Efficacy of the positioning system when measuring canine intraocular pressures with the Reichert® Tono-Vera® Vet rebound tonometer.

OBJECTIVE: To compare intraocular pressure (IOP) measurements in dogs taken with the Reichert® Tono-Vera® Vet rebound tonometer with and without the automatic positioning system. ANIMALS STUDIED: Measurements were taken on 49 eyes from 26 Beagle-derived dogs with variable genetics-four non-glaucomatous and 22 ADAMTS10-mutant dogs affected with different stages of open-angle glaucoma. Seventeen of the 26 dogs were measured 2-4 times on different days with variable intervals since IOP-lowering medications were administered. PROCEDURES: In each dog, tonometry was performed with the Tono-Vera® Vet using three different methods in a randomized order: (Method 1) Average of three readings with an automatic positioning system; (Method 2) one reading with an automatic positioning system; and (Method 3) average of three readings obtained without the automatic positioning system. Statistical analyses included one-way ANOVA, Tukey pairwise comparisons, and Bland-Altman plots (MiniTab®). RESULTS: With each of the three tonometry methods, 116 measurements were taken, resulting in 348 total IOP measurements with a range of 12.8-49.9 mmHg. The means and standard deviations for each method were 25.4 ± 6.9 mmHg (Method 1), 26.0 ± 7.2 mmHg (Method 2), and 26.9 ± 7.7 mmHg (Method 3), with no significant differences (p = .27). Mean IOP variances were also not significantly different between tonometry methods (p = .24 to .78). CONCLUSIONS: Because mean IOPs and their standard deviations were not statistically different between the three tonometry methods, we conclude that Tono-Vera® Vet measurements conducted without the aid of the positioning system still provide reliable results.

Validation of the eyeTelemed IOPvet indentation tonometer for use in dogs.

OBJECTIVE: To assess the accuracy of canine intraocular pressure (IOP) estimates from the eyeTelemed IOPvet indentation tonometer. ANIMALS STUDIED: Part 1 included 54 eyes from 28 Beagle dogs-23 ADAMTS10-mutants with open-angle glaucoma and 5 normals. Part 2 involved five normal canine ex vivo globes. PROCEDURE: Part 1 (in vivo) compared IOPvet estimates in normal and glaucomatous dogs to Reichert Tono-Vera® Vet rebound tonometry. The three IOPvet estimates were green (normal; <20 mmHg, according to the manufacturer), yellow (elevated; 20-30 mmHg), and red (high; >30 mmHg). In Part 2 (ex vivo), the pressure inside freshly enucleated normal canine eyes was progressively increased from 5 to 80 mmHg and compared to IOPvet estimates. Descriptive statistics compared IOPvet estimates to rebound tonometry and direct manometry, with the threshold from normal to glaucoma set at 30 mmHg. RESULTS: In Part 1 (in vivo), normal pressures (≤30 mmHg) were mainly identified correctly as green or yellow-110 of 111 estimates, corresponding to a specificity of 99%. Only 16 of 125 affected estimates were correctly displayed in the >30-mmHg range; the remaining 109 showed ≤30 mmHg, corresponding to a sensitivity of 13%. In Part 2 (ex vivo), all normal pressures were correctly estimated with green, but 64 of 88 manometric IOPs >30 mmHg were falsely estimated as 20-30 mmHg. CONCLUSIONS: The IOPvet is inaccurate in estimating canine IOP with a low sensitivity at identifying dogs with IOP > 30 mmHg. Canine-specific instrument revision is required to correctly identify elevated (yellow = 20-30 mmHg) and high (red >30 mmHg) IOPs.

Relative ability of aqueous humor from dogs with and without primary angle-closure glaucoma and ADAMTS10 open-angle glaucoma to catalyze or inhibit collagenolysis.

OBJECTIVE: The objective of the study was to compare the ability of aqueous humor (AH) from dogs with primary angle-closure glaucoma (CPACG), companion dogs without overt evidence of CPACG, and Beagles with and without ADAMTS10 open-angle glaucoma (ADAMTS10-OAG) to catalyze or inhibit collagenolysis. ANIMALS STUDIED: Seventeen normal pet dogs, 27 dogs with CPACG, 19 Beagles with ADAMTS10-OAG, and 4 unaffected Beagles. PROCEDURES: A fluorescein-based substrate degradation assay was used to assess AH proteolytic capacity. Samples were then assayed using the same substrate degradation assay, with recombinant activated matrix metalloproteinase-2 (MMP-2) added to measure protease inhibition effects. RESULTS: For the protease activity assay, relative fluorescence (RF) for AH from normal pet dogs was 13.28 ± 2.25% of control collagenase while RF for AH from dogs with CPACG was 17.47 ± 4.67%; RF was 8.57 ± 1.72% for ADAMTS10-OAG Beagles and 7.99 ± 1.15% for unaffected Beagles. For the MMP-2 inhibition assay, RF for AH from normal dogs was 34.96 ± 15.04% compared to MMP-2 controls, while RF from dogs with CPACG was 16.69 ± 7.95%; RF was 85.85 ± 13.23% for Beagles with ADAMTS10-OAG and 94.51 ± 8.36% for unaffected Beagles. Significant differences were found between dogs with CPACG and both normal pet dogs and dogs with ADAMTS10-OAG and between normal pet dogs and both groups of Beagles. CONCLUSIONS: AH from dogs with CPACG is significantly more able to catalyze proteolysis and inhibit MMP-2 than AH from normal dogs or dogs with ADAMTS10-OAG. Results suggest that pathogenesis may differ between CPACG and ADAMTS10-OAG.

Comparison of three rebound tonometers in normal and glaucomatous dogs.

OBJECTIVE: The objectives of the study were to compare intraocular pressure (IOP) readings across a wide range and obtained via three rebound tonometers in ADAMTS10-mutant Beagle-derived dogs with different stages of open-angle glaucoma (OAG) and normal control dogs and to investigate the effect of central corneal thickness (CCT). ANIMALS STUDIED: Measurements were performed on 99 eyes from 50 Beagle-derived dogs with variable genetics-16 non-glaucomatous and 34 with ADAMTS10-OAG. Seventeen OAG eyes were measured twice-with and without the use of IOP-lowering medications. PROCEDURES: IOP was measured in each eye using three tonometers with their "dog" setting-ICare® Tonovet (TV), ICare® Tonovet Plus® (TVP), and the novel Reichert® Tono-Vera® Vet (TVA)-in randomized order. CCT was measured with the Accutome® PachPen. Statistical analyses included one-way ANOVA, Tukey pairwise comparisons, and regression analyses of tonometer readings and pairwise IOP-CCT Pearson correlations (MiniTab®). RESULTS: A total of 116 IOP measurements were taken with each of the three tonometers. When comparing readings over a range of ~7-77 mmHg, mean IOPs from the TV were significantly lower compared with TVP (-4.6 mmHg, p < .001) and TVA (-3.7 mmHg, p = .001). We found no significant differences between TVA and TVP measurements (p = .695). There was a moderate positive correlation between CCT and IOP for TVA (r = 0.53, p < .001), TVP (r = 0.48, p < .001), and TV (r = 0.47, p < .001). CONCLUSIONS: Our data demonstrate strong agreement between TVP and TVA, suggesting that the TVA may similarly reflect true IOP values in canines. CCT influenced IOP measurements of all three tonometers.

Iris angiography in ADAMTS10 mutant dogs with open-angle glaucoma (ADAMTS10-OAG).

OBJECTIVE: To evaluate anterior segment angiographic findings in hypertensive ADAMTS10-open-angle glaucoma (ADAMTS10-OAG) eyes as compared to normotensive control eyes. ANIMALS STUDIED: Nine ADAMTS10-OAG beagles and four wild-type control dogs. PROCEDURES: Anterior segment angiography was performed under general anesthesia following intravenous injection of indocyanine green (ICG; 1 mg/kg) and sodium fluorescein (SF; 20 mg/kg) using a Heidelberg Spectralis® confocal scanning laser ophthalmoscope. Time to onset of iridal angiographic phases and the presence/severity of dye leakage into the iris stromal and/or aqueous humor were recorded. Group findings were compared, and multiple linear regression analysis was performed to identify potential factor associations with disease status. RESULTS: Time to onset of all angiographic phases visualized using ICG was significantly prolonged while time to onset of SF leakage into the aqueous humor was significantly reduced in glaucomatous eyes compared to controls. Only glaucomatous eyes (n = 9) demonstrated evidence of SF stromal leakage. Mean intraocular pressure (IOP) and age were significantly higher, while mean cardiac pulse was significantly lower in glaucomatous eyes compared to controls. Blood pressure and ocular perfusion pressure were not significantly different between groups. Multiple linear regression analysis, controlling for age, IOP, and pulse demonstrated glaucoma, was not predictive of the time to onset of any angiographic phase, stromal, or aqueous humor leakage. However, pulse was a significant factor contributing to the severity of aqueous humor leakage. CONCLUSIONS: A compromised vascular supply to the anterior segment exists in dogs with ADAMTS10-OAG. These observations warrant further exploration of what role altered perfusion and/or disruption to the blood-aqueous barrier may play.

Atypical chorioretinal lesions in Siberian Husky dogs with primary angle-closure glaucoma: a case series.

BACKGROUND: A number of etiologies for different canine chorioretinal lesions have been proved or suggested but some fundic lesions remain unclear in terms of an etiologic diagnosis, treatment options and prognosis. The purpose of this case series is to describe atypical chorioretinal lesions observed in dogs with primary angle-closure glaucoma (PACG). CASE PRESENTATION: Two spayed-female Siberian Huskies (3- and 4-year-old) and one Siberian Husky/Australian Shepherd mixed breed dog (11-month-old) that had multifocal depigmented retinal lesions and PACG were included. PROCEDURES: Ophthalmic examination, gross, and histopathologic examination findings are described. One of the dogs underwent further clinical diagnostics. Advanced clinical diagnostics on the fellow, presumed to be non-glaucomatous eye of a dog revealed: pectinate ligament dysplasia by gonioscopy, retinal thinning in the depigmented area and wedge shaped retinal thinning with delayed choroidal vascular perfusion by optical coherence tomography, confocal scanning laser ophthalmoscopy, fluorescein and indocyanine green angiography. Quantifiable maze testing for the same eye revealed mild nyctalopia but the full-field electroretinogram showed no generalized decrease of retinal function. Genetic testing for mutations within the retinitis pigmentosa GTPase regulator gene causing X-linked progressive retinal atrophy in Siberian Huskies was negative. Histopathologic evaluations on enucleated eyes in two dogs confirmed goniodysgenesis, PACG with optic nerve head cupping, and diffuse inner retinal atrophy. In addition, segmental profound retinal atrophy, loss of retinal pigment epithelium, and adhesion of the retina to Bruch's membrane was observed and coincided with multifocal depigmented lesions noted on fundic examination. CONCLUSIONS: To our knowledge, this is the first case series with clinical and histopathologic data of chorioretinal lesions, most likely caused by severely impaired choroidal perfusion. Further studies are warranted to elucidate the etiology and pathophysiology, including its possible association with PACG.

Outer retinal thickness and visibility of the choriocapillaris in four distinct retinal regions imaged with spectral domain optical coherence tomography in dogs and cats.

PURPOSE: To evaluate the outer retinal band thickness and choriocapillaris (CC) visibility in four distinct retinal regions in dogs and cats imaged with spectral domain optical coherence tomography (SD-OCT). To attempt delineation of a fovea-like region in canine and feline SD-OCT scans, aided by the identification of outer retinal thickness differences between retinal regions. METHODS: Spectralis® HRA + OCT SD-OCT scans from healthy, anesthetized dogs (n = 10) and cats (n = 12) were analyzed. Scanlines on which the CC was identifiable were counted and CC visibility was scored. Outer nuclear layer (ONL) thickness and the distances from external limiting membrane (ELM) to retinal pigment epithelium/Bruch's membrane complex (RPE/BM) and ELM to CC were measured in the area centralis (AC), a visually identified fovea-like region, and in regions superior and inferior to the optic nerve head (ONH). Measurements were analyzed using a multilevel regression. RESULTS: The CC was visible in over 90% of scanlines from dogs and cats. The ONL was consistently thinnest in the fovea-like region. The outer retina (ELM-RPE and ELM-CC) was thickest within the AC compared with superior and inferior to the ONH in dogs and cats (p < .001 for all comparisons). CONCLUSIONS: The CC appears a valid, albeit less than ideal outer retinal boundary marker in tapetal species. The AC can be objectively differentiated from the surrounding retina on SD-OCT images of dogs and cats; a fovea-like region was identified in dogs and its presence was suggested in cats. These findings allow targeted imaging and image evaluation of these regions of retinal specialization.

Localized alopecia and suppression of hypothalamic-pituitary-adrenal (HPA) axis in dogs following treatment with difluprednate 0.05% ophthalmic emulsion (Durezol®).

BACKGROUND: Despite the common use of topical ophthalmic corticosteroids in dogs, detailed reports on systemic and dermatologic adverse effects are limited. RESULTS: Nine purpose-bred research Beagles were treated with difluprednate 0.05% ophthalmic emulsion in one or both eyes 2-3 times daily. Some difluprednate treated dogs developed mild to severe alopecia of the periocular region, face, and distal pinna (5/9). The median duration of treatment prior to onset of dermatologic signs for difluprednate treated dogs was 550 days (453-1160 days). Diagnostic testing included complete blood count (CBC) and serum biochemistry, adrenocorticotropic hormone (ACTH) stimulation testing combined with endogenous ACTH measurement, and skin biopsy. The CBC and chemistry were within normal limits for all dogs. There were varying degrees of suppression of the hypothalamic-pituitary-adrenocortical (HPA) axis with difluprednate treatment. Dogs with the most profound alopecic changes had less pronounced HPA axis suppression compared to dogs with no integumentary changes. Skin biopsies demonstrated follicular atrophy and follicular keratosis. When topical difluprednate was reduced to unilateral therapy, the hair regrew on the untreated side of the face. In addition to the affected research dogs, a 7-year old female spayed Chihuahua that was being treated as a clinical patient with long-term difluprednate 0.05% ophthalmic emulsion developed generalized hypotrichosis on the head and body and a potbellied appearance. ACTH stimulation testing revealed suppression of the HPA axis with a mild increase in serum alkaline phosphatase (ALP) activity and a urine specific gravity of 1.016. The combination of clinical signs and laboratory abnormalities was supportive of iatrogenic hyperadrenocorticism. CONCLUSIONS: In dogs long-term use of difluprednate ophthalmic emulsion results in HPA axis suppression and in some cases iatrogenic hyperadrenocorticism. A novel pattern of localized alopecia is suspected to be related to dermal absorption and local action due to superior potency and penetration compared to other commonly utilized ophthalmic corticosteroids.

Safety and efficacy of topically administered netarsudil-latanoprost fixed dose combination (FDC; Rocklatan™) in normal and glaucomatous dogs with ADAMTS10-open-angle glaucoma (ADAMTS10-OAG).

OBJECTIVE: The aim of the study was to evaluate safety and efficacy of topically administered 0.02% netarsudil-0.005% latanoprost fixed-dose combination (FDC) (Rocklatan™; Aerie Pharmaceutical) in normal and glaucomatous dogs with ADAMTS10-open-angle glaucoma (ADAMTS10-OAG). ANIMALS STUDIED: Five normal and five glaucomatous beagle dogs with ADAMTS10-OAG were the study animals. PROCEDURES: In each dog, left (OS) or right eye (OD) was randomly selected for netarsudil-latanoprost FDC treatment. Contralateral eyes served as latanoprost-treated controls. The study was divided into four consecutive study periods: following a 4-day baseline period, two sequential 8-day study periods followed with once daily (q24h) and twice daily (q12h) treatments and ending with a washout period. Efficacy was measured by diurnal intraocular pressure (IOP) and pupil diameter. Safety was assessed by routine ophthalmic examination, gonioscopy, and pachymetry. Differences in least square means of quantitative outcome measures were compared between FDC and latanoprost treatments by using the linear Gaussian model. RESULTS: Baseline IOPs were 13.6 ± 0.7 mmHg (mean ± SEM) in normal and 28.3 ± 1.4 mmHg in OAG-affected dogs. There was a significant decrease in mean diurnal IOP following FDC administration in both normal (q24h: -2.1 mmHg; q12h: -4.1 mmHg) and glaucomatous dogs (q24h: -14.2 mmHg; q12h: -17.7 mmHg; p < .0001). There was no significant difference in the treatment effect when comparing FDC to latanoprost. Both FDC and latanoprost administration resulted in similarly significant pupil constriction (p < .0001). The FDC administration was well-tolerated but resulted in conjunctival hyperemia. CONCLUSIONS: Once or twice daily administration of netarsudil-latanoprost FDC (Rocklatan™) and latanoprost was equally effective in lowering IOP in normal and OAG-affected dogs. There was no netarsudil-related added treatment effect.

Safety and efficacy of topically administered netarsudil (Rhopressa™) in normal and glaucomatous dogs with ADAMTS10-open-angle glaucoma (ADAMTS10-OAG).

OBJECTIVE: To evaluate safety and efficacy of topically administered 0.02% netarsudil ophthalmic solution (Rhopressa™; Aerie Pharmaceutical) in normal and glaucomatous dogs with ADAMTS10-open-angle glaucoma (ADAMTS10-OAG). ANIMALS STUDIED: Five normal and 5 glaucomatous Beagle dogs with ADAMTS10-OAG. PROCEDURES: In each dog, left or right eye was randomly selected for netarsudil treatment. Contralateral eyes were sham-treated with balanced salt solution (BSS). Following a 1-week baseline period, dogs were treated once daily (q24h) during week 2, and twice daily (q12h) during week 3; week 4 served as washout period. Efficacy was measured by diurnal intraocular pressure (IOP) and pupil diameter. Safety was assessed by routine ophthalmic examination, gonioscopy, and pachymetry. Differences in least square means of quantitative outcome measures were compared between netarsudil and BSS sham-treated eyes by linear Gaussian model. RESULTS: Baseline IOPs were 18.5 ± 0.5 mm Hg (mean ± SEM) in normal and 27.8 ± 1.0 mm Hg in OAG dogs. Even though mean IOPs were lower in netarsudil- vs sham-treated eyes, the overall differences were neither significant nor clinically relevant, regardless of treatment frequency (q24h-normal: sham 16.4 ± 1.1 mm Hg vs treatment 15.6 ± 1.0 mm Hg; q24hr-OAG: sham 25.8 ± 2.3 mm Hg vs. treatment 25.7 ± 2.4 mm Hg; q12hr-normal: sham 15.4 ± 0.8 mm Hg vs. treatment 14.4 ± 0.8 mm Hg; q12hr-OAG: sham 26.3 ± 1.7 mm Hg vs. treatment 25.4 ± 1.8 mm Hg). Netarsudil administration was well tolerated but resulted in significant, moderate-to-severe conjunctival hyperemia (P < .001). CONCLUSIONS: Once or twice daily administration of netarsudil resulted in marginal and clinically irrelevant IOP decreases in normal and OAG-affected dogs. Except for conjunctival hyperemia, the drug was well tolerated.

A view of the E2-CD81 interface at the binding site of a neutralizing antibody against hepatitis C virus.

UNLABELLED: Hepatitis C virus (HCV) glycoprotein E2 is considered a major target for generating neutralizing antibodies against HCV, primarily due to its role of engaging host entry factors, such as CD81, a key cell surface protein associated with HCV entry. Based on a series of biochemical analyses in combination with molecular docking, we present a description of a potential binding interface formed between the E2 protein and CD81. The virus side of this interface includes a hydrophobic helix motif comprised of residues W(437)LAGLF(442), which encompasses the binding site of a neutralizing monoclonal antibody, mAb41. The helical conformation of this motif provides a structural framework for the positioning of residues F442 and Y443, serving as contact points for the interaction with CD81. The cell side of this interface likewise involves a surface-exposed hydrophobic helix, namely, the D-helix of CD81, which coincides with the binding site of 1D6, a monoclonal anti-CD81 antibody known to block HCV entry. Our illustration of this virus-host interface suggests an important role played by the W(437)LAGLF(442) helix of the E2 protein in the hydrophobic interaction with the D-helix of CD81, thereby facilitating our understanding of the mechanism for antibody-mediated neutralization of HCV. IMPORTANCE: Characterization of the interface established between a virus and host cells can provide important information that may be used for the control of virus infections. The interface that enables hepatitis C virus (HCV) to infect human liver cells has not been well understood because of the number of cell surface proteins, factors, and conditions found to be associated with the infection process. Based on a series of biochemical analyses in combination with molecular docking, we present such an interface, consisting of two hydrophobic helical structures, from the HCV E2 surface glycoprotein and the CD81 protein, a major host cell receptor recognized by all HCV strains. Our study reveals the critical role played by hydrophobic interactions in the formation of this virus-host interface, thereby contributing to our understanding of the mechanism for antibody-mediated neutralization of HCV.

Structural evidence for a bifurcated mode of action in the antibody-mediated neutralization of hepatitis C virus.

Hepatitis C virus (HCV) envelope glycoprotein E2 has been considered as a major target for vaccine design. Epitope II, mapped between residues 427-446 within the E2 protein, elicits antibodies that are either neutralizing or nonneutralizing. The fundamental mechanism of antibody-mediated neutralization at epitope II remains to be defined at the atomic level. Here we report the crystal structure of the epitope II peptide in complex with a monoclonal antibody (mAb#8) capable of neutralizing HCV. The complex structure revealed that this neutralizing antibody engages epitope II via interactions with both the C-terminal α-helix and the N-terminal loop using a bifurcated mode of action. Our structural insights into the key determinants for the antibody-mediated neutralization may contribute to the immune prophylaxis of HCV infection and the development of an effective HCV vaccine.

Development of Crystalline Corneal Opacities (Steroid Keratopathy) in Dogs After Treatment With Ophthalmic Corticosteroids.

PURPOSE: To retrospectively evaluate and describe the relationship between the use of topical corticosteroids and the development of crystalline corneal opacities (steroid keratopathy) in a colony of research Beagles and Beagle-derived dogs. METHODS: Medical records of 73 purpose-bred Beagles and Beagle-derived dogs were reviewed from June 2012 to May 2021. All dogs were treated with topical ophthalmic corticosteroids for at least 21 days. In addition to regular ophthalmic examination, some dogs also had a systemic lipid profile (n = 6) performed to work up further and characterize the crystalline corneal opacities. Globes of 3 dogs were examined histopathologically. RESULTS: Axial stromal crystalline corneal opacities were appreciated in 25 eyes of 14 dogs after a median of 141 days after initiating treatment (35-396 days). Multiple corticosteroids were used, including neomycin-polymyxin b-dexamethasone 0.1% ophthalmic ointment, prednisolone acetate 1% ophthalmic suspension, and difluprednate 0.05% ophthalmic emulsion (Durezol). Resolution of corneal opacity was documented in 4 of 25 eyes when ophthalmic corticosteroids were discontinued after a median of 406.5 days (271-416 days). Histopathologic examination revealed a dense band of acellular material, poorly staining with periodic acid-Schiff, subtending the corneal epithelium, and being surrounded by spindle cells. CONCLUSIONS: This case series documents the onset of steroid keratopathy in Beagles and Beagle-derived dogs after treatment with ophthalmic corticosteroids. Clinical resolution of steroid keratopathy lesions may be possible after discontinuation of ophthalmic corticosteroids.

Assessment of Early Glaucomatous Optic Neuropathy in the Dog by Spectral Domain Optical Coherence Tomography (SD-OCT).

BACKGROUND: Inherited primary open-angle glaucoma (POAG) in Beagle dogs is a well-established large animal model of glaucoma and is caused by a G661R missense mutation in the ADAMTS10 gene. Using this model, the study describes early clinical disease markers for canine glaucoma. METHODS: Spectral-domain optical coherence tomography (SD-OCT) was used to assess nine adult, ADAMTS10-mutant (median age 45.6 months, range 28.8-52.8 months; mean diurnal intraocular pressure (IOP): 29.9 +/- SEM 0.44 mmHg) and three related age-matched control Beagles (mean diurnal IOP: 18.0 +/- SEM 0.53 mmHg). RESULTS: Of all the optic nerve head (ONH) parameters evaluated, the loss of myelin peak height in the horizontal plane was most significant (from 154 +/- SEM 38.4 µm to 9.3 +/- SEM 22.1 µm; p < 0.01). There was a strong significant negative correlation between myelin peak height and IOP (Spearman correlation: -0.78; p < 0.003). There were no significant differences in the thickness of any retinal layers evaluated. CONCLUSIONS: SD-OCT is a useful tool to detect early glaucomatous damage to the ONH in dogs before vision loss. Loss in myelin peak height without inner retinal thinning was identified as an early clinical disease marker. This suggests that initial degenerative changes are mostly due to the loss of myelin.

Amino acid residue-specific neutralization and nonneutralization of hepatitis C virus by monoclonal antibodies to the E2 protein.

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W(437), L(438), L(441), and F(442), within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.

Ocular pulse amplitude (OPA) in canine ADAMTS10-open-angle glaucoma (ADAMTS10-OAG).

Introduction: The role of ocular rigidity and biomechanics remains incompletely understood in glaucoma, including assessing an individual's sensitivity to intraocular pressure (IOP). In this regard, the clinical assessment of ocular biomechanics represents an important need. The purpose of this study was to determine a possible relationship between the G661R missense mutation in the ADAMTS10 gene and the ocular pulse amplitude (OPA), the difference between diastolic and systolic intraocular pressure (IOP), in a well-established canine model of open-angle glaucoma (OAG). Methods: Animals studied included 39 ADAMTS10-mutant dogs with different stages of OAG and 14 unaffected control male and female dogs between 6 months and 12 years (median: 3.2 years). Dogs were sedated intravenously with butorphanol tartrate and midazolam HCl, and their IOPs were measured with the Icare® Tonovet rebound tonometer. The Reichert Model 30™ Pneumotonometer was used to measure OPA. Central corneal thickness (CCT) was measured via Accutome® PachPen, and A-scan biometry was assessed with DGH Technology Scanmate. All outcome measures of left and right eyes were averaged for each dog. Data analysis was conducted with ANOVA, ANCOVA, and regression models. Results: ADAMTS10-OAG-affected dogs displayed a greater IOP of 23.0 ± 7.0 mmHg (mean ± SD) compared to 15.3 ± 3.6 mmHg in normal dogs (p < 0.0001). Mutant dogs had a significantly lower OPA of 4.1 ± 2.0 mmHg compared to 6.5 ± 2.8 mmHg of normal dogs (p < 0.01). There was no significant age effect, but OPA was correlated with IOP in ADAMTS10-mutant dogs. Conclusion: The lower OPA in ADAMTS10-mutant dogs corresponds to the previously documented weaker and biochemically distinct posterior sclera, but a direct relationship remains to be confirmed. The OPA may be a valuable clinical tool to assess ocular stiffness and an individual's susceptibility to IOP elevation.

The specificity of ubiquitin binding to ubiquilin-1 is regulated by sequences besides its UBA domain.

UBQLN proteins regulate proteostasis by facilitating clearance of misfolded proteins through the proteasome and autophagy degradation pathways. Consistent with its proteasomal function, UBQLN proteins contain both UBL and UBA domains, which bind subunits of the proteasome, including the S5a subunit, and ubiquitin chains, respectively. Conclusions regarding the binding properties of UBQLN proteins have been derived principally through studies of its individual domains, not the full-length (FL) proteins. Here we describe the in vitro binding properties of FL-UBQLN1 with the S5a subunit of the proteasome and two different lysine-linked (K48 or K63) ubiquitin chains. We show that in contrast to its isolated UBA domain, which binds almost equally well with both K48 and K63 ubiquitin chains, FL UBQLN1 binds preferentially with K63 chains. Furthermore, we show that deletion of the UBL domain from UBQLN1 abrogates ubiquitin binding. Taken together these results suggest that sequences outside of the UBA domain in UBQLN1 function to regulate the specificity and binding with different ubiquitin moieties. We also show that the UBL domain of UBQLN1 is required for S5a binding and that its binding to UBQLN1, in turn, enhances K48 ubiquitin chain binding to the complex. We discuss the implications of our findings with the known function of UBQLN proteins in protein degradation.

BDNF preserves the dendritic morphology of alpha and beta ganglion cells in the cat retina after optic nerve injury.

PURPOSE: To examine whether brain-derived neurotrophic factor (BDNF), a potent neuroprotectant in the mammalian retina, also plays a role in preserving the dendritic integrity of the surviving ganglion cells after optic nerve injury. METHODS: Single ganglion cells from cats that underwent unilateral optic nerve crush and received no treatment or nerve crush combined with intravitreous treatment of the affected eye with BDNF were labeled intracellularly, reconstructed using confocal microscopy, and compared quantitatively. RESULTS: Optic nerve injury produced a significant decrease in the soma, dendritic field size, and dendritic complexity of alpha cells. beta Cells also displayed a significant decrease in soma size, but their dendritic fields were not affected as severely as those of alpha cells. Intravitreous treatment of the eye with BDNF at the time of injury preserved the normal somal and dendritic morphologies of both alpha and beta cells. CONCLUSIONS: BDNF, in addition to promoting ganglion cell survival, plays an important role in preserving the somal and dendritic morphologies of the surviving ganglion cells, a necessary precursor to maintaining normal visual function. Ganglion cells, however, are not created equal with respect to their responses to nerve injury or to treatment of the eye with BDNF. Thus, development of effective treatment strategies for preserving ganglion cell function in optic nerve-related diseases mandates a clearer understanding of the cellular response characteristics of the specific neurons involved.

Assessment of Rod, Cone, and Intrinsically Photosensitive Retinal Ganglion Cell Contributions to the Canine Chromatic Pupillary Response.

Purpose: The purpose of this study was to evaluate a chromatic pupillometry protocol for specific functional assessment of rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs) in dogs. Methods: Chromatic pupillometry was tested and compared in 37 dogs in different stages of primary loss of rod, cone, and combined rod/cone and optic nerve function, and in 5 wild-type (WT) dogs. Eyes were stimulated with 1-s flashes of dim (1 cd/m2) and bright (400 cd/m2) blue light (for scotopic conditions) or bright red (400 cd/m2) light with 25-cd/m2 blue background (for photopic conditions). Canine retinal melanopsin/Opn4 was cloned, and its expression was evaluated using real-time quantitative reverse transcription-PCR and immunohistochemistry. Results: Mean ± SD percentage of pupil constriction amplitudes induced by scotopic dim blue (scDB), scotopic bright blue (scBB), and photopic bright red (phBR) lights in WT dogs were 21.3% ± 10.6%, 50.0% ± 17.5%, and 19.4% ± 7.4%, respectively. Melanopsin-mediated responses to scBB persisted for several minutes (7.7 ± 4.6 min) after stimulus offset. In dogs with inherited retinal degeneration, loss of rod function resulted in absent scDB responses, followed by decreased phBR responses with disease progression and loss of cone function. Primary loss of cone function abolished phBR responses but preserved those responses to blue light (scDB and scBB). Although melanopsin/Opn4 expression was diminished with retinal degeneration, melanopsin-expressing ipRGCs were identified for the first time in both WT and degenerated canine retinas. Conclusions: Pupil responses elicited by light stimuli of different colors and intensities allowed differential functional assessment of canine rods, cones, and ipRGCs. Chromatic pupillometry offers an effective tool for diagnosing retinal and optic nerve diseases.

Safety and Efficacy of AAV5 Vectors Expressing Human or Canine CNGB3 in CNGB3-Mutant Dogs.

Achromatopsia is an inherited retinal disorder of cone photoreceptors characterized by markedly reduced visual acuity, extreme light sensitivity, and absence of color discrimination. Approximately 50% of cases are caused by mutations in the cone photoreceptor-specific cyclic nucleotide gated channel beta subunit (CNGB3) gene. Studies in CNGB3-mutant dogs showed that subretinal injection of an AAV vector expressing human CNGB3, which has 76% amino acid identity with canine CNGB3, driven by a 2.1 kb human red cone opsin promoter (PR2.1) and packaged in AAV5 capsids (AAV5-PR2.1-hCNGB3) rescued cone photoreceptor function, but at high doses was associated with an inflammatory response (focal chorioretinitis) consistent with immune-mediated toxicity. AAV vectors containing the PR2.1 promoter packaged in AAV5 capsids and expressing either the native canine CNGB3 (AAV5-PR2.1-cCNGB3) or the human CNGB3 (AAV5-PR2.1-hCNGB3) were evaluated at different dose levels in CNGB3-mutant dogs. The vector expressing canine CNGB3 achieved somewhat better rescue of cone function but unexpectedly was associated with a greater degree of retinal toxicity than the vector expressing human CNGB3. Very low-level T-cell immune responses to some AAV or CNGB3 peptides were observed in animals that received the higher vector dose. There was a more than twofold increase in serum neutralizing antibodies to AAV in one of three animals in the low-dose group and in two of three animals in the high-dose group. No serum anti-hCNGB3 antibodies were detected in any animal. The results of this study do not support the hypothesis that the focal chorioretinitis seen with high doses of AAV5-PR2.1-hCNGB3 in the initial studies was due to an immune response to human CNGB3.