DISPOSITION AND TOXICITY OF A MIXED BACKBONE ANTISENSE OLIGONUCLEOTIDE, TARGETED AGAINST HUMAN CYTOMEGALOVIRUS, AFTER INTRAVITREAL INJECTION OF ESCALATING SINGLE DOSES IN THE RABBIT

This paper is available online at http://www.dmd.org ABSTRACT: The ocular disposition and toxicity of GEM132, a mixed backbone phosphorothioate oligonucleotide developed for the treatment of cytomegalovirus-induced retinitis, were studied in rabbits for 6 months following single intravitreal injection of 5, 20, or 100 g/eye (toxicity) and 3.7, 15.7, or 78.5 g/eye (disposition). Intraocular pressure, electroretinograms, and ophthalmoscopy were evaluated in the toxicity arm as well as gross and microscopic pathology at the termination of the study. Vitreous humor, retina, and the remaining ocular tissues were collected from all animals in the disposition arm. No toxicities were observed in the low-dose group. Intraocular pressure was transiently mildly increased in the mid-and high-dose groups; macroscopic findings were mild and infrequent. Changes in electroretinograms and histopathological findings attributed to GEM132 were observed by 4 weeks postdose in the high-dose group. Area under the curve values in all ocular tissues sampled were proportional to dose, suggesting GEM132 disposition exhibited first-order kinetics. Vitreous humor concentrations decreased in a multiphasic manner, consistent with rapid distribution. Polyacrylamide gel electrophoresis analysis of retinal extracts indicated that, at 4 weeks postdose, 90% of the radioactivity was associated with parent compound. At 8 weeks postdose, this had decreased to 70%, and subsequently to 50% at 21 weeks postdose. In retina, GEM132 reached concentrations >5 times IC 90 by 1 week postdose, with maximum concentrations 4 to 8 weeks postdose. Retinal concentrations of intact GEM132 then declined at a very slow rate. Microautoradiography suggested that radioactivity was distributed throughout the retinal layers, the largest amount being located in the middle layers. The advancement of antisense oligonucleotides to human clinical trials as anticancer Human cytomegalovirus (HCMV), a common opportunistic infection in immunocompromised persons, is a major cause of life-threatening disease. CMV-induced retinitis, if left untreated, will cause severe, irreversible damage to the retina, resulting in progressive loss of vision in the involved eye(s). Currently available therapies for CMV-induced retinitis include ganciclovir, foscamet, cidofovir, and the recently approved PS antisense oligonucleotide, fomivirsen (ISIS 2922). GEM132 is a 20-mer mixed backbone oligonucleotide (MBO) with 2Ј-O-methylribonucleosides at the two 3Ј-and four 5Ј-terminal nucleotides. It is antisense, complementary to the intron-exon boundary of the UL35 and UL37 premRNA transcripts of HCMV, and has shown antiviral activity in vitro with an IC 90 of about 0.1 M against standard laboratory strains of the virus Although studies have been published on the pharmacokinetics of PS oligonucleotides after intravitreal dosing Materials and Methods Synthesis and Purification of GEM132. GEM132 is a 20-base phosphorothioate MBO (5Ј-UGGGGCTTACCTTGCGAACA-3Ј), 6605-Da molecular 1255 mass (free acid), in which two deoxynucleosides at the 5Ј end and four deoxynucleosides at the 3Ј end (underlined) are substituted with 2Ј-O-methylribonucleosides. GEM132 was chemically synthesized using deoxynucleoside phosphoramidites (Milligen, Milford, MA) and 2Ј-O-methylribonucleoside phosphoramidites (Glen Research, Sterling, VA) on an automated synthesizer as previously described Experimental Design. Male Dutch Belted rabbits were given a single intravitreal injection (50 l) of GEM132 in both eyes. Control animals received saline only. For the disposition study, [ 14 C]GEM132 was administered; eyes and selected tissues were harvested at various times postdosing (n ϭ 3 eyes/time point). The oligonucleotide was dissolved in 0.9% saline, and the doses administered were 3.7 g/eye (0.07 Ci), 15.7 g/eye (0.3 Ci), and 78.5 g/eye (1.5 Ci). For the toxicity study, unlabeled GEM132 was administered at doses of 5, 20, or 100 g/eye. Electroretinograms (ERGs). Electroretinograms were obtained from all animals in the study at regular intervals. Light stimuli included scotopic blue, red, and white single flash and 30 Hz white flicker. The animals were anesthetized with ketamine (50 mg/kg)/xylazine (5 mg/kg) and dark-adapted at least 30 min before recording of the ERG. A mydriatic agent was applied to the eyes to dilate the pupils before testing. Sample Processing and Storage Conditions. At each time point, the left eye from the even-numbered animals and both eyes from the odd-numbered animals were dissected, and retina, vitreous humor, and remaining ocular tissue were removed and weighed. The weighed vitreous humor (0.724 Ϯ 0.031 g, mean Ϯ S.D.) and retina (0.0283 Ϯ 0.0003 g) samples were homogenized in 2 ml of 0.9% saline in preweighed glass tubes with a Teflon probe. After homogenization, aliquots of retina were solubilized in 35% tetraethylammonium hydroxide. Ten milliliters of Hionic Fluor scintillation fluid (Canberra Packard, Ontario, Canada) was added to vitreous humor and solubilized retina, and radioactivity was measured by liquid scintillation spectroscopy. Remaining ocular tissue samples (1.47 Ϯ 0.18 g) were minced and digested in toto in 35% tetraethylammonium hydroxide. Duplicate aliquots of the digest were added to 10 ml of scintillation fluid, and radioactivity was determined. Samples having a radioactivity level of less than or equal to double background were considered below the limit of quantitation and considered zero. Autoradiography and Microautoradiography Conditions. At each time point, the right eye of each even-numbered animal was removed and fixed in a 2.5% gluteraldehyde solution, stored, refrigerated, and transferred within 24 h to 10% neutral buffered Formalin where they remained for at least 24 h before trimming. Eyes were then trimmed, dehydrated, infiltrated, and imbedded in paraffin within 8 days after necropsy. Six-micrometer-thick sections were prepared. The following procedures were conducted under reduced safety light conditions: sections were subjected to deparaffination, dipped in diluted and warmed Kodak NTB-2 nuclear track emulsion, followed by a 30-min rinse in hot distilled water and controlled drying. After drying, the emulsion-coated slides were stored in lightproof plastic boxes at 4 -8°C. All slides were developed after 15 days of exposure using a solution of Kodak D-19 developer, washed, fixed in Kodak Rapid Fixer, and washed. Slides were stained with H&E, mounted, and evaluated for the localization and relative concentration of radioactivity (visualized as small, reduced silver grains lying on cellular surfaces). A four-grade (1, slight; 2, mild; 3, moderate; and 4, severe) grading scheme was used to semiquantify the amount of radioactivity. The amount of radioactivity present in underlying sclera was used to establish a threshold between a negative and a slight deposition of radioactivity in the retina. Polyacrylamide Gel Electrophoresis (PAGE) and Phosphor Image Analysis. Retinal homogenates were subjected to protein digestion by incubation with proteinase K enzyme (0.25 ml of a 20 mg/ml solution) containing 20 mM Tris EDTA for 3 h at 60°C. Samples were extracted twice with Tris ETDA-saturated phenol-chloroform solution (1:1, v/v) and once with chloroform to remove proteinase K, digested protein, and lipids from nucleic acids. After extraction, an aliquot of the organic and aqueous phase from each sample was removed, and total radioactivity was determined by liquid scintillation spectroscopy. This allowed for the determination of extraction recovery. The average recoveries (ϮS.D.) were 79.7 Ϯ 13.2% (quality control standards), 74.3 Ϯ 19.1% (day 7), 75.7 Ϯ 6.4% (day 14), 72.4 Ϯ 10.3% (day 28), 74.1 Ϯ 8.2% (day 56), 87.3 Ϯ 3.6% (day 114), and 87.4 Ϯ 12.0% (day 149). No radioactivity (Ն2ϫ background) was measurable in the organic phase, indicating that all the GEM132-associated radioactivities were retained in the aqueous phase following extraction. Loss of radioactivity was believed to be due to the difficulty in completely separating the two phases; a thin layer of the aqueous phase was always left behind. An 8-l aliquot of the aqueous phase was loaded on a 20% polyacrylamide gel containing 7.5 M urea and subjected to electrophoresis (70 -290 V, 3-14 mA, 60 -153 min). Quality control standards (0.11, 0.60, and 4.1 mg/ml, corresponding to 780, 4,500, and 21,000 dpm) were prepared by adding known amounts of radiolabeled GEM132 to retinal homogenates obtained from control rabbits. Duplicate standards were loaded on each of the two outer channels of the gel. After electrophoresis, Southern blots of the gels were performed on 0.45-nitrocellulose membranes over a period of about 12 h. The nitrocellulose membranes were then removed and allowed to dry at room temperature. Membranes were exposed to the phosphor screen for 25 to 288 h (Molecular Dynamics Storage Phosphor Screen, Sunnyvale, CA) at room temperature, after which the phosphor screen was scanned by a phosphor imager (Molecular Dynamics Phosphor Imager SI) to obtain radioactivity profiles. The lower limit of detection was determined to be an area of Ն15 phosphor image units. Concentrations of intact GEM132 were calculated by multiplying the measured total radioactivity in the retina (total MBO) by the fraction eluting with the same mobility as the standards, as determined by PAGE/phosphor image analysis and correcting for purity (90%). Intergel variability and intragel variability were determined for phosphor image analysis. Intergel variability ranged from 8.6 to 5.3%. For intragel variability, the %CV for four samples processed on the same gel ranged from 5.5 to 11.1%. The limit of quantification for phosphor image analysis, determined for four different exposure times, was as follows: 295 dpm (24 h), 200 dpm (48 and 96 h), and 100 dpm (144 h). Results Ophthalmic Examinations. Signs of ocular irritation attributed to the injection of GEM132 were infrequent, mild, and transient. Mild cellular infiltrate in the anterior vitreous occurred almost exclusively in the high-dose animals from 2 to 16 weeks postdose. Signs of iritis were also mild and infrequent. Retinal lesions were observed almost exclusively in the high-dose animals. Changes in intraocular pressures were mild and transient and considered only potentially related to treatment. Electroretinograms. Representative data for ERGs are shown in Histopathology. Histopathological findings attributed to the administration of GEM132 were seen in the retina, lens, and optic nerve of a few animals in the mid-dose group at 8 and 21 weeks and the high-dose group at 4, 8, and 16 weeks postdose. Slight retinal degen- DVORCHIK AND MARQUIS eration characterized by partial disorganization of the inner and outer plexiform layer and the ganglion cell layer was observed in one high-dose animal at week 4 and in three high-dose animals at week 8. This finding was also observed in one mid-dose animal at weeks 8 and 21 and in one control and two low-dose animals at week 21. Retinal detachment was observed in one high-dose animal at week 8. A basophilic granular material was observed in the retina of one highdose animal at week 4, in three high-dose animals at week 8, and in two high-dose animals at week 16. One mid-dose animal at week 8 also presented with this finding. A slight to mild mononuclear infiltration was seen in the optic nerves of some high-dose animals at weeks 4 and 6 and in one high-dose animal at week 16. This was also observed in some mid-dose animals at week 8

Theranostic SPECT reconstruction for improved resolution: application to radionuclide therapy dosimetry

BACKGROUND: SPECT-derived dose estimates in tissues of diameter less than 3× system resolution are subject to significant losses due to the limited spatial resolution of the gamma camera. Incorporating resolution modelling (RM) into the SPECT reconstruction has been proposed as a possible solution; however, the images produced are prone to noise amplification and Gibbs artefacts. We propose a novel approach to SPECT reconstruction in a theranostic setting, which we term SPECTRE (single photon emission computed theranostic reconstruction); using a diagnostic PET image, with its superior resolution, to guide the SPECT reconstruction of the therapeutic equivalent. This report demonstrates a proof in principle of this approach. METHODS: We have employed the hybrid kernelised expectation maximisation (HKEM) algorithm implemented in STIR, with the aim of producing SPECT images with PET-equivalent resolution. We demonstrate its application in both a dual 68Ga/177Lu IEC phantom study and a clinical example using 64Cu/67Cu. RESULTS: SPECTRE is shown to produce images comparable in accuracy and recovery to PET with minimal introduction of artefacts and amplification of noise. CONCLUSION: The SPECTRE approach to image reconstruction shows improved quantitative accuracy with a reduction in noise amplification. SPECTRE shows great promise as a method of improving SPECT radioactivity concentrations, directly leading to more accurate dosimetry estimates in small structures and target lesions. Further investigation and optimisation of the algorithm parameters is needed before this reconstruction method can be utilised in a clinical setting

A qualitative study of Parent to Parent support for parents of children with special needs. Consortium to evaluate Parent to Parent.

OBJECTIVE: To examine qualitatively the experiences of parents participating in Parent to Parent programs. METHOD: Twenty-four parents of children with special needs, a subset of subjects in a larger quantitative study, participated in a semi-structured telephone interview to explore the impact and meaning of being matched with a trained supporting parent. RESULTS: Qualitative analysis reveals a successful match is contingent upon creation of a reliable ally in the supporting parent, comprised of four main components: (1) perceived sameness, (2) situational comparisons that enable learning and growth, (3) round-the-clock availability of support, and (4) mutuality of support. CONCLUSIONS: Parent to Parent support creates a community of similar others trained to listen and be supportive and provides an opportunity for matched parents to experience equality and mutuality in their relationship. Findings also identify the need for quality control in Parent to Parent programs and the importance of such programs as an adjunct to traditional professional services

Reasons, Coherence, and Group Rationality

Philosophy and Phenomenological Research, EarlyView

Propositional update operators based on formula/literal dependence

International audienceWe present and study a general family of belief update operators in a propositional setting. Its operators are based on formula/literal dependence, which is more fine-grained than the notion of formula/variable dependence that was proposed in the literature: formula/variable dependence is a particular case of formula/literal dependence. Our update operators are defined according to the "forget-then-conjoin" scheme: updating a belief base by an input formula consists in first forgetting in the base every literal on which the input formula has a negative influence, and then conjoining the resulting base with the input formula. The operators of our family differ by the underlying notion of formula/literal dependence, which may be defined syntactically or semantically, and which may or may not exploit further information like known persistent literals and pre-set dependencies. We argue that this allows to handle the frame problem and the ramification problem in a more appropriate way. We evaluate the update operators of our family w.r.t. two important dimensions: the logical dimension, by checking the status of the Katsuno-Mendelzon postulates for update, and the computational dimension, by identifying the complexity of a number of decision problems (including model checking, consistency and inference), both in the general case and in some restricted cases, as well as by studying compactability issues. It follows that several operators of our family are interesting alternatives to previous belief update operators

Novel cyclic di-GMP effectors of the YajQ protein family control bacterial virulence

Bis-(3 ',5 ') cyclic di-guanylate (cyclic di-GMP) is a key bacterial second messenger that is implicated in the regulation of many critical processes that include motility, biofilm formation and virulence. Cyclic di-GMP influences diverse functions through interaction with a range of effectors. Our knowledge of these effectors and their different regulatory actions is far from complete, however. Here we have used an affinity pull-down assay using cyclic di-GMP-coupled magnetic beads to identify cyclic di-GMP binding proteins in the plant pathogen Xanthomonas campestris pv. campestris (Xcc). This analysis identified XC_3703, a protein of the YajQ family, as a potential cyclic di-GMP receptor. Isothermal titration calorimetry showed that the purified XC_3703 protein bound cyclic di-GMP with a high affinity (K-d similar to 2 mu M). Mutation of XC_3703 led to reduced virulence of Xcc to plants and alteration in biofilm formation. Yeast two-hybrid and far-western analyses showed that XC_3703 was able to interact with XC_2801, a transcription factor of the LysR family. Mutation of XC_2801 and XC_3703 had partially overlapping effects on the transcriptome of Xcc, and both affected virulence. Electromobility shift assays showed that XC_3703 positively affected the binding of XC_2801 to the promoters of target virulence genes, an effect that was reversed by cyclic di-GMP. Genetic and functional analysis of YajQ family members from the human pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia showed that they also specifically bound cyclic di-GMP and contributed to virulence in model systems. The findings thus identify a new class of cyclic di-GMP effector that regulates bacterial virulence

Multi-Periodic Oscillations in Cepheids and RR Lyrae-Type Stars

Classical Cepheids and RR Lyrae-type stars are usually considered to be textbook examples of purely radial, strictly periodic pulsators. Not all the variables, however, conform to this simple picture. In this review I discuss different forms of multi-periodicity observed in Cepheids and RR Lyrae stars, including Blazhko effect and various types of radial and nonradial multi-mode oscillations.Comment: Proceedings of the 20th Stellar Pulsation Conference Series: "Impact of new instrumentation & new insights in stellar pulsations", 5-9 September 2011, Granada, Spai

Quantification of the effects of audible rattle and source type on the human response to environmental vibration

The present research quantifies the influence of source type and the presence of audible vibration-induced rattle on annoyance caused by vibration in residential environments. The sources of vibration considered are railway and the construction of a light rail system. Data were measured in the United Kingdom using a socio-vibration survey (N = 1281). These data are analyzed using ordinal logit models to produce exposure{response relationships describing community annoyance as a function of vibration exposure. The influence of source type and the presence of audible vibration-induced rattle on annoyance are investigated using dummy variable analysis, and quantified using odds-ratios and community tolerance levels (CTL). It is concluded that the sample population is more likely to express higher levels of annoyance if the vibration source is construction compared to railway, and if vibration-induced rattle is audible

Parental transfer of the antimicrobial protein LBP/BPI protects Biomphalaria glabrata eggs against oomycete infections

Copyright: © 2013 Baron et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was funded by ANR (ANR-07-BLAN-0214 and ANR-12-EMMA-00O7-01), CNRS and INRA. PvW was financially supported by the BBSRC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Statistical mechanics of voting

Decision procedures aggregating the preferences of multiple agents can produce cycles and hence outcomes which have been described heuristically as `chaotic'. We make this description precise by constructing an explicit dynamical system from the agents' preferences and a voting rule. The dynamics form a one dimensional statistical mechanics model; this suggests the use of the topological entropy to quantify the complexity of the system. We formulate natural political/social questions about the expected complexity of a voting rule and degree of cohesion/diversity among agents in terms of random matrix models---ensembles of statistical mechanics models---and compute quantitative answers in some representative cases.Comment: 9 pages, plain TeX, 2 PostScript figures included with epsf.tex (ignore the under/overfull \vbox error messages