Novel pathogenic mutations and skin biopsy analysis in Knobloch syndrome

Purpose: To facilitate future diagnosis of Knobloch syndrome (KS) and better understand its etiology, we sought to identify not yet described COL18A1 mutations in KS patients. In addition, we tested whether mutations in this gene lead to absence of the COL18A1 gene product and attempted to better characterize the functional effect of a previously reported missense mutation. Methods: Direct sequencing of COL18A1 exons was performed in KS patients from four unrelated pedigrees. We used immunofluorescent histochemistry in skin biopsies to evaluate the presence of type XVIII collagen in four KS patients carrying two already described mutations: c. 3277C>T, a nonsense mutation, and c. 3601G>A, a missense mutation. Furthermore, we determined the binding properties of the mutated endostatin domain p.A1381T (c.3601G>A) to extracellular matrix proteins using ELISA and surface plasmon resonance assays. Results: We identified four novel mutations in COL18A1, including a large deletion involving exon 41. Skin biopsies from KS patients revealed lack of type XVIII collagen in epithelial basement membranes and blood vessels. We also found a reduced affinity of p.A1381T endostatin to some extracellular matrix components. Conclusions: COL18A1 mutations involved in Knobloch syndrome have a distribution bias toward the coding exons of the C-terminal end. Large deletions must also be considered when point mutations are not identified in patients with characteristic KS phenotype. We report, for the first time, lack of type XVIII collagen in KS patients by immunofluorescent histochemistry in skin biopsy samples. As a final point, we suggest the employment of this technique as a preliminary and complementary test for diagnosis of KS in cases when mutation screening either does not detect mutations or reveals mutations of uncertain effect, such as the p.A1381T change.

Toxicity of High-Dose Intravitreal Adalimumab (Humira) in the Rabbit

Purpose: To evaluate the ocular toxicity of escalating doses of intravitreous adalimumab (Humira (R)) in the rabbit eye. Methods: Thirty New Zealand albino rabbits received intravitreous injections of 0.5mg (6 eyes), 1.0mg (6 eyes), 2.5mg (6 eyes), 5mg (6 eyes), and 10mg (6 eyes) adalimumab. Slit lamp biomicroscopy and fundoscopy were carried out at baseline, day 7, and day 14 after intravitreous injection, whereas electroretinography (ERG) was carried out at baseline and day 14. Animals were euthanized on day 14, and histopathological examination of the eyes was performed. Results: Slit lamp biomicroscopy and fundoscopy were normal in all eyes receiving doses up to 5mg. In the 10mg group, 3 of 6 eyes showed mild anterior chamber inflammatory reaction on day 7. Similarly, scotopic and photopic a- and b-wave ERG amplitudes at baseline and day 14 were similar in all groups up to 5mg, but there was a significant decrease in the photopic-wave ERG response in the 10mg group (P = 0.046). Finally, histopathology demonstrated no differences among eyes receiving balanced salt solution, 0.5, 1.0, 2.5, 5.0, or 10mg of adalimumab. Conclusions: Intravitreous adalimumab exhibited no associated ocular short-term toxicity in rabbit eyes up to the 5mg dose. In the 10mg group mild clinical findings and ERG amplitude reduction could reflect early toxicity.

Photoprotective effect of Pothomorphe umbellata on UVB radiation-induced biomarkers involved in carcinogenesis of hairless mouse epidermis

In this study we assessed the protective effect of topical application of Pothomorphe umbellata extract on ultraviolet B (UVB)-induced skin lesion parameters in hairless mouse epidermis. A single dose of UVB irradiation (0.23 kJ/m(2)) resulted in a significant decrease in thymine dimer-positive cells and apoptotic sunburn cells, with an increase in p53 and proliferating cell nuclear antigen-positive cells in the epidermis. After 5 weeks (total dose 13.17 kJ/m(2)) and 15 weeks (total dose 55.51 kJ/m(2)) of irradiation, P. umbellata treatment inhibited the hyperplasic response and induced an increase in p53-positive cells. These findings suggest that P. umbellata extract affords protection against UVB-induced skin lesions.

Ascorbic acid concentration is reduced in the secondary aqueous humour of glaucomatous patients

P>Background: We aimed to evaluate the ascorbic acid concentration in secondary aqueous humour (AH) from glaucomatous patients and to compare it with primary AH from primary open-angle glaucoma patients and non-glaucomatous patients. Methods: Primary AH samples were prospectively obtained from clinically uncontrolled primary open-angle glaucoma patients and senile cataract patients (controls) prior to trabeculectomy and cataract surgery. Secondary AH samples were obtained from eyes with previous intraocular surgery, prior to trabeculectomy or cataract surgery. AH (0.1 mL) was aspirated by inserting a 26-gauge needle into the anterior chamber just before surgery and then immediately stored at -80 degrees C. The ascorbic acid concentration was determined in a masked fashion by high-pressure liquid chromatography. Results: A total of 18 patients with senile cataract, 16 glaucomatous patients with primary AH (no previous intraocular surgery) and 11 glaucomatous patients with secondary AH (previous intraocular surgery) were included. There was no difference in mean age between groups (P = 0.15). The mean +/- standard deviation concentration of ascorbic acid in the secondary AH from glaucomatous patients (504 +/- 213 mu mol/L [95% confidence interval {CI}, 383-624]) was significantly lower than the concentration of ascorbic acid found in the primary aqueous of primary open-angle glaucoma (919 +/- 427 mu mol/L [95% CI, 709-1128]) and control patients (1049 +/- 433 mu mol/L [95% CI, 848-1249]; P < 0.01, Kruskal-Wallis test). Conclusions: The ascorbic acid concentration in secondary AH of glaucomatous patients was approximately twofold lower in comparison with primary AH of glaucomatous and cataract patients. The implications of a reduced concentration of ascorbic acid in the secondary AH deserve further investigation.

Determination of Vecuronium Bromide in Pharmaceuticals: Development, Validation and Comparative Study of HPLC and CZE Analytical Methods

Vecuronium bromide is a neuromuscular blocking agent used for anesthesia to induce skeletal muscle relaxation. HPLC and CZE analytical methods were developed and validated for the quantitative determination of vecuronium bromide. The HPLC method was achieved on an amino column (Luna 150 x 4.6 mm, 5 mu m) using UV detection at 205 nm. The mobile phase was composed of acetonitrile:water containing 25.0 mmol L(-1) of sodium phosphate monobasic (50:50 v/v), pH 4.6 and flow rate of 1.0 mL min(-1). The CZE method was achieved on an uncoated fused-silica capillary (40.0 cm total length, 31.5 cm effective length and 50 mu m i.d.) using indirect UV detection at 230 nm. The electrolyte comprised 1.0 mmol L(-1) of quinine sulfate dihydrate at pH 3.3 and 8.0% of acetonitrile. The results were used to compare both techniques. No significant differences were observed (p > 0.05).

Excised Porcine Cornea Integrity Evaluation in an in vitro Model of Iontophoretic Ocular Research

Background/Aims: It is a challenge to adapt traditional in vitro diffusion experiments to ocular tissue. Thus, the aim of this work was to present experimental evidence on the integrity of the porcine cornea, barrier function and maintenance of electrical properties for 6 h of experiment when the tissue is mounted on an inexpensive and easy-to-use in vitro model for ocular iontophoresis. Methods: A modified Franz diffusion cell containing two ports for the insertion of the electrodes and a receiving compartment that does not need gassing with carbogen was used in the studies. Corneal electron transmission microscopy images were obtained, and diffusion experiments with fluorescent markers were performed to examine the integrity of the barrier function. The preservation of the negatively charged corneal epithelium was verified by the determination of the electro-osmotic flow of a hydrophilic and non-ionized molecule. Results: The diffusion cell was able to maintain the temperature, homogenization, porcine epithelial corneal structure integrity, barrier function and electrical characteristics throughout the 6 h of permeation experiment, without requiring CO(2) gassing when the receiving chamber was filled with 25 m M of HEPES buffer solution. Conclusion: The system described here is inexpensive, easy to handle and reliable as an in vitro model for iontophoretic ocular delivery studies. Copyright (C) 2010 S. Karger AG, Basel

Comparative Study of Analgesic Effectiveness Using Three Different Anesthetic Techniques for Intravitreal Injection of Bevacizumab

BACKGROUND AND OBJECTIVE: To compare the analgesic effectiveness and aesthetic appearance associated with topical, subconjunctival, and peribulbar anesthesia for intravitreal bevacizumab injection. PATIENTS AND METHODS: Sixty consecutive patients undergoing their first intravitreal bevacizumab injection were randomized to receive one of three forms of anesthesia: proxymetacaine eye drops, subconjunctival injection of 2% xylocaine, and peribulbar injection of 2% xylocaine. Pain associated with the intravitreal injection and with the entire procedure (including anesthesia administration) was recorded using a Visual Analog Scale 15 minutes after intravitreal injection. Anterior segment evaluation was performed 24 hours after injection to measure the number of clock hours of subconjunctival hemorrhage. RESULTS: Median injection-related pain score was significantly lower in the peribulbar group compared with the topical and subconjunctival groups (P < .05). Median entire procedure pain score was significantly higher In the peribulbar group compared with the topical and subconjunctival groups (P < .05). The median extent of subconjunctival hemorrhage was significantly lower in the topical group compared with the other groups (P < .05). CONCLUSION: Among the three anesthetic techniques, peribulbar anesthesia was associated with greater effectiveness in controlling injection-related pain but was least effective in controlling entire procedure pain. There was no significant difference in pain scores between the topical and subconjunctival groups, and topical anesthesia was associated with less subconjunctival hemorrhage.

Differences in the early biomechanical effects of hyperopic and myopic laser in situ keratomileusis

PURPOSE: To compare changes in corneal hysteresis (CH) and the corneal resistance factor (CRF) in myopic and hyperopic laser in situ keratomileusis (LASIK) and evaluate their relationship to the number of photoablative pulses delivered, a surrogate for ablation volume. SETTING: Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, USA. METHODS: Preoperative and 1-week postoperative Ocular Response Analyzer measurements in eyes that had femtosecond-assisted LASIK were studied retrospectively. Changes in CH and CRF were compared and tested for correlation with the number of excimer laser pulses. RESULTS: Thirteen myopic eyes and 11 hyperopic eyes were evaluated. Preoperative corneal thickness, CH, CRF, programmed correction magnitude, flap thickness, and total number of fixed spotsize photoablative pulses were similar in the 2 groups (P>.1). Decreases in CH and CRF were greater after myopic LASIK than after hyperopic LASIK (P<.005), and changes in CRF were correlated with the number of excimer laser pulses in the myopic group only (r = -0.63, P = .02). Regardless of ablation profile, changes in CH were more strongly correlated with preoperative CH values than with attempted ablation volume. CONCLUSIONS: With comparable flap thickness and attempted ablation volumes, myopic photoablation profiles were associated with greater decreases in CRF and CH than hyperopic profiles. Results indicate that preoperative corneal biomechanical status, ablation volume, and the spatial distribution of ablation are important factors that affect corneal resistance and viscous dissipative properties differently. Preferential tissue removal in the natively thicker paracentral cornea in hyperopia may partially account for the rarity of ectasia after hyperopic LASIK.

Prostatic Artery Embolization as a Primary Treatment for Benign Prostatic Hyperplasia: Preliminary Results in Two Patients

Symptomatic benign prostatic hyperplasia (BPH) typically occurs in the sixth and seventh decades, and the most frequent obstructive urinary symptoms are hesitancy, decreased urinary stream, sensation of incomplete emptying, nocturia, frequency, and urgency. Various medications, specifically 5-alpha-reductase inhibitors and selective alpha-blockers, can decrease the severity of the symptoms secondary to BPH, but prostatectomy is still considered to be the traditional method of management. We report the preliminary results for two patients with acute urinary retention due to BPH, successfully treated by prostate artery embolization (PAE). The patients were investigated using the International Prostate Symptom Score, by digital rectal examination, urodynamic testing, prostate biopsy, transrectal ultrasound (US), and magnetic resonance imaging (MRI). Uroflowmetry and postvoid residual urine volume complemented the investigation at 30, 90, and 180 days after PAE. The procedure was performed under local anesthesia; embolization of the prostate arteries was performed with a microcatheter and 300- to 500-mu m microspheres using complete stasis as the end point. One patient was subjected to bilateral PAE and the other to unilateral PAE; they urinated spontaneously after removal of the urethral catheter, 15 and 10 days after the procedure, respectively. At 6-month follow-up, US and MRI revealed a prostate reduction of 39.7% and 47.8%, respectively, for the bilateral PAE and 25.5 and 27.8%, respectively, for the patient submitted to unilateral PAE. The early results, at 6-month follow-up, for the two patients with BPH show a promising potential alternative for treatment with PAE.

Oculometric parameters of hyperopia in children with esotropic amblyopia

Purpose: To study the oculometric parameters of hyperopia in children with esotropic amblyopia, comparing amblyopic eyes with fellow eyes. Methods: Thirty-seven patients (5-8 years old) with bilateral hyperopia and esotropic amblyopia underwent a comprehensive ophthalmic examination, including cycloplegic refraction, keratometry and A-scan ultrasonography. Anterior chamber depth, lens thickness, vitreous chamber depth and total axial length were recorded. The refractive power of the crystalline lens was calculated using Bennett`s equations. Paired Student`s t-tests were used to compare ocular biometric measurements between amblyopic eyes and their fellow eyes. The associations of biometric parameters with refractive errors were assessed using Pearson correlation coefficients and linear regression. Multivariable models including axial length, corneal power and lens power were also constructed. Results: Amblyopic eyes were found to have significantly more hyperopic refraction, less corneal power, greater lens power, shorter vitreous chamber depth and shorter axial length, despite similar anterior chamber depth and lens thickness. The strongest correlation with refractive error was observed for the axial length/corneal radius ratio (r(36) = -0.92, p < 0.001 for amblyopic and r(36) = 0.87, p < 0.001 for fellow eyes). Axial length accounted for 39.2% (R(2)) of the refractive error variance in amblyopic eyes and 35.5% in fellow eyes. Adding corneal power to the model increased R(2) to 85.7% and 79.6%, respectively. A statistically significant correlation was found between axial length and corneal power, indicating decreasing corneal power with increasing axial length, and they were similar for amblyopic eyes (r(36) = 0.53,p < 0.001) and fellow eyes (r(36) = -0.57, p < 0.001). A statistically significant correlation was also found between axial length and lens power, indicating decreasing lens power with increasing axial length (r(36) = -0.72, p < 0.001 for amblyopic eyes and r(36) = -0.69, p < 0.001 for fellow eyes). Conclusions: We observed that the correlation among the major oculometric parameters and their individual contribution to hyperopia in esotropic children were similar in amblyopic and non-amblyopic eyes. This finding suggests that the counterbalancing effect of greater corneal and lens power associated with shorter axial length is similar in both eyes of patients with esotropic amblyopia.

DISCORDANCE FOR RETINITIS PIGMENTOSA IN TWO MONOZYGOTIC TWIN PAIRS

Background: Retinitis pigmentosa (RP) is a group of genetically heterogeneous diseases with progressive degeneration of the retina. The condition can be inherited as an autosomal dominant, autosomal recessive, and X-linked trait. Methods: We report on two female twin pairs. One twin of each pair is affected with RP, the other twin is unaffected, both clinically and functionally. Molecular analysis in both twins included zygosity determination, arrayed primer extension chip analysis for autosomal recessive and dominant RP, sequencing of the entire RPGR gene, and analysis of X-chromosome inactivation status. Results: Both unrelated twin pairs were genetically identical. Of the potential pathogenetic mechanisms, skewed X-inactivation was excluded on leukocytes. Autosomal recessive RP and autosomal dominant RP arrayed primer extension chip analysis result was completely normal, excluding known mutations in known genes as the cause of disease in the affected twins. Sequencing excluded mutations in RPGR. A postzygotic recessive or dominant genetic mutation of an RP gene is not impossible. A postfertilization error as a potential cause of uniparental isodisomy is unlikely albeit not entirely impossible. Conclusion: The authors report on the second and third unrelated identical twin pair discordant for RP. The exact cause of the condition and the explanation of the clinical discordance remain elusive. RETINA 31:1164-1169, 2011

Ocular toxicity of intravitreous adalimumab (Humira) in the rabbit

Purpose To evaluate the ocular toxicity of escalating doses of intravitreous adalimumab (Humira) in the rabbit eye. Methods Twelve New Zealand albino rabbits received unilateral intravitreous injections of 0.1 ml of adalimumab 0.25 mg (three eyes), 0.50 mg (three eyes), 1.0 mg (three eyes) or 0.1 ml balanced salt solution (BSS, threeeyes). Slit-lamp biomicroscopy and fundoscopy were carried out at baseline, day 1, 7 and 14 following intravitreous injection, while electroretinography (ERG) was carried out at baseline and day 14. Animals were euthanized on day 14, and histopathological examination of the eyes was performed. Results Slit-lamp biomicroscopy and fundoscopy were normal in eyes having received BSS, 0.25 mg or 0.50 mg adalimumab; however, inflammation was present in two of three eyes having received 1.0 mg adalimumab. Similarly, comparison of scotopic and photopic ERG light at baseline and day 14 demonstrated no changes in eyes receiving BSS, 0.25 mg or 0.50 mg adalimumab, but two of three eyes having received 1.0 mg adalimumab showed a greater than 30% reduction in a and b wave. Finally, histopathology demonstrated no differences between eyes receiving BSS, 0.25 mg or 0.50 mg of adalimumab, but two of three eyes injected with 1.0 mg demonstrated inflammatory cell infiltration of the vitreous and anterior chamber, with one of these eyes demonstrating retinal necrosis. Conclusions Escalating doses of intravitreous adalimumab in rabbit eyes caused no detectable functional or structural ocular toxicity up to a dose of 0.50 mg. Administration of 1.0 mg in 0.1 ml was associated with an inflammatory reaction and retinal necrosis.

Optical Quality in Eyes Implanted With Aspheric and Spherical Intraocular Lenses Assessed by NIDEK OPD-Scan: A Randomized, Bilateral, Clinical Trial

PURPOSE: To determine whether implantation of an intraocular lens (IOL) with an aspheric surface (Akreos AO, Bausch & Lomb Inc) results in reduced ocular aberrations (spherical aberration) and improved Strehl ratio and modulation transfer function (MTF) after cataract surgery. METHODS: In an intraindividual, randomized, double-masked, prospective study of 50 eyes (25 patients) with bilateral cataract, an IOL with modified anterior and posterior surfaces (Akreos AO) was implanted in one eye and a biconvex IOL with spherical surfaces (Akreos Fit, Bausch & Lomb Inc) implanted in the fellow eye. Ocular aberrations, Strehl ratio, and MTF curve with 4.5-, 5.0-, and 6.0-mm pupils were measured with a NIDEK OPD-Scan dynamic retinoscopy aberrometer 3 months after surgery. Uncorrected and corrected distance visual acuity (UDVA and CDVA, respectively) were also measured. RESULTS: No statistically significant difference was noted between eyes in postoperative UDVA and CDVA at 1 month. At 3 months, the Akreos AO IOL group obtained statistically significant lower values of higher order and spherical aberrations with 4.5-, 5.0-, and 6.0-mm pupil diameters than the Akreos Fit IOL group (P<.05). The value of Strehl ratio was statistically significantly higher in eyes with the Akreos AO IOL for 4.5- and 6.0-mm pupils (P<.05). The MTF curve was better in the Akreos AO IOL group in 4.5-, 5.0-, and 6.0-mm pupils (P<.05). CONCLUSIONS: The aspheric Akreos AO IOL induced significantly less spherical aberration than the Akreos Fit IOL for 4.5-, 5.0-, and 6.0-mm pupils. Modulation transfer function and Strehl ratio were also better in eyes implanted with the Akreos AO IOL than the Akreos Fit. [J Refract Surg. 2011;27(4):287-292.] doi:10.3928/1081597X-20100714-01

Central Corneal Thickness in Patients With Systemic Sclerosis: A Controlled Study

Purpose: To compare the central corneal thickness (CCT) of patients with systemic sclerosis (SSc) and control subjects. Methods: The study group comprised 37 consecutive patients with SSc, and the control group comprised 23 healthy individuals similar in age and sex. CCT value was measured by ultrasound pachymetry. Results: In the SSc group, the mean CCT in the right eye was 534.9 +/- 33.5 mu m and 536.9 +/- 32.4 mu m in the left eye. In the control group, the mean CCT was 533.0 +/- 32.9 mu m in the right eye and 533.1 +/- 33.6 mu m in the left eye. The mean CCT was not significantly different in the SSc group compared with the control group for both the right (P = 0.83) and left (P = 0.67) eyes. Conclusions: CCT measurements do not significantly differ in patients with SSc compared with healthy control subjects.

Correlation between macular and retinal nerve fibre layer Fourier-domain OCT measurements and visual field loss in chiasmal compression

Purpose The aim of this study was to test the correlation between Fourier-domain (FD) optical coherence tomography (OCT) macular and retinal nerve fibre layer (RNFL) thickness and visual field (VF) loss on standard automated perimetry (SAP) in chiasmal compression. Methods A total of 35 eyes with permanent temporal VF defects and 35 controls underwent SAP and FD-OCT (3D OCT-1000; Topcon Corp.) examinations. Macular thickness measurements were averaged for the central area and for each quadrant and half of that area, whereas RNFL thickness was determined for six sectors around the optic disc. VF loss was estimated in six sectors of the VF and in the central 16 test points in the VF. The correlation between VF loss and OCT measurements was tested with Spearman`s correlation coefficients and with linear regression analysis. Results Macular and RNFL thickness parameters correlated strongly with SAP VF loss. Correlations were generally stronger between VF loss and quadrantic or hemianopic macular thickness than with sectoral RNFL thickness. For the macular parameters, we observed the strongest correlation between macular thickness in the inferonasal quadrant and VF loss in the superior temporal central quadrant (rho=0.78; P<0.001) whereas for the RNFL parameters the strongest correlation was observed between the superonasal optic disc sector and the central temporal VF defect (rho=0.60; P<0.001).