[Measurement of the central corneal thickness using optical reflectometry and ultrasound]. / Messung der zentralen Hornhautdicke mittels optischer Reflektometrie im Vergleich zur Ultraschall-Pachymetrie.

AIM: The aim of this study was to evaluate measurements of the central corneal thickness using OLCR and ultrasound pachymetry (IOPac). MATERIALS AND METHODS: In a retrospective observational study, fifty patients were assessed. Central corneal thickness was measured using OLCR and ultrasound. RESULTS: The IOPac system shows results for the central corneal thickness between 419 µm and 613 µm. The OLCR values ranged between 421 and 598 µm. The coefficient of variation was 1.12 % in the case of the IOPac and 0.97 % in the case of the OLCR. The paired Student's t-test showed no significant differences between the two methods. The agreement between the two methods was high with r = 0.929. CONCLUSIONS: The agreement between the results for the central corneal thickness using OLCR and ultrasound is high. The OLCR is a non-touch technology that does not require local anaesthesia, thus further reducing the risk of infection or mechanical trauma. Especially in surgical applications or glaucoma assessments, movement artefacts need to be ruled out, which potentially could cause wrong values and thus lead to wrong decisions.

Reproducibility of high-resolution optical coherence tomography measurements of the nerve fibre layer with the new Heidelberg Spectralis optical coherence tomography.

AIM: Conventional time-domain OCT technology for detection of retinal nerve fibre layer (RNFL) neurodegeneration suffers from technical inaccuracy owing to a lack of exact scan centring around the optic disc as well as a true follow-up possibility. In this study, the authors evaluated a novel high-resolution spectral-domain OCT device (SD-OCT) with an incorporated eye-tracking feature in its ability to objectively measure the RNFL thickness (RNFLT) by testing intraobserver reproducibility in a series of healthy volunteers. METHODS: Triplicate circumferential RNFL scans of six peripapillary sectors were obtained from both eyes of all 31 participants. The authors compared the measurements of RNFLT during three separate examination days under miotic (Mi) and mydriatic (My) pupil conditions using a high-speed (HS) and high-resolution (HR) scan-acquisition mode. To examine the intersession reproducibility of the SD-OCT measurements, the mean, SD and coefficient of variation (COV) were calculated. RESULTS: No significant differences were found in all groups, independent of the mode of image acquisition and examination day (p always >0,05). Under all conditions, low COVs between 0.545% and 3.97% (intrasession COV on baseline) were found. The intersession COV with activated follow-up mode ranged between 0.29% and 1.07%. In both settings, the temporal sector showed the highest COV values. CONCLUSIONS: True follow-up measurement of identical peripapillary regions may enable clinicians to detect discrete levels of retinal thickness change over time. This constitutes a crucial prerequisite for a reliable monitoring of subtle RNFL changes in neurodegenerative disorders.

[Comparison of spectral- and time-domain OCT in regard of the automatic detection of central retinal thickness]. / Vergleich von Spektral- und Zeit-Domänen-OCT hinsichtlich der automatisierten Erfassung der zentralen Netzhautdicke.

BACKGROUND: The aim of this study was to compare macular thickness measurements obtained from time-domain optical coherence tomography (OCT) and spectral-domain OCT to evaluate their agreement. MATERIALS AND METHODS: Fifty randomly assigned subjects were included. Twenty-five were male, twenty-five female. In all cases both eyes were measured. Serial measurements were obtained from time-domain (StratusOCT, Zeiss) and spectral-domain OCT (Heidelberg Engineering, Germany). Total and regional macular thicknesses were obtained from both machines. Their agreement was estimated by Bland Altman plots. RESULTS: The foveal and total macular thicknesses measured by 3D OCT were significantly greater than those measured by StratusOCT (p < 0.001). CONCLUSIONS: While both time-domain and spectral-domain OCTs are reliable for macular thickness measurements, spectral-domain OCT delivers significantly higher values for macular thickness compared to the time-domain OCT. Macular measurements obtained from the two OCT systems may not be used interchangeably.

[Potential endogenous immunological self-protection of uveal melanoma by indoleamine 2,3-dioxygenase]. / Potenzieller endogener, immunologischer Schutzmechanismus uvealer Melanome durch Indoleamine 2,3-Dioxygenase.

BACKGROUND: Uveal melanomas are the most common intraocular tumours in the adult. Although they represent less than 1% of all tumour cases, uveal melanomas are considerd to be rather aggressive due to early hepatic metastases. Indoleamine 2,3-dioxygenase (IDO) is the principle enzyme in the degradation of the essential amino acid L-tryptophan to L-kynurenine. MATERIALS AND METHODS: In this study, the L-kynurenine production of six uveal melanoma cell lines was examined and immunohistochemistry performed for detection of IDO expression within uveal melanomas. RESULTS: In all the examined cell lines, a basal degradation of tryptophan to L-kynurenine was detectable. Supplementation with interferon-gamma could up-regulate this basal L-kynurenine production. The expression of IDO using immunohistochemistry was demonstrated within all examined tumours. CONCLUSIONS: The expression of IDO within the tumours may shed light on an important adaptive mechanism which allows the melanoma cells to escape T-cell-dependent immunosurveillance as soon as these cells metastise and enter non-immunologically privileged tissue. As competitive inhibition of IDO by 1-methyl-tryptophan is possible, a new therapeutic pathway might be available.

Upregulation of chemokine expression in murine cornea due to mechanical trauma or endotoxin.

BACKGROUND AND AIMS: Allograft rejection is the commonest cause of corneal transplant failure and is significantly higher in high-risk patients. Corneal tissue is reported to produce chemokines in response to stress/inflammation. Expression of chemokines is central to the recruitment of leucocytes during inflammatory events. This study was designed to evaluate the effects of surgical trauma or storage conditions on chemokine expression. METHODS: Murine corneas were manipulated by incubation in different conditions for up to 24 h, by the addition of endotoxin or by surgical trauma. The ex vivo production of chemokines was assessed using a real-time reverse-transcriptase PCR (RT-PCR) assay to measure mRNA encoding MIP-1alpha, MIP-1beta and MIP-1gamma, MCP1, IP-10, lymphotactin, fractalkine, RANTES, eotaxin, MIG, MIP2 and the cytokine MIF. The expression of RANTES was also determined by ELISA, and the ability of supernatant from corneas on chemotaxis of cells was also determined. Finally, we compared the survival of corneal grafts that had (or had not) been treated with endotoxin. RESULTS: We found that on incubation in corneal storage medium, expression of mRNA for the majority of these chemokines greatly increased. Upregulation of chemokine mRNA expression was also seen following the mechanical trauma of suture insertion and exposure of the cornea to endotoxin. In the case of mechanical trauma, functional activity of the chemokines was demonstrated using a chemotaxis assay. Orthotopic transplantation of LPS-treated corneas, in which chemokine expression was elevated, resulted in increased infiltration by leucocytes and more rapid rejection of allogeneic grafts. CONCLUSION: Our results indicate that ex vivo storage and manipulation of murine corneas can influence the expression of chemokines in corneas, and can result in earlier graft rejection. This may be of importance when considering procedures for manipulation and ex vivo storage of donor corneas prior to transplantation, as well as the surgical procedure itself.

Expression of indoleamine 2,3-dioxygenase (IDO) by endothelial cells: implications for the control of alloresponses.

Indoleamine 2,3-dioxygenase (IDO) is an important enzyme in the regulation of immune responses; cells that express IDO can suppress T-cell responses and promote tolerance. Because of the critical role of endothelial cells in graft rejection, we have investigated the role of IDO expression by vascular endothelial cells and its consequence on immunoregulation. We compared the expression of IDO by primary human umbilical vein endothelial cells (HUVECs), human saphenous vein endothelial cells (HSVECs) and arterially derived endothelial cells using reverse transcriptase PCR, Western blotting and assays for enzymatic activity. In HUVECs IDO is upregulated by incubation with cytokines or in mycoplasma-infected cells. On the other hand HSVECs and arterially derived endothelial cells express little IDO, which is poorly upregulated upon activation (except by mycoplasma). Inhibition of IDO activity improved the ability of HUVECs to stimulate allogeneic T-cell responses. If either HUVECs or HSVECs are transfected with the gene encoding IDO, then they are incapable of stimulating allogeneic T-cell responses and induce anergy in allospecific T cells (which can also act as regulatory cells). The variable expression of IDO in different endothelial cells is important not only in understanding the role of endothelial cells in the regulation of graft rejection, but also as a potential therapeutic strategy.

[Comparison of several viral vectors for gene therapy of corneal endothelial cells]. / Vergleich verschiedener viraler Vektoren zur Gentherapie von Hornhautendothelzellen.

AIM: In this paper we compare the transduction efficiency, toxicity, and safety of retroviral vectors [equine infectious anemia virus (EIAV), human immunodeficiency virus-1 (HIV-1), human foamy virus (PFV] and adenovirus (Ad) for potential use in gene therapy of corneal endothelial cells. METHOD: Murine corneal endothelial cells were transduced with EIAV, HIV-1, PFV, and Ad, resulting in the overexpression of a green fluorescent protein (eGFP) transgene marker. The transduction efficiency was assessed by flow cytometry, while cytotoxicity and apoptosis rate were detected by annexin V/propidium iodide (PI) stain. RESULTS: Ad had the highest transduction efficiency with 99% of the cells expressing the transgene, followed by EIAV (95%), HIV-1 (75%), and PFV (43%). However, the high transduction efficiency of Ad also resulted in the highest apoptosis rate (25%) in the corneal endothelial cells. There was no detectable difference in the toxicity between PFV and HIV-1 (10%). EIAV transduction had the lowest cytotoxicity, with only 3% of the cells being annexin V/PI positive. CONCLUSION: Compared to other vectors EIAV exhibited high transduction efficiency combined with low toxicity to corneal endothelial cells. Therefore, it is a powerful tool for gene therapy applications in selected corneal endothelial diseases.

[Reduction of lipid peroxidation and apoptosis in corneal endothelial cells by vitamin A]. / Verminderung der Lipidperoxidation und der Apoptoserate in kornealen Endothelzellen durch Vitamin A.

PURPOSE: The goal of this study was to determine the effects of lipid peroxidation-mediated toxicity of iron ions on corneal endothelial cells leading to apoptosis. METHODS: Murine corneal endothelial cells were maintained in tissue culture medium supplemented with free iron ions, known to lead to increased lipid peroxidation. Retinoic acid in the cell supernatant and cytoplasm of these cells was determined using HPLC. The rate of apoptosis was assessed by quantification of caspase-3-like activity. The lipid peroxidation was measured using the malondialdehyde method. Supplementation of retinoic acid was tested in the setting of apoptosis. RESULTS: Free iron ions led to a rapid loss of retinoic acid in the supernatant and the corneal endothelial cells. This was correlated with rising levels of malondialdehyde following oxidative stress and increased apoptosis. Supplementation of retinoic acid alone significantly reduced oxidative stress and apoptosis in the respective cells. CONCLUSION: In this study the authors present a novel in vitro model to test the direct influence of pro-oxidative species on corneal endothelial cells. The authors also prove that supplementing corneal endothelial cells with retinoic acid sufficiently prevents free radical injury and apoptosis.

[Immunohistochemical detection of enhanced deposition of isoprostane 8-epi-PGF2alpha in vascular lesions of temporal arteritis]. / Immunhistochemischer Nachweis einer verstärkten Anreicherung von Isoprostan 8-epi-PGF2alpha in den Gefässläsionen bei Arteriitis temporalis.

BACKGROUND AND PURPOSE: Oxidative injury caused by lipid oxidation is considered a major factor in the development of several ocular disorders including temporal arteritis. This study investigates the role of 8-epi-PGF2alpha as a marker of oxidative stress in vivo. PATIENTS AND METHODS: Sections from isolated human temporal arteries, obtained from patients suspected of having giant cell arteritis ( n=22), were analyzed by semiquantitative immunohistochemistry and planimetry. RESULTS: Immunoreactivity for 8-epi-PGF2alpha was significantly higher in patients with temporal arteritis (AT) compared to healthy temporal arteries (Co). The percentage of the areas stained positive in immunohistochemistry was higher in the AT group compared to the control group (Co). The enrichment values in the intimal layer containing the endothelium of the same temporal arteries were also significantly higher ( p<0.01) in temporal arteries of the AT group. CONCLUSIONS: Our findings of an enhanced accumulation of 8-epi-PGF2alpha in areas of the arterial wall subjected to extensive vascular tissue destruction suggest that lipid peroxidation products may also play a role in the pathogenesis of this disease.

[Immunohistochemical detection of altered low density lipoprotein (ox-LDL) in the vessel walls of patients with giant cell arteritis]. / Immunhistochemischer Nachweis veränderter Low-density-Lipoproteine (ox-LDL) in der Gefässwand bei Patienten mit Arteriitis temporalis.

BACKGROUND AND PURPOSE: Recent data indicate that lipid peroxidation is implicated in the pathogenesis of giant cell arteritis with a close anatomic relationship between reactive oxygen species and oxidatively injured vascular tissue. PATIENTS AND METHODS: Immunohistochemistry utilizing anti-ox-LDL was performed on paraffin sections of isolated temporal arteries obtained from patients (n=23) suspected of having temporal arteritis. Enrichment as well as staining intensity of ox-LDL in vascular tissue was analysed by digital image planimetry. RESULTS: Temporal arteries with biopsy proven temporal arteritis (n=11) presented with significantly higher enrichment of ox-LDL in the intima (16.9+/-4.2% vs. 11.25+/-2.3%; p<0.01) and mean (9.6+/-2.4% vs. 6.75+/-1.8%; p<0.01) as compared to healthy controls. Comparable results for the staining intensity were found in the intimal (2.8+/-0.5 eU vs. 1.7+/-0.4 eU; p<0.01) and medial layer (1.55+/-0.5 eU vs. 1.04+/-0.6 eU; p<0.01) of diseased patients compared to controls. CONCLUSIONS: Accumulation of ox-LDL in the intimal layer, especially at the intima-media-border, was closely related to disruption of the elastica interna and adjacent vascular tissue, presumably contributing to the underlying process of intimal hyperplasia through unimpeded migration of smooth muscle and accumulation inflammatory cells.