Harry O. Orlans

In vitro antibiotic susceptibility patterns of bacterial keratitis isolates in Oxford, UK: a 10-year review

PurposeTo analyse the spectrum of bacterial keratitis isolates and their in vitroantibiotic susceptibilities over a 10-year period in Oxford, UK; and to compare the in vitroefficacy of ciprofloxacin with that of the combination of gentamicin and cefuroxime over the same period.MethodsAll culture-positive corneal scrapes received from the Oxford Eye Hospital between July 1999 and June 2009 were identified retrospectively using a local microbiology database. For analysis of trends over time, the data was split into two equal 5-year periods. Statistical analysis was done using the χ 2 and Fisher exact tests.ResultsOver the 10-year study period, 467 corneal scrapes were performed of which 252 (54.0%) had positive bacterial cultures, growing a total of 267 organisms. The most commonly isolated bacteria were Staphylococci(40.1%) followed by Pseudomonasspecies (28.5%), other Gram-negative species (17.2%), Streptococci(7.1%), and Corynebacteria(6.0%). Between the first and second time periods there was an increase in the number of coagulase-negative Staphylococciand an increased resistance of the non-Pseudomonas Gram-negative group to chloramphenicol. Of the 189 isolates tested for sensitivity to both empirical antibiotic regimens, 176 (93.2%) were susceptible to ciprofloxacin whereas 188 (99.5%) were susceptible to either gentamicin or cefuroxime (P=0.0015).ConclusionsThe spectrum of bacterial keratitis isolates and their in vitroantibiotic sensitivity patterns have generally remained stable over time. The combination of gentamicin and cefuroxime provides a broader spectrum of antimicrobial cover than ciprofloxacin monotherapy in Oxford, although both regimens continue to be appropriate choices for the initial management of this condition.

Anti-VEGF Therapy and the Retina: An Update

Ocular angiogenesis and macular oedema are major causes of sight loss across the world. Aberrant neovascularisation, which may arise secondary to numerous disease processes, can result in reduced vision as a result of oedema, haemorrhage, and scarring. The development of antivascular endothelial growth factor (anti-VEGF) agents has revolutionised the treatment of retinal vasogenic conditions. These drugs are now commonly employed for the treatment of a plethora of ocular pathologies including choroidal neovascularisation, diabetic macular oedema, and retinal vein occlusion to name a few. In this paper, we will explore the current use of anti-VEGF in a variety of retinal diseases and the impact that these medications have had on visual outcome for patients.

Secretory cells of the supraoptic nucleus have central as well as neurohypophysial projections

Conventional neuroanatomical methods may fail to demonstrate the presence of axons that are finer than 1 µm in diameter because such processes are near or below the limit of resolution of the light microscope. The presence of such axons can, however, be readily demonstrated by recording. The most easily interpreted type of recording for this purpose is the demonstration of antidromic activation of the cell body following stimulation of the region through which the axon passes. We have exploited this technique in the hypothalamus and have demonstrated the presence of double axonal projections or axons branching very near the cell bodies of the secretory cells of the neurohypophysial system in the rat supraoptic nucleus. We found that a small proportion of supraoptic magnocellular cells could be antidromically activated both from the neural stalk and from elsewhere in the hypothalamus, including the suprachiasmatic nucleus (8 cells of a total of 182) and the antero‐ventral third ventricular region (AV3V; 4 of 182 cells) near the organum vasculosum of the lamina terminalis (OVLT). Collision of antidromic and orthodromic spikes showed that the cells were clearly antidromically (rather than synaptically, or orthodromically) activated from both sites. A stimulus applied to one of the axons prevented propagation of a spike evoked by a pulse delivered to the other axon until sufficient time had elapsed after the first stimulus for the resultant spike to have propagated from the first stimulus site along one cell process (towards the cell body or branch point), and from this point along the other axonal branch to the second stimulus site (there was also a short additional delay period during which the axon at the site of the second stimulus recovered from its absolute refractory period). If the interval between the stimuli was progressively reduced, there came a point where the second spike failed. Such a clear demonstration of dual projections in a system where the cells were previously thought to have only a single axon raises the possibility that many nerve cells in the CNS have previously unsuspected projections.

Inclusion of the Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances AAV2-Driven Transduction of Mouse and Human Retina

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) has been included in the transgene cassette of adeno-associated virus (AAV) in several gene therapy clinical trials, including those for inherited retinal diseases. However, the extent to which WPRE increases transgene expression in the retina is still unclear. To address this question, AAV2 vectors containing a reporter gene with and without WPRE were initially compared in vitro and subsequently in vivo by subretinal delivery in mice. In both instances, the presence of WPRE led to significantly higher levels of transgene expression as measured by fundus fluorescence, western blot, and immunohistochemistry. The two vectors were further compared in human retinal explants derived from patients undergoing clinically indicated retinectomy, where again the presence of WPRE resulted in an enhancement of reporter gene expression. Finally, an analogous approach using a transgene currently employed in a clinical trial for choroideremia delivered similar results both in vitro and in vivo, confirming that the WPRE effect is transgene independent. Our data fully support the inclusion of WPRE in ongoing and future AAV retinal gene therapy trials, where it may allow a therapeutic effect to be achieved at an overall lower dose of vector.

Osmotic modulation of stimulus-evoked responses in the rat supraoptic nucleus.

Neural information is conveyed by action potentials along axons to downstream synaptic targets. Synapses permit functionally relevant modulation of the information transmitted by converging inputs. Previous studies have measured the amount of information associated with a given stimulus based either on spike counts or on the relative frequencies of spike sequences represented as binary strings. Here we apply information theory to the phase–interval stimulus histogram (PhISH) to measure the extent of the stimulus‐evoked response using the statistical relationship between each interspike interval and its phase within the stimulus cycle. We used the PhISH as a novel approach to investigate how different osmotic states affect the flow of information through the osmoreceptor complex of the hypothalamus. The amount of information conveyed from one (afferent) element of the complex, the anteroventral region of the third ventricle (AV3V), to another (an efferent element), the supraoptic nucleus, was increased by hypertonic stimulation (intravenous mannitol, z = 4.39, P < 0.001) and decreased by hypotonic stimulation (intragastric water, z = −3.37, P < 0.001). Supraoptic responses to AV3V stimulation differed from those that follow stimulation of a hypothalamic element outside the osmoreceptor complex, the suprachiasmatic nucleus (SCN), which also projects to the supraoptic nucleus. Thus osmosensitive gain control mechanisms differentially modulate osmotically dependent and osmotically independent inputs, and enhance the osmoresponsiveness of supraoptic cells within a physiological range. The value of the novel approach is that its use is not limited to the osmoreceptor ensemble but it can be used to investigate the flow of information throughout the central nervous system.

Impact of Vital Dyes on Cell Viability and Transduction Efficiency of AAV Vectors Used in Retinal Gene Therapy Surgery: An In Vitro and In Vivo Analysis

Purpose Treatment of inherited retinal degenerations using adeno-associated viral (AAV) vectors involves delivery by subretinal injection. In the latter stages, alteration of normal anatomy may cause difficulty in visualizing the retinotomy, retinal detachment extension, and vector diffusion. Vital dyes may be useful surgical adjuncts, but their safety and impact on AAV transduction are largely unknown. Methods The effects of Sodium Fluorescein (SF), Membrane Blue (MB), and Membrane Blue Dual (DB) at a range of dilutions were assessed on human embryonic kidney cells in vitro using an AAV2-green fluorescent protein (GFP) reporter at different multiplicities of infection. Flow cytometry analysis was performed to assess both cell viability and transduction efficiency. The effect on quantitative (q)PCR titer was determined. Balanced salt solution (BSS) or dilute DB (1:5 in BSS) were delivered subretinally into left/right eyes of C57BL/6J mice (n = 12). Retinal structure and function were analyzed by optical coherence tomography, autofluorescence, dark-and light-adapted full-field electroretinography. Results DB and MB were not toxic at any concentration tested, SF only when undiluted. The presence of dyes did not adversely affect the genomic titer. DB even increased the values, due to presence of surfactant in the formulation. AAV2-GFP transduction efficiency was not reduced by the dyes. No structural and functional toxic effects were observed following subretinal delivery of DB. Conclusions Only undiluted SF affected cell viability. No effects on qPCR titer and transduction efficiency were observed. DB does not appear toxic when delivered subretinally and improves titer accuracy. DB may therefore be a safe and helpful adjunct during gene therapy surgery. Translational Relevance This paper might be of interest to the retinal gene therapy community: it is a “bench to bedside” research paper about the potential use of dyes as a surgical adjunct during the gene therapy surgery. We have tested the potential toxicity and impact on transduction efficiency in an in vitro and in vivo model.

Slowly progressive retinitis pigmentosa caused by two novel mutations in the MAK gene.

ABSTRACT Background: The growing number of clinical trials currently underway for inherited retinal diseases has highlighted the importance of achieving a molecular diagnosis for all new cases presenting to hospital eye services. The male germ cell-associated kinase (MAK) gene encodes a cilium-associated protein selectively expressed in the retina and testis, and has recently been implicated in autosomal recessive retinitis pigmentosa (RP). Whole exome sequencing has previously identified a homozygous Alu insertion in probands with recessive RP and nonsense and missense mutations have also been reported. Materials and methods: Here we describe two novel mutations in different alleles of the MAK gene in a 75-year-old British female, who had a clinical diagnosis of RP () with onset in the fourth decade and no relevant family history. The mutations were established through next generation sequencing of a panel of 111 genes associated with RP and RP-like phenotypes. Results: Two novel null mutations were identified within the MAK gene. The first c.1195_1196delAC p.(Thr399fs), was a two base-pair deletion creating a frame-shift in exon 9 predicted to result in nonsense-mediated decay. The second, c.279-2A>G, involved the splice acceptor consensus site upstream of exon 4, predicted to lead to aberrant splicing. Conclusions: The natural history of this individual’s RP is consistent with previously described MAK mutations, being significantly milder than that associated with other photoreceptor ciliopathies. We suggest inclusion of MAK as part of wider genetic testing in all individuals presenting with RP.

Human Retinal Explant Culture for Ex Vivo Validation of AAV Gene Therapy

Recombinant adeno-associated viral (AAV) vectors have been successfully employed as the mode of gene delivery in several clinical trials for the treatment of inherited retinal diseases to date. The design of such vectors is critical in determining cellular tropism and level of subsequent gene expression that may be achieved following viral delivery. Here we describe a system for living retinal tissue extraction, ex vivo culture, viral transduction and assessment of transgene expression that may be used to assess viral constructs for gene therapy in the human retina at a preclinical stage.

Beneficial effects on vision in patients undergoing retinal gene therapy for choroideremia

Retinal gene therapy is increasingly recognized as a novel molecular intervention that has huge potential in treating common causes of blindness, the majority of which have a genetic aetiology1–5. Choroideremia is a chronic X-linked retinal degeneration that was first described in 18726. It leads to progressive blindness due to deficiency of Rab-escort protein 1 (REP1). We designed an adeno-associated viral vector to express REP1 and assessed it in a gene therapy clinical trial by subretinal injection in 14 patients with choroideremia. The primary endpoint was vision change in treated eyes 2 years after surgery compared to unoperated fellow eyes. Despite complications in two patients, visual acuity improved in the 14 treated eyes over controls (median 4.5 letter gain, versus 1.5 letter loss, P = 0.04), with 6 treated eyes gaining more than one line of vision (>5 letters). The results suggest that retinal gene therapy can sustain and improve visual acuity in a cohort of predominantly late-stage choroideremia patients in whom rapid visual acuity loss would ordinarily be predicted.The long-term follow-up results of a phase 1/2 retinal gene therapy clinical trial for choroideremia (NCT01461213) support the safety and efficacy of the treatment.

Direct ophthalmoscopy should be taught within the context of its limitations.

It is with great interest that we read your controversy regarding whether or not direct ophthalmoscopy should be taught to medical students.1,2 We feel that direct ophthalmoscopy is a fundamental skill that all doctors should be able to perform. However, the skills in ophthalmoscopy must be complemented by a complete ocular examination and not consist of ophthalmoscopy alone (particularly visual acuity measurement, examination of pupils, visual fields and basic eye movements). For example, a doctor may feel that an optic disc appears to be swollen; however, it is only after complementing this assessment by proving the presence of an RAPD that their strength of conviction would grow. Direct ophthalmoscopy can form part of final year examination assessment at Medical School and several post-graduate membership examinations. Eye problems represent 1.5% of presentations to GPs3 and consultation rates for GP and eye casualty have been recorded at 71.8 per 1000 population per year.4 Bruner5 developed the theory of the spiral curriculum whereby complex ideas can be taught at simple levels early on and then re-visited at more complex levels later on. This idea of spiral learning underpins many medical school curriculums and encourages independent problem solving. Therefore, one can be exposed to the technique of direct ophthalmoscopy early on during medical school clinical teaching and re-visit situations when the direct ophthalmoscope would aid diagnosis and management in later clinical years. This technique currently underpins much of current clinical teaching today. For example, most medical students learn the ‘nut-and-bolts’ of a cardio-respiratory examination in the first year of medical school but only contextualise this in later years. Ideally, when examining a fundus we would want to dilate the pupil and this is rarely done outside of the ophthalmology clinic, because of the fear of inducing angle closure glaucoma. Knowing that the risk of such an event occurring with Tropicamide eye drops is negligible should re-assure doctors. Guidance needs to be integrated within the curriculum allowing use of mydriatics to allow adequate examination. Not doing this would be akin to expecting detection of a murmur through multiple layers of clothing.