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Aljoscha S Neubauer

Publications and source records attributed to Aljoscha S Neubauer.

28 records · Page 2Linked to original sources
PubMed2003

Tissue transglutaminase as a modifying enzyme of the extracellular matrix in PVR membranes.

PURPOSE: Proliferative vitreoretinopathy (PVR) is characterized by the development of epi- and subretinal fibrocellular membranes containing modified retinal pigment epithelial (RPE) cells among others. In the present study, the role of transglutaminases in accumulation of extracellular matrix (ECM) proteins in these membranes was investigated. Transglutaminases are enzymes capable of cross-linking ECM proteins to proteolysis-resistant complexes. METHODS: PVR membranes were incubated with dansyl-cadaverine to demonstrate active transglutaminase. Localization of tissue transglutaminase (tTgase), its reaction product epsilon-(gamma-glutamyl)-lysine, and fibronectin was investigated immunohistochemically. Colocalization was studied with a confocal laser scanning microscope. PVR membranes were also analyzed by RT-PCR for the presence of tTgase mRNA. In vitro, RPE cells were treated with transforming growth factor-beta2 (TGF-beta2), basic fibroblast growth factor, interleukin-6, and interleukin-1beta. Their effect was studied using immunohistochemistry and Northern and Western blot analyses. RESULTS: Transglutaminase activity and expression of tTgase were present in all PVR membranes. Staining was most prominent at the rim of the membranes. The enzyme was colocalized with epsilon-(gamma-glutamyl)-lysine and fibronectin. No staining differences were found between epi- and subretinal membranes. Although native RPE cells contained only a basal level of tTgase mRNA, the expression and activity of tTgase was increased under culture conditions and further stimulated by TGF-beta2 treatment. CONCLUSIONS: The findings demonstrate that in PVR membranes tTgase is present and functionally active. The amount and activity of this enzyme appears to be related to the differentiation state of the RPE cells and their stimulation by TGF-beta2, a growth factor known to be increased in the vitreous of PVR. Intervention at this pathway may open a new approach for PVR prevention and therapy.

Adolescent↗
PubMed2003

Alkylphosphocholines inhibit proliferation of human retinal pigment epithelial cells.

PURPOSE: To investigate the effect and mechanism of action of alkylphosphocholines (APCs) on proliferation of human retinal pigment epithelium (RPE) cells and RPE-mediated collagen matrix contraction in vitro. METHODS: Cultured RPE cells of five human donors were treated with four APCs in the presence of fetal calf serum. Proliferation was assessed by the tetrazolium dye-reduction (MTT) assay and by counting the number of cells dividing in culture. The effect of APCs on RPE-mediated matrix contraction was determined in three-dimensional collagen gels. Cell viability was tested by the trypan blue exclusion assay. As a possible mechanism of APC action, protein kinase C (PKC) activity was quantified by scintillation counting of (32)P-labeled phosphate transferred to a PKC-specific substrate. RESULTS: All APCs inhibited RPE proliferation and RPE-mediated collagen matrix contraction in a dose-dependent manner in vitro. The antiproliferative and anticontractile effect of APCs increased with elongation of the fatty acid chain beyond C20. IC(50)s of all APCs varied between 8.5 micro M (erucyl-phosphocholine, C22:1-PC), 9.0 micro M (Z)-12-heneicosenyl-phosphocholine, C21:1-PC), 11.0 micro M (Z)-10-eicosenyl-phosphocholine, C20:1-PC), and 26.5 micro M (oleyl-phosphocholine, C18:1-PC). Trypan blue staining revealed a toxicity below 5% for all APCs within the concentration interval tested. PKC activity was significantly reduced by all four APCs, with C22:1-PC being the most effective. CONCLUSIONS: APCs inhibit proliferation of RPE cells and RPE-mediated matrix contraction in vitro at nontoxic concentrations. This effect seems to be exerted through inhibition of PKC activity. Therefore, APCs are promising candidates for treatment of RPE-mediated proliferative processes such as proliferative vitreoretinopathy.

Adolescent↗
PubMed2003

Comparative proteome analysis of native differentiated and cultured dedifferentiated human RPE cells.

PURPOSE: Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells. METHODS: Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3-10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins. RESULTS: One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A. CONCLUSIONS: Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.

Cell Differentiation↗
PubMed2003

Tele-screening for diabetic retinopathy with the retinal thickness analyzer.

OBJECTIVE: To compare the effectiveness of tele-screening using a novel enhanced retinal thickness analyzer (RTA) with onsite routine ophthalmologic examination for diabetic retinopathy. RESEARCH DESIGN AND METHODS: A consecutive series of 31 eyes from diabetic patients were included. All underwent ophthalmologic examination, including stereoscopic dilated funduscopy and scanning with the RTA. The RTA reports consisted of a wide-angle, red-free fundus photograph and a macular-region retinal thickness map. Reports were graded by three independent graders in a masked manner. The diagnoses of proliferative retinopathy, macular edema, and treatment decisions made by the RTA graders and the clinical examiner were compared. RESULTS: On clinical examination 5 of 31 eyes were diagnosed with proliferative diabetic retinopathy (PDR). All five were referred for treatment by two graders and four eyes by one grader. All eyes with PDR and 12 of the 26 eyes with nonproliferative diabetic retinopathy showed severe macular edema. Seven of the 12 eyes with macular edema were clinically eligible for focal laser treatment, and all of them were detected by all RTA graders. Macular thickening was detected in eight eyes by RTA where no treatment was necessary, as judged by clinical examination. Thus, sensitivity was 93% (mean) for detecting PDR and 100% for detecting macular edema, with a specificity of 58-96% depending on the grader. The RTA did not allow valid assessment due to poor image quality in only one case. CONCLUSIONS: Screening for diabetic retinopathy with a combination of wide-angle fundus photography and macular thickness mapping by an objective method, such as optical coherence tomography or the RTA, offers the prerequisites for establishing a successful tele-screening program.

Aged↗
PubMed2002

Consenting to cornea donation: influencing factors.

BACKGROUND: The purpose of the study was to determine factors influencing the decision of the next relative being asked for consent to cornea donation in a prospective, non-comparative case series. METHODS: Interviews with the relatives of 264 potential cornea donors to the eye bank of the Ludwig-Maximilians-Universität, Munich, were documented following a standardized protocol. The influence of sociological factors on the frequency of obtained consents was investigated by uni- and multivariate analysis. RESULTS: During a 9-month period, 214 relatives of 264 possible donors were interviewed for consent to cornea donation. In total, 144 consents (67%) and 70 refusals (33%) were obtained; 50 relatives could not be reached. Analysis showed a higher consent rate for donors with a university degree (72%) versus those without (68%). Consent was more often obtained from relatives of donors that were divorced (71%) than from unmarried ones (63%). Husband and wife (71%) consented more frequently to cornea donation than children of age (62%) or parents (58%). Examination of residential area postal codes indicated more frequent refusals from donors of rural (56% consent rate) than urban Munich populations (67% consent rate). The situation was the opposite concerning the residential area of the consenting relative. Postal codes of both the donor and the relative were statistically significant factors in a multiple logistic regression model. There was relatively high willingness to donate in suicides (70%), but not significantly different from other causes like natural deaths (70%) and traffic accidents (66%). CONCLUSIONS: Some socioeconomic factors such as - in particular - the permanent address of the potential donors and their relatives reflecting a different background and status play an important role in the willingness to donate. A better understanding of this decision making can help the physician to ask more empathically for consent and inform the public more specifically about the donation process.

Adolescent↗
PubMed2002

Rapid direct antibiotic susceptibility testing in endophthalmitis.

OBJECTIVE: To compare a new rapid antibiotic susceptibility test (RAST) to the conventional method in patients with endophthalmitis. DESIGN: Prospective nonrandomized comparative trial. PARTICIPANTS: Intraocular aspirates from 24 consecutive patients with endophthalmitis were tested. METHODS: Approximately 0.25 ml of vitreous or anterior chamber aspirate was obtained and tested for antibiotic sensitivity using the Kirby-Bauer agar disk diffusion method. Using this conventional testing method, the aspirates were cultured for bacterial growth before antibiotic susceptibility testing (AST). In contrast, for direct RAST, the aspirates were inoculated directly onto agar plates with antibiotic disks without first culturing for bacterial growth. RESULTS: Of the 24 endophthalmitis aspirates studied, 4 (17%) could not be tested by direct RAST because either more than one bacterial species was present or no bacteria were cultured. The remaining 20 cases provided 467 antibiotic-microorganism combinations. The two methods yielded identical susceptibility results in 409 (88%) of the 467 tests. In 42 tests (9%), a minor discrepancy occurred between the conventional method and RAST, in which one resulted in intermediate susceptibility, whereas the other was either resistant or sensitive. Significant discrepancy, defined as a bacterial sample reported as sensitive in one method yet resistant in the other, occurred with 16 samples (3%). Results of the RAST were available within 6 to 10 hours compared with more than 24 hours for the conventional method. Of clinical significance, the results of the RAST revealed that in 11 cases of epidemic Stenotrophomonas maltophilia endophthalmitis, the bacteria were resistant to the antibiotics prescribed, and change of antibiotic therapy was made on the basis of the RAST results. CONCLUSIONS: RAST provided accurate antibiotic susceptibility results in a much shorter time than did the conventional method. We suggest that conventional AST be performed for confirmation of the RAST results, but initial antibiotic therapy can quite reliably be based on the results of RAST.

Anti-Bacterial Agents↗
PubMed2002

Sterile structural imaging of donor cornea by optical coherence tomography.

PURPOSE: The purpose of the study was to demonstrate a new noncontact method for sterile measurement of structure and thickness of donor cornea with use of optical coherence tomography (OCT). METHODS: A commercially available OCT instrument designed for retinal measurements was used for noncontact assessment of human corneas. Structural changes occurring during organ culture were evaluated in 29 corneas. Comparison with histology was performed, and the ability of OCT to detect corneal scars and corneal thickness was investigated. RESULTS: Corneal epithelium, stroma, and posterior curvature, as well as thickness, can be measured by standard OCT while the cornea remains in its storage bottle. Epithelial changes leading to a reduction of epithelial thickness, stromal structural changes, and hydration folds can be visualized. OCT scans correlate well with histology. Preexisting and developing corneal scars can be detected by OCT. CONCLUSIONS: Corneal structural imaging can be performed under sterile conditions by OCT. This provides a method for improvement of corneal storage and a screening method for signs of photorefractive surgery and scarring in donor cornea.

Cornea↗
PubMed2002

Retinal pigment epithelium is protected against apoptosis by alphaB-crystallin.

PURPOSE: The degeneration of retinal pigment epithelial (RPE) cells is considered to be a crucial event in the pathophysiology of age-related macular degeneration (AMD). Cumulative oxidative damage has been implicated in the development of the changes seen in AMD. The present study was undertaken to evaluate the expression of the small heat shock protein alphaB-crystallin in the RPE in response to oxidative stress and to explore whether alphaB-crystallin expression confers an antiapoptotic cytoprotective effect on RPE cells. METHODS: Native human RPE cells from the macula and retinal periphery were analyzed by RT-PCR and Western blot analysis for expression of alphaB-crystallin. Monolayer cultures of human RPE cells were stressed by heat shock (42 degrees C for 20 minutes) or oxidant-mediated injury (50-300 micro M H(2)O(2) for 1 hour). Induction of alphaB-crystallin and the corresponding mRNA was assessed by Western and Northern blot analyses. To study the cytoprotective effect of alphaB-crystallin, human RPE cells were transfected with either a neomycin-selectable expression vector containing alphaB-crystallin cDNA or a control vector without alphaB-crystallin cDNA. Caspase-3 activity was determined by observing the cleavage of a colorimetric peptide substrate. Cell viability was quantified by combined propidium iodide and Hoechst 33342 staining. RESULTS: alphaB-crystallin is constitutively expressed in RPE under in vivo and in vitro conditions. Western blot analysis of freshly isolated RPE showed greater baseline expression levels in RPE derived from the macular area than in that from the more peripheral regions. Heat shock treatment and oxidative stress caused a significant increase in alphaB-crystallin mRNA and protein. Oxidant-mediated injury in RPE cells with baseline expression levels of alphaB-crystallin resulted in apoptotic cell death, as measured by caspase-3 activity, whereas RPE cells that had been stably transfected with alphaB-crystallin were more resistant to H(2)O(2)-induced cellular injury. CONCLUSIONS: alphaB-crystallin may function as a stress-inducible antiapoptotic protein in human RPE and is inducible by oxidative stress, a condition implicated in the pathogenesis of AMD. Overexpression of alphaB-crystallin may be an important mechanism for the RPE to prevent apoptotic cell death in response to cellular stress.

Adolescent↗
PubMedDate not supplied

The multifocal pattern electroretinogram in chloroquine retinopathy.

PURPOSE: Optimal screening for ocular toxicity caused by chloroquine and hydroxychloroquine is still controversial. With the multifocal pattern electroretinogram (mfPERG), a new electrophysiological technique has recently become available to detect early changes of ganglion cells. In this study this new technique is applied to a series of 10 patients seen consecutively receiving long-term chloroquine medication. METHODS: In 10 patients receiving chloroquine medication, clinical examination, Amsler visual field testing and computerized color vision testing were performed. If toxicity was suspected, automated perimetry was carried out. In addition, in all patients conventional pattern electroretinogram (PERG) and mfPERG testing were performed. RESULTS: On clinical examination 8 patients showed no chloroquine-associated maculopathy, while 2 patients did. Of these 2, only 1 reported abnormalities when viewing the Amsler chart, while automated perimetry showed typical, ring-like paracentral scotomas in both affected patients and color vision was significantly abnormal. In the normal patients, 4 of 8 had a mild color vision disturbance, which correlated to age-related macular changes. The amplitudes of the PERG and the central (approximately 10 degrees ) responses of the mfPERG were markedly reduced in chloroquine maculopathy, while the latencies were unchanged. The peripheral rings of mfPERG (ranging to 48 degrees ) were not affected by chloroquine toxicity. Both PERG and mfPERG were less affected by age-related macular changes. CONCLUSIONS: The reduction of PERG and central mfPERG responses in chloroquine maculopathy may help with the early detection of toxicity.

Adult↗