Outer retinal transduction by AAV2-7m8 following intravitreal injection in a sheep model of CNGA3 achromatopsia.

Sheep carrying a mutated CNGA3 gene exhibit diminished cone function and provide a naturally occurring large animal model of achromatopsia. Subretinal injection of a vector carrying the CNGA3 transgene resulted in long-term recovery of cone function and photopic vision in these sheep. Research is underway to develop efficacious vectors that would enable safer transgene delivery, while avoiding potential drawbacks of subretinal injections. The current study evaluated two modified vectors, adeno-associated virus 2-7m8 (AAV2-7m8) and AAV9-7m8. Intravitreal injection of AAV2-7m8 carrying enhanced green fluorescent protein under a cone-specific promoter resulted in moderate photoreceptor transduction in wild-type sheep, whereas peripheral subretinal delivery of AAV9-7m8 resulted in the radial spread of the vector beyond the point of deposition. Intravitreal injection of AAV2-7m8 carrying human CNGA3 in mutant sheep resulted in mild photoreceptor transduction, but did not lead to the clinical rescue of photopic vision, while day-blind sheep treated with a subretinal injection exhibited functional recovery of photopic vision. Transgene messenger RNA levels in retinas of intravitreally treated eyes amounted to 4-23% of the endogenous CNGA3 levels, indicating that expression levels >23% are needed to achieve clinical rescue. Overall, our results indicate intravitreal injections of AAV2.7m8 transduce ovine photoreceptors, but not with sufficient efficacy to achieve clinical rescue in CNGA3 mutant sheep.

Mutations in PNPLA6 are linked to photoreceptor degeneration and various forms of childhood blindness.

Blindness due to retinal degeneration affects millions of people worldwide, but many disease-causing mutations remain unknown. PNPLA6 encodes the patatin-like phospholipase domain containing protein 6, also known as neuropathy target esterase (NTE), which is the target of toxic organophosphates that induce human paralysis due to severe axonopathy of large neurons. Mutations in PNPLA6 also cause human spastic paraplegia characterized by motor neuron degeneration. Here we identify PNPLA6 mutations in childhood blindness in seven families with retinal degeneration, including Leber congenital amaurosis and Oliver McFarlane syndrome. PNPLA6 localizes mostly at the inner segment plasma membrane in photoreceptors and mutations in Drosophila PNPLA6 lead to photoreceptor cell death. We also report that lysophosphatidylcholine and lysophosphatidic acid levels are elevated in mutant Drosophila. These findings show a role for PNPLA6 in photoreceptor survival and identify phospholipid metabolism as a potential therapeutic target for some forms of blindness.

Fellow eye effect of unilateral intravitreal bevacizumab injection in eyes with diabetic macular edema.

AIMS: Anti-vascular endothelial growth factor compounds are routinely used for the treatment of diabetic macular edema (DME). We aim to evaluate for the existence and magnitude of treatment effect on fellow un-injected eyes. METHODS: A consecutive group of patients with bilateral DME who received unilateral bevacizumab injections was retrospectively evaluated. Data collected included demographics, ophthalmic and systemic findings, and optical coherence tomography (OCT) measurements of macular thickness. RESULTS: Thirty-five patients were evaluated. Mean follow-up was 245 days (range: 30-800), and the mean number of bevacizumab injections was 3.6 (range: 1-11). At end of follow-up, the mean (SD) OCT central subfield thickness reduced by 72 ± 112 micron in the injected eye (from 469 ± 139 to 397 ± 120 micron; P=0.001), while in the non-injected eye it reduced by 49 ± 75 micron (from 380 ± 130 to 331 ± 106 micron; P<0.001). Sixteen injected eyes (45.7%) showed central subfield thickness reduction of ≥50 micron while 10 (28.6%) non-injected eyes showed such thickness reduction. Improved VA following treatment was detected in 14 (40%) injected eyes and in 15 (43%) non-injected eyes. CONCLUSIONS: Unilateral bevacizumab injections in patients with bilateral DME are often associated with bilateral response. SUMMARY STATEMENT: Anti-vascular endothelial growth factor compounds are routinely used for the treatment of diabetic macular edema (DME). In this retrospective study, we show that unilateral bevacizumab injections often result in reduction of the macular thickness in the fellow un-injected eye.

Draft genome sequence of an extremely drug-resistant KPC-producing Klebsiella pneumoniae ST258 epidemic strain.

Extremely drug-resistant (XDR) Klebsiella pneumoniae carbapenemase-producing clone ST258 has rapidly disseminated worldwide. We report here the draft genome sequence of the K. pneumoniae ST258 XDR clinical strain from Israel.

Reduced susceptibility to chlorhexidine among extremely-drug-resistant strains of Klebsiella pneumoniae.

BACKGROUND: Over the last decade, extremely-drug-resistant (XDR) strains of Klebsiella pneumoniae have emerged worldwide, mainly as a result of patient-to-patient spread. The predominant clone, sequence type 258 (ST258), is associated with high morbidity and mortality, and is a worldwide threat to public health. It was hypothesized that reduced susceptibility to chlorhexidine, the most widely used hospital disinfectant, may contribute to the endemic nature of this strain. AIM: To characterize and compare the susceptibility of the epidemic K. pneumoniae clone ST258 and non-epidemic K. pneumoniae clones to chlorhexidine. METHODS: The minimum inhibitory concentration (MIC) of chlorhexidine was determined in 126 XDR K. pneumoniae clinical isolates using agar dilution. Expression of three different efflux pumps -cepA, acrA and kdeA - was investigated in the absence and presence of chlorhexidine using quantitative real-time polymerase chain reaction. Heteroresistance to chlorhexidine was identified using population analysis. FINDINGS: The MIC of chlorhexidine was higher for K. pneumoniae ST258 (N = 70) than other K. pneumoniae sequence types (N = 56); 99% of ST258 isolates had MICs >32 µg/mL, compared with 52% of other K. pneumoniae sequence types (P < 0.0001). Reduced susceptibility to chlorhexidine appeared to be independent of the expression of cepA, acrA and kdeA efflux pumps. Chlorhexidine-resistant subpopulations were observed independent of the bacterial sequence type or the MIC. CONCLUSIONS: Reduced susceptibility to chlorhexidine may contribute to the success of XDR K. pneumoniae as a nosocomial pathogen, and may provide a selective advantage to the international epidemic strain K. pneumoniae ST258. The heterogeneous nature of chlorhexidine-resistant subpopulations suggests that this phenomenon might not be rendered genetically.

Intravitreal bevacizumab therapy for neovascular age-related macular degeneration associated with poor initial visual acuity.

AIM: The aim of the study was to assess the efficacy of intravitreal bevacizumab injections for eyes with neovascular age-related macular degeneration (NVAMD) and poor initial visual acuity (VA). METHODS: A retrospective study of 44 consecutive treatment-naïve eyes with NVAMD who had initial VA of 0.1 decimal or worse, and that were treated with intravitreal bevacizumab injections, was undertaken. Charts, optical coherence tomography (OCT) and fluorescein angiograms (FAs) were reviewed for the purpose of the study. RESULTS: Mean lesion size was 3375 (SD 2116) microm, all lesions showed sub- or intra-retinal fluid in OCT, and active neovascularisation comprised 41.6 (SD 17.7)% (range 10-90%) of the lesion area according to FA. The mean follow-up time was 3.9 (SD 5.8) (range 1-21) months. Patients received a mean of 2.6 (SD 2.4) bevacizumab injections (range 1-14), and mean VA improved from 1.85 (SD 0.64) to 1.52 (SD 0.77) LogMAR (p = 0.002). At final examination, nine eyes (20%) had reduced VA, ten eyes (23%) had stable VA and 25 eyes (57%) had improved VA compared with baseline. Following treatment, mean macular thickness was reduced from 332 (SD 98) to 248 (SD 79) microm (p<0.0001). CONCLUSIONS: Poor initial VA should not prevent use of bevacizumab in eyes with NVAMD. Selection of patients with signs of active neovascularisation based on ophthalmoscopy, OCT and FA may increase the likelihood of a favourable response to treatment.

Lack of benefit of early awareness to age-related macular degeneration.

AIMS: To assess the rate of early awareness to the presence of age-related macular degeneration (AMD) and whether it enables early detection of transition to neovascular AMD (NVAMD) as compared with patients whose first presentation to an ophthalmologist is already at the neovascular stage of disease. METHODS: A retrospective analysis of 268 eyes of 268 consecutive patients with newly diagnosed NVAMD that were treated in a tertiary referral centre was performed. Patients were classified into those who were unaware (Group 1), or aware (Group 2), of the fact that they had AMD before diagnosis of NVAMD. Visual acuity, lesion size and composition, and demographics were compared between both groups. RESULTS: In all, 185 patients (69%) and 83 patients (31%) were classified to Groups 1 and 2, respectively. Patients in Groups 1 and 2 had similar demographic characteristics, presenting visual acuity and lesion size, and lesion compositions. Group 1 patients were more likely to have a positive history for smoking (41 vs26% in Group 2, P=0.03), whereas Group 2 patients were more likely to have positive family history for AMD (20 vs10%, P=0.02). CONCLUSIONS: These data suggest that current screening methods fail to identify the majority of patients with AMD before the development of NVAMD. The findings also demonstrate that in the routine clinical setting, prior awareness of AMD may not facilitate early detection of treatable choroidal neovascularization lesions.

Vibrio shiloi sp. nov., the causative agent of bleaching of the coral Oculina patagonica.

The aetiological agent of bleaching of the coral Oculina patagonica was characterized as a new Vibrio species on the basis of 16S rDNA sequence, DNA-DNA hybridization data and phenotypic properties, including the cellular fatty acid profile. Based on its 16S rDNA and DNA-DNA hybridization, the new Vibrio species is closely related to Vibrio mediterranei. The name Vibrio shiloi sp. nov. is proposed for the new coral-bleaching species, the type strain being AK1T (= ATCC BAA-91T = DSM 13774T).

Gene transfer by viral vectors into blood vessels in a rat model of retinopathy of prematurity.

AIMS: To test the feasibility of gene transfer into hyaloid blood vessels and into preretinal neovascularisation in a rat model of retinopathy of prematurity (ROP), using different viral vectors. METHODS: Newborn rats were exposed to alternating hypoxic and hyperoxic conditions in order to induce ocular neovascularisation (ROP rats). Adenovirus, herpes simplex, vaccinia, and retroviral (MuLV based) vectors, all carrying the beta galactosidase (beta-gal) gene, were injected intravitreally on postnatal day 18 (P18). Two sets of controls were also examined: P18 ROP rats injected with saline and P18 rats that were raised in room air before the viral vectors or saline were injected. Two days after injection, the rats were killed, eyes enucleated, and beta-gal expression was examined by X-gal staining in whole mounts and in histological sections. RESULTS: Intravitreal injection of the adenovirus and vaccinia vectors yielded marked beta-gal expression in hyaloid blood vessels in the rat ROP model. Retinal expression of beta-gal with these vectors was limited almost exclusively to the vicinity of the injection site. Injection of herpes simplex yielded a punctuate pattern of beta-gal expression in the retina but not in blood vessels. No significant beta-gal expression occurred in rat eyes injected with the retroviral vector. CONCLUSIONS: Adenovirus is an efficient vector for gene transfer into blood vessels in an animal model of ROP. This may be a first step towards utilising gene transfer as a tool for modulating ocular neovascularisation for experimental and therapeutic purposes.

Role of endosymbiotic zooxanthellae and coral mucus in the adhesion of the coral-bleaching pathogen Vibrio shiloi to its host.

Vibrio shiloi, the causative agent of bleaching the coral Oculina patagonica in the Mediterranean Sea, adheres to its coral host by a beta-D-galactopyranoside-containing receptor on the coral surface. The receptor is present in the coral mucus, since V. shiloi adhered avidly to mucus-coated ELISA plates. Adhesion was inhibited by methyl-beta-D-galactopyranoside. Removal of the mucus from O. patagonica resulted in a delay in adhesion of V. shiloi to the coral, corresponding to regeneration of the mucus. DCMU inhibited the recovery of adhesion of the bacteria to the mucus-depleted corals, indicating that active photosynthesis by the endosymbiotic zooxanthellae was necessary for the synthesis or secretion of the receptor. Further evidence of the role of the zooxanthellae in producing the receptor came from a study of adhesion of V. shiloi to different species of corals. The bacteria failed to adhere to bleached corals and white (azooxanthellate) O. patagonica cave corals, both of which lacked the algae. In addition, V. shiloi adhered to two Mediterranean corals (Madracis and Cladocora) that contained zooxanthellae and did not adhere to two azooxanthellate Mediterranean corals (Phyllangia and Polycyathus). V. shiloi demonstrated positive chemotaxis towards the mucus of O. patagonica. The data demonstrate that endosymbiotic zooxanthellae contribute to the production of coral mucus and that V. shiloi infects only mucus-containing, zooxanthellate corals.

Proline-rich peptide from the coral pathogen Vibrio shiloi that inhibits photosynthesis of Zooxanthellae.

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29 degrees C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH(4)Cl, pure natural or synthetic toxin P (10 microM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH(4)Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH(4)Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH(3) into the cell. It is known that uptake of NH(3) into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.

Long-term assessment of combined vitamin A and E treatment for the prevention of retinal degeneration in abetalipoproteinaemia and hypobetalipoproteinaemia patients.

PURPOSE: To assess the long-term efficacy of combined vitamin A and E treatment in preventing retinal degeneration in patients with abetalipoproteinaemia (ABL) or homozygous hypobetalipoproteinaemia (HBL). METHODS: Ten patients with ABL and 3 with homozygous HBL who were treated with oral supplements of vitamins A and E were studied. Systemic, ophthalmological and electroretinographic follow-up for a mean of 11.7 years (range 4-20 years) after onset of treatment was evaluated. RESULTS: Despite vitamin A and E treatment, 7 of 10 patients who began treatment prior to 2 years of age and all 3 patients who began treatment later in life manifested unusual fundoscopic pigmentary changes over time. At the end of follow-up, 11 of 13 patients had subnormal mixed cone-rod electroretinogram amplitudes. Seven of 10 patients for whom perimetry was available had mild to severe constriction of the visual fields. CONCLUSIONS: Combined oral vitamin A and E supplementation that is initiated prior to 2 years of age can markedly attenuate the severe retinal degeneration that is associated with untreated ABL or homozygous HBL. Yet, fundoscopic and functional retinal changes do occur despite early initiation of vitamin treatment. Therefore, the adequacy of the present treatment protocol for ABL and homozygous HBL should be re-evaluated.

Visual impairment in children with epilepsy treated with vigabatrin.

Vigabatrin is an anti-epileptic drug particularly useful for drug-resistant partial seizures and infantile spasms. Recently, vigabatrin-induced visual field constriction (VFC) and abnormal ocular electrophysiological studies were reported. In this study, we assessed visual fields, visual evoked potentials (VEPs), and electroretinography (ERG) in children treated with vigabatrin. Twenty-four visually asymptomatic children underwent a clinical ophthalmological examination, perimetry when appropriate, and VEP and ERG. Thirteen patients had at least one abnormal study. VFC was seen in 11 of 17 patients who had perimetry; 5 of 15 patients who underwent VEP testing and 4 of 11 who underwent ERG testing had abnormal examinations. For the most part, abnormal VEPs and ERGs were found in children who also had VFC. There was a consistent trend for longer treatment periods to correlate with VFC, abnormal ERGs, and VEPs. In summary, over half of the children treated with vigabatrin demonstrated VFC or abnormal ocular electrophysiological studies. Perimetry seemed to be the most sensitive modality for identifying vigabatrin toxicity. Abnormal ERGs and VEPs were primarily seen in children with VFC and may be useful in monitoring children who are not appropriate candidates for perimetry. Although the incidence of vigabatrin-induced VFC is worrisome, in the context of intractable seizures or infantile spasms, therapeutic benefits must be weighed against risks.

Penetration of the coral-bleaching bacterium Vibrio shiloi into Oculina patagonica.

Inoculation of the coral-bleaching bacterium Vibrio shiloi into seawater containing its host Oculina patagonica led to adhesion of the bacteria to the coral surface via a beta-D-galactose receptor, followed by penetration of the bacteria into the coral tissue. The internalized V. shiloi cells were observed inside the exodermal layer of the coral by electron microscopy and fluorescence microscopy using specific anti-V. shiloi antibodies to stain the intracellular bacteria. At 29 degrees C, 80% of the bacteria bound to the coral within 8 h. Penetration, measured by the viable count (gentamicin invasion assay) inside the coral tissue, was 5.6, 20.9, and 21.7% of the initial inoculum at 8, 12, and 24 h, respectively. The viable count in the coral tissue decreased to 5.3% at 48 h, and none could be detected at 72 h. Determination of V. shiloi total counts (using the anti-V. shiloi antibodies) in the coral tissue showed results similar to viable counts for the first 12 h of infection. After 12 h, however, the total count more than doubled from 12 to 24 h and continued to rise, reaching a value 6 times that of the initial inoculum at 72 h. Thus, the intracellular V. shiloi organisms were transformed into a form that could multiply inside the coral tissue but did not form colonies on agar medium. Internalization of the bacteria was accompanied by the production of high concentrations of V. shiloi toxin P activity in the coral tissue. Internalization and multiplication of V. shiloi are discussed in terms of the mechanism of bacterial bleaching of corals.

Retinitis pigmentosa associated with Fuchs' heterochromic uveitis.

OBJECTIVE: To investigate whether the combination of Fuchs' heterochromic uveitis (FHU) and retinitis pigmentosa (RP) in the same patient is coincidental or represents a true association. METHODS: We have examined the frequency of FHU in 338 patients with RP and in 1984 patients who were seen in our primary care ophthalmic clinic because of reasons other than RP. RESULTS: Of 338 patients with RP, 4 (1.2%) had the typical findings of FHU. Three of them had Usher syndrome type II, and 1 had RP simplex. By contrast, only 1 patient in the control group had FHU (5%), and the difference in the frequency of FHU between the 2 groups was significant (P=.002, Fisher exact test). CONCLUSIONS: Fuchs' heterochromic uveitis is associated with RP. Since autoimmune phenomena have been previously described in patients with RP, it is conceivable that RP predisposes to the development of FHU. Arch Ophthalmol. 2000;118:800-802

Gallium-desferrioxamine protects the cat retina against injury after ischemia and reperfusion.

This study sought to determine whether gallium-desferrioxamine (Ga/DFO) can curb free radical formation and mitigate biochemical and electrophysiological parameters of injury in the cat retina subjected to ischemia followed by reperfusion. For the biochemical studies, cat eyes were subjected to 90 min of retinal ischemia followed by 5 min of reperfusion, and enucleation of one eye of each cat was used to measure retinal reperfusion injury. Before enucleation of fellow eyes, 2.5 mg/kg Ga/DFO was injected intravenously 5 min before reperfusion. The flux of hydroxyl radicals, as measured directly by conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid (2,3- and 2,5-DHBA), was significantly lower in Ga/DFO-treated eyes. The mean normalized level of 2,3-DHBA (considered a specific marker of hydroxyl radicals) was 3.5 times higher in untreated eyes. Ga/DFO caused a significant reduction, by 2.56-fold, in lipid peroxidation, as reflected by levels of malondialdehyde. Ascorbic acid, a natural antioxidant present in the retina, is severely depleted in untreated eyes. In contrast, in Ga/DFO-treated eyes, levels were 10 times higher than the control. Energy charge was 2.38 times higher in treated eyes. Levels of purine catabolites (hypoxanthine, xanthine, and uric acid) that reflect excessive metabolism of purine nucleotides were approximately twice higher in untreated retinas. Electroretionographic studies, performed on a different subset of animals, substantiated the biochemical results. In Ga/DFO-treated eyes the amplitude of the mixed cone-rod response b-wave (as compared with fellow nonischemic eyes) fully recovered within 24 h after ischemia (b-wave ratio 1.04 +/- 0.09, [mean +/- SEM]) whereas ischemic/reperfused and nontreated eyes recovered to only 0.33 +/- 0. 05. The results show that severe biochemical and functional retinal injury occurs in cat eyes subjected to ischemia and reperfusion. These severe changes were significantly reduced by a single administration of Ga/DFO just before reperfusion. We hypothesize that the protection afforded by Ga/DFO is due to a combined effect of "Push-Pull" mechanisms interfering with transition metal-dependent and free radical-mediated injurious processes.

Retinitis pigmentosa, Coats disease and uveitis.

PURPOSE: To study the anamnestic immune response to retinal specific antigens of two patients suffering from a rare triad of retinitis pigmentosa, Coats disease and uveitis. PATIENTS: 17-year-old girl presented with an acute episode of panuveitis, and her 19-year-old brother suffered from chronic uveitis. On examination, both patients showed retinal vascular changes and subretinal exudations typical of Coats disease, with bone-spicule pigmentary changes as observed in retinitis pigmentosa. RESULTS: All routine examinations were unrevealing. However, the peripheral lymphocytes from these two siblings gave a specific anamnestic response to retinal antigens in vitro. A stimulation index of 4.6 was obtained when the sister's lymphocytes were stimulated with interphotoreceptor binding protein, IRBP--during the acute stage of the uveitis. The brother's lymphocytes showed a stimulation index of 2.7 towards S-Ag during the chronic phase of his uveitic condition. CONCLUSIONS: These results indicate that autoimmunity towards retinal antigens may play some role in specific types of retinitis pigmentosa. Whether these autoimmune reactions are a primary pathological mechanism or are secondary to the extensive destruction of the photoreceptor layer resulting from the retinitis pigmentosa remains debatable.