of children with and without orthokeratology (ortho-k) treatment, and the associations between carriage of Staphylococcus aureus and Gram-negative rods with contamination of contact lenses and lens cases in ortho-k subjects and with spectacles of control subjects. METHODS: Twenty-three ortho-k subjects (treatment >12 months) and 20 control myopic subjects aged 7-14 years were recruited. Samples were collected from four sites surrounding the left eye (lower conjunctiva, upper and lower eyelids, and eyelashes) for all subjects, spectacles for control subjects and contact lens accessories for ortho-k subjects. Samples were cultured, total numbers enumerated, and isolates identified using chromogenic agars. RESULTS: Ortho-k subjects had significantly less total isolates in the conjunctiva than controls (p = 0.009). Otherwise, carriage rates in normal flora levels of the peri-orbital tissues were not significantly different between the two groups (p > 0.19). The total isolates and carriage rates of normal flora on spectacles, contact lenses, and lens cases were similar to those identified on the skin tissues. Small numbers of bacteria were identified from the multipurpose solution of two ortho-k subjects. Although the association between the carriage of S. aureus with contamination of accessories was statistically significant only in control subjects (p = 0.03), ortho-k subjects not yielding S. aureus and Gram-negative rods from samples of their peri-orbital tissues tended to be less likely to have these organisms in their accessories. No Pseudomonas was isolated from any of the sites sampled and no Acinetobacter was isolated from any of the accessories. CONCLUSIONS: Ortho-k may lower the total number of bacteria in conjunctiva due to the use of solution and lenses, but the physiologic effect of this treatment on the types of normal flora in children was minimal and should not increase the risk of microbial keratitis in children with good compliance.

Does the presence of QAC genes in staphylococci affect the efficacy of disinfecting solutions used by orthokeratology lens wearers?

BACKGROUND/AIM: There has been increasing evidence of the emergence of antiseptic resistance mediated by quaternary ammonium compound (QAC) resistance genes, which may reduce the efficacy of disinfection. Although the presence of QAC-positive staphylococci has been shown to be elevated in contact lens wearers, the efficacy of multipurpose solutions (MPS) against such isolates has not been determined. This study investigated the efficacy of four MPS for rigid gas permeable (RGP) lenses against staphylococci-harbouring QAC genes. METHODS: Ability to reduce viability by three or more log reductions of four MPS for RGP lenses was tested against 60 disinfectant-resistant gene-positive staphylococci, comprising 38 coagulase-negative staphylococci (CNS) (17 ITALIC! qacA/B, 7 ITALIC! smr, 5 ITALIC! qacH, 9 habouring two or more genes) and 22 ITALIC! Staphylococcus aureus (16 ITALIC! qacA/B, 4 ITALIC! smr, 2 ITALIC! qacA/B+ ITALIC! smr)). 60 gene-negative isolates of staphylococci (30 CNS and 30 ITALIC! S aureus) were used as controls. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these four MPS were determined. RESULTS: Although there was some variation between solutions, all failed to achieve a 3-log reduction in some ITALIC! S aureus and CNS isolates. Strains harbouring disinfectant-resistant genes were significantly less likely to be reduced by 3 logs by three of the solutions. Overall, the MIC and MBC of the four MPS against gene-positive clinical isolates were significantly higher than those of gene-negative isolates. CONCLUSION: The efficacy of MPS solutions for RGP lenses against staphylococci varied. The presence of disinfectant-resistance genes significantly adversely affected disinfecting capacity of RGP solutions.

Inhibitory Effects of 2,2'-Dipyridyl and 1,2,3,4,6-Penta-O-Galloyl-b-D-Glucopyranose on Biofilm Formation in Contact Lens Cases.

PURPOSE: This study observed biofilm formation by Pseudomonas aeruginosa and Staphylococcus aureus in contact lens cases and investigated the inhibitory effects of 2,2'-dipyridyl (2DP) and 1,2,3,4,6-penta-O-galloyl-b-D-glucopyranose (PGG). METHODS: Biofilm formation of P. aeruginosa ATCC 9027 and S. aureus ATCC 25923 and ATCC 6538 in contact lens cases was determined for a range of initial inocula and incubation times using crystal violet staining. The effects of 2DP and PGG on biofilm were evaluated alone and in combination by their incorporation into the media at commencement of incubation. RESULTS: At 24 hours, biofilm production was related to initial concentration. However, with extended incubation, higher initial concentrations affected formation in S. aureus. Presence of 312 µM 2DP significantly inhibited P. aeruginosa biofilm formation, but had little effect on that of S. aureus. In contrast, PGG (50 µM) inhibited S. aureus biofilm formation, but had much less effect on that of P. aeruginosa. Combination of the agents effectively inhibited biofilm formation of all three organisms throughout a week-long incubation period with OD levels barely exceeding cell-free controls. CONCLUSIONS: Biofilm formation of P. aeruginosa could be prevented by 2DP, while biofilm formation of S. aureus was inhibited by PGG. However, combining these agents showed better inhibition of biofilm production than use of either agent alone on both species. This combination may be useful in prevention of biofilm in contact lens cases, thereby reducing infection risk due to poor compliance with lens case cleaning and replacement. Further work is needed to confirm compatibility with multipurpose solutions and investigate cytotoxicity to ocular tissues.

Prevalence of antiseptic-resistance genes in staphylococci isolated from orthokeratology lens and spectacle wearers in Hong Kong.

PURPOSE: To compare isolation of staphylococci from periorbital tissues and accessories of orthokeratology (ortho-k) lens and spectacle wearers and investigate prevalence of antiseptic-resistance (QAC) genes. To determine minimum inhibitory concentrations (MIC) of antiseptics and antibiotic susceptibility of isolates. METHODS: Staphylococci were isolated from eyelids, eyelashes, and conjunctival sacs of 23 ortho-k lens wearers and 20 spectacle wearers. Samples were also collected from ortho-k lenses, lens cases, and spectacle frames. Isolations of Staphylococcus aureus were compared between ortho-k subjects and controls for all samples and for coagulase-negative staphylococci (CNS) from conjunctival sacs. QAC genes were amplified in 110 S. aureus and 59 CNS isolates and prevalence compared in isolates from ortho-k lens and spectacle wearers. Associations were assessed between presence of QAC genes and antibiotic and antiseptic susceptibility. RESULTS: Although isolation of S. aureus did not differ significantly in periorbital samples from ortho-k and control subjects, QAC genes were significantly more common in both S. aureus and CNS from ortho-k subjects (odds ratio 4.4 and 10.74, respectively). Overall, qacA/B was the predominant gene detected, being present in 26.5% CNS and 11% S. aureus. smr and qacH were present in 12% of CNS, but were less common in S. aureus. QAC gene-positive isolates had higher MICs to benzalkonium chloride and chlorhexidine digluconate. CONCLUSIONS: Our results suggest that long-term use of multipurpose solutions containing quaternary ammonium compounds may select for carriage of organisms harboring QAC genes. As these genes are associated with antibiotic resistance, their increased prevalence in isolates from contact lens wearers is a concern.

Effect of multipurpose solutions against Acinetobacter carrying QAC genes.

PURPOSE: Acinetobacter has low virulence but causes infections in subjects with reduced immunity. It has been reported in ocular infections including those of patients using contact lenses. Treatment is difficult because Acinetobacter is frequently multidrug resistant. Antibiotic-resistant strains frequently also harbor genes for antiseptic resistance (quaternary ammonium compound [QAC]) genes. Because Acinetobacter is part of the normal flora, it may contaminate contact lens and accessories. This study aims to investigate carriage rates of QAC genes in household and clinical isolates of Acinetobacter and to determine the effectiveness of two multipurpose solutions (MPSs) for soft lenses against organisms carrying QAC genes. METHODS: DNA was extracted from 11 bathroom isolates and 15 clinical isolates and amplified by polymerase chain reaction to determine the presence of qacEΔ1. Gene-positive and gene-negative control strains were used to challenge the two MPSs, and minimum inhibitory concentrations (MICs) of these organisms to benzalkonium chloride and chlorhexidine gluconate were determined. RESULTS: More than 90% of isolates carried qacEΔ1. The MICs of clinical isolates were higher than those of isolates of bathrooms. Both MPSs were able to produce a 3-log reduction in the numbers of all isolates. CONCLUSIONS: Although most isolates carried qacEΔ1 and elevated MICs to benzalkonium chloride and chlorhexidine gluconate were observed, all were susceptible to both MPSs tested. However, if there were to be poor compliance with care procedures, it is probable that such organisms could survive in the presence of diluted or expired solutions.

Amoebicidal effects of contact lens disinfecting solutions.

PURPOSE: To compare the traditional manual hemacytometer method and an automated counter (Vi-cell) to enumerate and distinguish between viable and non-viable amoeba, and to determine the efficacies of contact lens (CL) disinfecting solutions against three species of Acanthamoeba. The efficacies in the presence of a bacterial food source and bovine serum albumin (BSA) were investigated. METHODS: Four brands of multipurpose solutions and a hydrogen peroxide disinfecting system (Oxysept) for soft CLs, and four disinfecting solutions for Rigid Gas Permeable (RGP) lenses were tested against three species of Acanthamoeba. Page's amoebic saline was included as a negative control and standard solutions of disinfecting agents, 6% hydrogen peroxide and 0.5% chlorhexidine, as positive controls. The effects of the presence of Pseudomonas aeruginosa and BSA on effectiveness were assessed. RESULTS: None of the CL solutions tested achieved a 1-log reduction in viability of all three Acanthamoeba species within the manufacturer's recommended disinfection times. The presence of P. aeruginosa did not significantly affect disinfecting capacity of multipurpose solution solutions but reduced activity of RGP solutions and the hydrogen peroxide system. BSA reduced trophozoicidal activity of all solutions. Bland and Altman analysis showed good agreement between Vi-cell and hemacytometer. CONCLUSIONS: The Vi-Cell analyzer offers a simple and effective method of determining amoebicidal activity. Our results show that the CL solutions tested could not satisfactorily kill Acanthamoeba.

Adherence of acanthamoeba to lens cases and effects of drying on survival.

PURPOSE: To compare the effects of drying the lens case with tissue on the presence of Acanthamoeba with cases left wet and to determine adherence to the lens case of varying concentrations of Acanthamoeba suspensions. The effect of drying on viability of Acanthamoeba in new, used, and soiled lens cases was compared over a 24 h period. METHODS: New (16) and scratched (16) lens cases were rinsed with a range of Acanthamoeba suspensions. Eight of each group were dried with tissue and the presence of Acanthamoeba was determined in all cases using polymerase chain reaction. To examine effects of drying, forty-two lens case wells were scratched to simulate use and 21 of these were artificially soiled with serum Bovine albumin. These cases and a further 21 unused wells were contaminated with Acanthamoeba (×10/ml) and then left to dry in a cool, dry environment. Three wells of each group were sampled at time 0, 2, 4, 6, 8, 12, and 24 h, and the number of viable Acanthamoeba were determined. RESULTS: Acanthamoeba were more likely to adhere to used than unused lens cases (p < 0.05). Detection of Acanthamoeba in wiped lens cases was at 2-log dilutions less than in cases left wet for both new and used lens cases. Adherence were significantly different between rinse and rinse/dried cases (p = 0.015). Air drying significantly reduced the numbers of viable amoebic cysts and trophozoites and the effect was time dependent. Survival was significantly higher in used and soiled wells. CONCLUSIONS: Drying with tissue after rinsing significantly reduces numbers of adhering Acanthamoeba. Acanthamoeba were found to be able to adhere even to new unused cases, so the importance of proper cleaning and disinfection of lens cases cannot be underestimated. Air drying reduces viability but some viable cells were present at 24 h in soiled cases, confirming the role of biofilm in protecting organisms from desiccation.