Lose the Nintendo and thou shall be healed! Restoring vision in malingering.

Non-organic visual loss can be hard to prove or explain to the parents of affected children at times. Here, we describe a simple yet effective approach that may help solve both issues by ensuring that the patient refrains from visual stimuli.

MITOGEN-ACTIVATED PROTEIN INHIBITORS: FULL-FIELD ELECTRORETINOGRAM DEMONSTRATING GENERALIZED RETINAL DYSFUNCTION.

PURPOSE: To report a patient with generalized retinal toxicity to mitogen-activated protein inhibitors. METHODS: Retrospective case report. RESULTS: Full-field electroretinogram findings indicate a generalized toxicity to the use of the mitogen-activated protein inhibitor trametinib. There was an improved response and resolution of serous detachments after decreasing the dose. CONCLUSION: Mitogen-activated protein inhibitors may affect global retinal function, as opposed to the serous detachments that are concentrated in the posterior pole. This may be of importance in further understanding the underlying pathologic mechanisms.

Biomarkers of disease in human nails: a comprehensive review.

Diagnostic, monitoring, response, predictive, risk, and prognostic biomarkers of disease are all widely studied, for the most part in biological fluids or tissues, but there is steadily growing interest in alternative matrices such as nails. Here we comprehensively review studies dealing with molecular or elemental biomarkers of disease, as opposed to semiological, pharmacological, toxicological, or biomonitoring studies. Nails have a long history of use in medicine as indicators of pathological processes and have also been used extensively as a matrix for monitoring exposure to environmental pollution. Nail clippings are simple to collect noninvasively as well as to transport and store, and the matrix itself is relatively stable. Nails incorporate, and are influenced by, circulating molecules and elements over their several months of growth, and it is widely held that markers of biological processes will remain in the nail, even when their levels in blood have declined. Nails thus offer the possibility to not only look back into a subject's metabolic history but also to study biomarkers of processes that operate over a longer time scale such as the post-translational modification of proteins. Reports on ungual biomarkers of metabolic and endocrine diseases, cancer, and psychological and neurological disorders will be presented, and an overview of the sampling and analytical techniques provided.

Analysis of protein glycation in human fingernail clippings with near-infrared (NIR) spectroscopy as an alternative technique for the diagnosis of diabetes mellitus.

BACKGROUND: Glycated keratin allows the monitoring of average tissue glucose exposure over previous weeks. In the present study, we wanted to explore if near-infrared (NIR) spectroscopy could be used as a non-invasive diagnostic tool for assessing glycation in diabetes mellitus. METHODS: A total of 52 patients with diabetes mellitus and 107 healthy subjects were enrolled in this study. A limited number (n=21) of nails of healthy subjects were glycated in vitro with 0.278 mol/L, 0.556 mol/L and 0.833 mol/L glucose solution to study the effect of glucose on the nail spectrum. Consequently, the nail clippings of the patients were analyzed using a Thermo Fisher Antaris II Near-IR Analyzer Spectrometer and near infrared (NIR) chemical imaging. Spectral classification (patients with diabetes mellitus vs. healthy subjects) was performed using partial least square discriminant analysis (PLS-DA). RESULTS: In vitro glycation resulted in peak sharpening between 4300 and 4400 cm-1 and spectral variations at 5270 cm-1 and between 6600 and 7500 cm-1. Similar regions encountered spectral deviations during analysis of the patients' nails. Optimization of the spectral collection parameters was necessary in order to distinguish a large dataset. Spectra had to be collected at 16 cm-1, 128 scans, region 4000-7500 cm-1. Using standard normal variate, Savitsky-Golay smoothing (7 points) and first derivative preprocessing allowed for the prediction of the test set with 100% correct assignments utilizing a PLS-DA model. CONCLUSIONS: Analysis of protein glycation in human fingernail clippings with NIR spectroscopy could be an alternative affordable technique for the diagnosis of diabetes mellitus.

Glycation in human fingernail clippings using ATR-FTIR spectrometry, a new marker for the diagnosis and monitoring of diabetes mellitus.

OBJECTIVES: Although HbA1c is a good diagnostic tool for diabetes, the precarity of the health system and the costs limit the use of this biomarker in developing countries. Fingernail clippings contain ±85% of keratins, which are prone to glycation. Nail keratin glycation may reflect the average glycemia over the last months. We explored if attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) can be used as a non-invasive tool for assessing glycation in diabetes. DESIGN AND METHODS: Using ATR-FTIR spectroscopy, glycation and deglycation experiments with fructosamine 3-kinase allowed to identify the spectrum that corresponds with keratin glycation in fingernail clippings. Clippings of 105 healthy subjects and 127 diabetics were subjected to the standardized ATR-FTIR spectroscopy method. RESULTS: In vitro glycation resulted in an increased absorption at 1047cm-1. Following enzymatic deglycation, this peak diminished significantly, proving that the AUC between 970 and 1140cm-1 corresponded with glycated proteins. Within-run CV of the assay was 3%. Storage of nail clippings at 37°C for 2weeks did not significantly change results. In diabetics, glycated nail protein concentrations (median: 1.51µmol/g protein, IQR: 1.37-1.85µmol/g protein) were significantly higher than in the controls (median: 1.19µmol/g protein, IQR: 1.09-1.26µmol/g protein) (p<0.0001). ROC analysis yielded an AUC of 0.92 at a cut-off point of 1.28µmol/g nail (specificity: 82%; sensitivity: 90%). No correlation was observed between the glycated nail protein concentrations and HbA1c. CONCLUSIONS: Protein glycation analysis in fingernails with ATR-FTIR spectroscopy could be an alternative affordable technique for diagnosing and monitoring diabetes. As the test does not consume reagents, and the preanalytical phase is extremely robust, the test could be particularly useful in developing countries.

Glycation of nail proteins: from basic biochemical findings to a representative marker for diabetic glycation-associated target organ damage.

BACKGROUND: Although assessment of glycated nail proteins may be a useful marker for monitoring of diabetes, their nature and formation are still poorly understood. Besides a detailed anatomical analysis of keratin glycation, the usefulness of glycated nail protein assessment for monitoring diabetic complications was investigated. METHODS: 216 patients (94 males, 122 females; mean age ± standard deviation: 75.0 ± 8.7 years) were enrolled. Glycation of nail and eye lens proteins was assessed using a photometric nitroblue tetrazolium-based assay. Following chromatographic separation of extracted nail proteins, binding and nonbinding fractions were analyzed using one-dimensional gel electrophoresis. Using a hand piece containing a latch-type-bur, a meticulous cutting of the nail plate into superficial and deep layers was performed, followed by a differential analysis of fructosamine. RESULTS: Using SDS PAGE, four and two bands were identified among the nonglycated and glycated nail fraction respectively. Significantly lower fructosamine concentrations were found in the superficial nail layer (mean: 2.16 ± 1.37 µmol/g nails) in comparison with the deep layer (mean: 4.36 ± 2.55 µmol/g nails) (P<0.05). A significant higher amount of glycated eye lens proteins was found in diabetes mellitus patients (mean: 3.80 ± 1.57 µmol/g eye lens) in comparison with nondiabetics (mean: 3.35 ± 1.34 µmol/g eye lens) (P<0.05). A marked correlation was found between glycated nail and glycated eye lens proteins [y (glycated nail proteins) = 0.39 + 0.99 x (eye lens glycated proteins); r2 = 0.58, P<0.001]. The concentration of glycated eye lens proteins and the HbA1c level were found to be predictors of the concentration of glycated nail proteins. CONCLUSIONS: Glycation of nail proteins takes place in the deep layer of finger nails, which is in close contact with blood vessels and interstitial fluid. Glycation of nail proteins can be regarded as a representative marker for diabetic glycation-associated target organ damage.

Glycated nail proteins: a new approach for detecting diabetes in developing countries.

OBJECTIVE: To assess glycation of nail proteins as a tool in the diagnosis of diabetes. METHODS: Glycation of nail proteins was assessed using a modified photometric nitroblue tetrazolium-based assay, which provides information about average glucose values of the last 6-9 months. Analysis is possible on 10 mg of nail clippings with a within-run coefficient of variation (CV) of 11%. The analyte is extremely stable. The reference range for glycated nail protein (0.55-3.60 µmol/g nail) increases upon ageing. RESULTS: In diabetics (n = 112), values for glycated nail protein are significantly higher (median: 4.07 µmol/g nail, IQR: 2.37-6.89 µmol/g nail, P < 0.0001) than in non-diabetics (n = 116). ROC analysis shows an AUC of 0.848 (specificity 93.1%; sensitivity 68.9%). CONCLUSION: This affordable method is a simple alternative for diagnosing diabetes in remote areas as the pre-analytical phase (including all processes from the time a laboratory request is made by a physician until the sample is ready for testing) is extremely robust.

Orbital cellulitis as complication of endophthalmitis after cataract surgery.

A 74-year-old man presented with light perception and presumed early bacterial endophthalmitis in the left eye after cataract surgery. Vitreous tap biopsy and core vitrectomy were performed immediately, along with injection of antibiotic agents (ceftazidime and vancomycin). Culture of the vitreous tap revealed Pseudomonas aeruginosa sensitive to ceftazidime. The eye remained inflamed despite 2 additional intravitreal ceftazidime injections. Orbital cellulitis with perforation of the globe was suspected and confirmed on magnetic resonance imaging, and enucleation was performed. Endophthalmitis due to P aeruginosa is associated with poor visual outcomes despite prompt treatment with appropriate intravitreal antibiotic agents. Progression to orbital cellulitis in immunocompetent patients is extremely rare. Careful monitoring of patients with endophthalmitis after cataract surgery is recommended. .

Trypan blue not toxic for retinal pigment epithelium in vitro.

PURPOSE: To investigate whether trypan blue has a toxic effect on cultured retinal pigment epithelial (retinal pigment epithelium) cells. DESIGN: Experimental study with a direct live/dead cell staining technique using fluorescent dyes. METHODS: Cultured human retinal pigment epithelium cells were exposed for 5 minutes to various concentrations of trypan blue (0.06%, 0.15%, 0.30%), and cell viability was confocally measured. RESULTS: No increased cell death was found in cultures incubated in any of the trypan blue concentrations used. CONCLUSION: These findings indicate that a short exposure of trypan blue does not have a toxic effect on cultured retinal pigment epithelium cells.

Invasion of retinal pigment epithelial cells: N-cadherin, hepatocyte growth factor, and focal adhesion kinase.

PURPOSE: To investigate the role of N-cadherin and hepatocyte growth factor (HGF) in the invasion of collagen type I by human retinal pigment epithelial (RPE) cells. METHODS: RPE sheets from eight human eyes were used for characterization through Western blot analysis of the expression of cadherin in total lysates or after immunoprecipitation with anti beta-catenin antibody. First-passage primary cultures of RPE sheets were successfully established from 28 of 56 human eyes. First-passage primary RPE cell cultures on glass substrate, consisting of patches of cells, were used for immunocytochemistry. Fifteen first-passage primary RPE cell cultures in culture vessels were grown to confluence. Four of the 15 first-passage primary RPE cell cultures were investigated for cadherin expression by immunocytochemistry, and the other 11 were further subcultured for two to six passages. These 11 cultures were used for functional assays and investigated for expression of cadherin at regular time intervals. Cells from passage-3 to -4 primary RPE cell cultures were tested for invasion into collagen type I gels, with or without neutralizing antibodies for HGF and N-cadherin, respectively. Activation of the c-Met receptor for HGF and of focal adhesion kinase (FAK) was investigated by immunoprecipitation with anti-phosphotyrosine antibody, gel electrophoresis, and immunostaining on Western blot. Levels of HGF in conditioned medium (CM) of RPE cells were determined by ELISA. RESULTS: RPE cells in culture displayed two phenotypes: Both fibroblast-like and epithelioid cells were present in all 15 first-passage primary cultures and in further passaged cultures derived therefrom. When seeded on collagen, all RPE cells acquired a fibroblast-like phenotype and invaded the collagen type I gel. RPE cells also stimulated branching morphogenesis of MDCK/AZ epithelial canine kidney cell colonies inside collagen. HGF was found in RPE CM, suggesting an autocrine loop for invasion through its c-Met receptor. HGF-neutralizing antibody inhibited invasion of collagen. The major cadherin expressed by RPE cells in culture was N(euronal)-cadherin. Invasion of collagen by RPE cells was also inhibited by an N-cadherin-neutralizing antibody. N-cadherin and c-Met coimmunoprecipitated in RPE cells. FAK and c-Met were both phosphorylated in RPE cells in culture, and phosphorylation was inhibited by antibodies neutralizing either N-cadherin or HGF. CONCLUSIONS: The present investigation provides evidence for an autocrine HGF/c-Met loop that stimulates RPE cell invasion into collagen through FAK. The invasion-stimulatory molecule N-cadherin also activates FAK in invasive RPE cells.

Toxic effect of indocyanine green on retinal pigment epithelium related to osmotic effects of the solvent.

PURPOSE: To investigate whether the toxic effect on cultured retinal pigment epithelial (RPE) cells after application of indocyanine green is related to the osmolarity of the solvent or to toxic effects of the dye and evaluate whether these changes also occur using infracyanine green. DESIGN: Experimental study with a direct live/dead cell staining technique using fluorescent dyes. METHODS: Cultured human RPE cells were exposed to various solutions and cell viability was confocally measured. RESULTS: Increased cell death was found in cultures incubated in the hypoosmotic solvent that is generally used for indocyanine green (P <.001, n = 12). Addition of indocyanine green did not alter this observation (P <.001, n = 12). In cultures exposed to a 5% glucose solution, no increased cell death was found (P =.94, n = 12), nor when infracyanine green was added (P =.13, n = 12). CONCLUSION: The observed toxicity of indocyanine green on RPE cells is probably related to the hypo-osmolarity of the solvent and may be avoided by using infracyanine green dissolved in glucose 5%.

Structure and function of the N-cadherin/catenin complex in retinoblastoma.

PURPOSE: To identify in human retinoblastoma and normal retinal tissue the type of cadherin, its relationship with cytoplasmic catenins, and its participation in invasion. METHODS: The cadherin/catenin complex was characterized in surgical retinoblastoma specimens from five patients and human retinas from four donor eyes by immunocytochemistry, flow cytometry, and coimmunoprecipitation with antibodies against N-cadherin, alpha-catenin, and beta-catenin, followed by Western blot analysis or autoradiography. Y79 and WERI-Rb-1 retinoblastoma cell lines serve the evaluation of the cadherin/catenin complex in aggregation and collagen type I invasion in vitro. The association of the cadherin/catenin complex with the cytoskeleton was examined by an antibody-capping assay. RESULTS: In retinoblastoma and normal retina N-cadherin associated with alpha-catenin and beta-catenin but not E- or P-cadherin. The N-cadherin/catenin complex formed a regular, linear, and continuous honeycomb pattern in normal retina that was irregular, clustered, and interrupted in retinoblastoma. The N-cadherin/catenin complex was found also in the retinoblastoma cell lines WERI-Rb and Y79, in which it also showed an irregular pattern. Both cell lines were invasive in collagen type I, and invasion was inhibited by the GC-4 antibody, which functionally neutralizes N-cadherin. Less GC-4 antibody was needed to inhibit invasion of Y79 cells, which expressed N-cadherin at a lower level, than to inhibit invasion of WERI-Rb-1 cells. In both cell lines, antibody capping of the N-cadherin/catenin complex indicated that its linkage with the cytoskeleton were weak or absent. CONCLUSIONS: Retinoblastoma cells, in contrast with normal retina, express an N-cadherin/catenin complex that is irregularly distributed and weakly linked to the cytoskeleton. In retinoblastoma, this complex acts as an invasion promoter.