Joining Chain–Expressing and–Nonexpressing B Cell Populations in the Mouse

The diphtheria toxin A chain (DTA) was gene targeted into the Joining chain (J chain) locus to create a mouse strain selecting against J chain–expressing cells, JDTA mice. Serum immunoglobulin (Ig)M and serum IgG were reduced six to eightfold, while serum IgA was elevated 14-fold in these mice. JDTA mice were immune competent although the serum Ig response compared with wild-type mice was reduced sixfold at day 14 but only fourfold at day 45 after immunization. Exchanging the DTA gene with a cDNA for c-myc resulted in mice with a distinct phenotype with increased Ig production and enhanced humoral immune responses. Analysis of single B cells stimulated by lipopolysaccharide in vitro using reverse transcription–polymerase chain reaction showed that J chain–nonexpressing B cells could be detected that had a secretory phenotype as determined by an abundance of transcript for secretory IgM. Finally, limiting dilution analysis of peripheral B cells showed that J chain expression was a clonal property already established in naive, peripheral B lymphocytes

Dynamic in vivo quantification of rod photoreceptor degeneration using fluorescent reporter mouse models of retinitis pigmentosa

Imaging techniques have revolutionised the assessment of retinal disease in humans and animal models. Here we describe a novel technique for the in vivo visualisation of rod photoreceptors which permits semiquantitative assessment of outer retinal degeneration, and validate this approach in two mouse models of retinitis pigmentosa (RP). Transgenic mice carrying an Nrl-EGFP allele and homozygous for either knock-out of rhodopsin (Nrl-EGFP, Rho-/-) or heterozygous for knock-in of P23H mutant rhodopsin (Nrl-EGFP, RhoP23H/+) were used in this study. These novel strains have green fluorescent rods which undergo a progressive degeneration. Fundus imaging was performed at three-weekly intervals by near infrared reflectance (NIR) and blue light autofluorescence (BAF) confocal scanning laser ophthalmoscopy (cSLO). Mean grey values (mGV), which quantify fluorescence levels within such images, were compared for degenerate and age-matched non-degenerate (Nrl-EGFP, Rho+/+) controls. Mean grey value significantly decreased over time in the Rho-/- and RhoP23H/+ groups but was maintained in Rho+/+ mice (P < 0.001, two-way ANOVA). This corresponded to outer nuclear layer (ONL) thinning as observed by histology. The mGV of superior retina was significantly greater than that of inferior retina in RhoP23H/+ (P = 0.0024) but not in age-matched Rho+/+ (P = 0.45) or Rho-/- (P = 0.65) mice reflecting histological findings. Focal loss of rods could be visualised and mapped in vivo with this technique following a toxic insult, with thinning of the ONL being confirmed in hypofluorescent regions by spectral domain ocular coherence tomography (OCT). Fluorescence labelling of rods permits in vivo characterisation of models of RP and may provide new insights into patterns of degeneration, or rescue effect after treatment. mGV can be used in such cases as a semiquantitative metric of ONL degeneration, and can be used to identify regional variations in photoreceptor loss

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

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 &lt; 0.001) and decreased by hypotonic stimulation (intragastric water, z = −3.37, P &lt; 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

Microbiology and visual outcomes of culture-positive bacterial endophthalmitis in Oxford, UK

Purpose: To review the microbiology of culture-positive cases of bacterial endophthalmitis, and to correlate this with visual outcomes. Method: Case notes were reviewed for culture-positive cases of bacterial endophthalmitis over a period from November 1999 to June 2012. Cases were identified retrospectively using a local database. The Fisher exact test was used for statistical analysis. Results: Of the 47 cases of culture-positive bacterial endophthalmitis identified, 81 % occurred postoperatively, 11 % followed intravitreal injection, 6 % had an endogenous source and 2 % followed ocular trauma. Eighty-seven percent of bacteria cultured were Gram-positive. The most commonly identified organisms were coagulase-negative Staphylococci (47 %) and Streptococcus spp. (30 %). Patients were treated with intravitreal vancomycin and either amikacin or ceftazidime. All Gram-negative isolates were sensitive to aminoglycosides and ceftazidime, and all Gram-positive isolates were vancomycin-sensitive. Final visual acuity (VA) was 6/12 or better in 41 % of cases and counting fingers (CF) or worse in 30 %. Endophthalmitis caused by Streptococcus spp. was associated with a poorer final VA (OR for CF or worse = 14.9, P &lt; 0.01). Cases caused by coagulase-negative Staphylococci had a better visual outcome (OR for VA of 6/12 or better = 5.7, P = 0.013). Five eyes were eviscerated or enucleated. Infection with Haemophilus influenzae was strongly associated with this outcome (OR = 57, P &lt; 0.01). Conclusion: Over the time period of this study there was no evidence of emerging resistance to empirical antibiotics which are commonly used for the treatment of bacterial endophthalmitis. Infection with coagulase-negative Staphylococci was associated with a good visual outcome, whilst infection with Streptococcus spp. or Haemophilus influenzae was associated with a poor visual outcome

Microbiology and visual outcomes of culture-positive bacterial endophthalmitis in Oxford, UK

Purpose: To review the microbiology of culture-positive cases of bacterial endophthalmitis, and to correlate this with visual outcomes. Method: Case notes were reviewed for culture-positive cases of bacterial endophthalmitis over a period from November 1999 to June 2012. Cases were identified retrospectively using a local database. The Fisher exact test was used for statistical analysis. Results: Of the 47 cases of culture-positive bacterial endophthalmitis identified, 81 % occurred postoperatively, 11 % followed intravitreal injection, 6 % had an endogenous source and 2 % followed ocular trauma. Eighty-seven percent of bacteria cultured were Gram-positive. The most commonly identified organisms were coagulase-negative Staphylococci (47 %) and Streptococcus spp. (30 %). Patients were treated with intravitreal vancomycin and either amikacin or ceftazidime. All Gram-negative isolates were sensitive to aminoglycosides and ceftazidime, and all Gram-positive isolates were vancomycin-sensitive. Final visual acuity (VA) was 6/12 or better in 41 % of cases and counting fingers (CF) or worse in 30 %. Endophthalmitis caused by Streptococcus spp. was associated with a poorer final VA (OR for CF or worse = 14.9, P < 0.01). Cases caused by coagulase-negative Staphylococci had a better visual outcome (OR for VA of 6/12 or better = 5.7, P = 0.013). Five eyes were eviscerated or enucleated. Infection with Haemophilus influenzae was strongly associated with this outcome (OR = 57, P < 0.01). Conclusion: Over the time period of this study there was no evidence of emerging resistance to empirical antibiotics which are commonly used for the treatment of bacterial endophthalmitis. Infection with coagulase-negative Staphylococci was associated with a good visual outcome, whilst infection with Streptococcus spp. or Haemophilus influenzae was associated with a poor visual outcome

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

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.</p

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.</p