[Uveal Melanoma Cell Under Oxidative Stress - Influence of VEGF and VEGF-Inhibitors]. / Uveale Melanomzellen unter oxidativem Stress ­ Einfluss von VEGF und VEGF-Inhibitoren.

BACKGROUND: The role of oxidative stress in cancer is complex. While the pathological alterations induced by oxidative stress may be involved in the induction of tumours, in the late stages of tumour development, it can facilitate the loss of tumour cells and might even prevent metastasis. Tumour cells show metabolic alterations, often inducing an increased production of reactive oxygen species, which makes these cells particularly vulnerable to additional oxidative stress. This is an important mode of action in the use of many chemotherapeutics and in the application of ionizing radiation. Uveal melanoma is the most frequent primary tumour in the adult eye. For metastasis of this tumour, which affects about 50 % of the patients, no appropriate treatment is currently available. However, the primary tumour can efficiently be treated with ionizing radiation. A frequent side effect of this treatment is radiation retinopathy, which is treated with vascular endothelial growth factor (VEGF) antagonists. A therapy of the primary tumour with VEGF antagonists is under discussion. So far, little data is available on this subject, however, a paradoxical worsening of the situation has been found in a mouse model of uveal melanoma treated with bevacizumab. METHODS: We have investigated the effect of VEGF and of the VEGF-antagonist bevacizumab on the survival of five different melanoma cell lines under oxidative stress treatment with hydrogen peroxide. In addition, we investigated the expression of relevant proteins and the effect of bevacizumab on the proliferation of the cells as well as its effect on the angiogenic behaviour of endothelial cells, co-cultured with uveal melanoma cells. RESULTS: Our study showed that not only VEGF but also, paradoxically, the VEGF-antagonist bevacizumab is able to protect uveal melanoma cells from oxidative stress-induced cell death. Bevacizumab did not influence the proliferation of the cells and showed only limited effectiveness to reduce angiogenic structures. CONCLUSION: Considering that oxidative stress is the mode of action for ionizing radiation to induce cell death, a protective effect of bevacizumab on uveal melanoma cells against oxidative stress is worrisome and argues against the use of VEGF in uveal melanoma.

Long-term follow-up of lamellar macular holes and pseudoholes over at least 5 years.

PURPOSE: To assess morphological and functional changes of lamellar macular holes and pseudoholes with or without vitrectomy and membrane peeling with at least 5 years follow-up. METHODS: Retrospective study of 73 eyes with lamellar macular hole (LH, n = 28), macular pseudohole (PH, n = 31), and pseudohole with cleaved edges (cleavedPH, n = 14). Forty-six eyes were merely observed without vitreoretinal intervention (observation group), and 27 eyes underwent vitrectomy with membrane peeling (vitrectomy group). Outcome measures were best corrected visual acuity (BCVA) and morphological retinal parameters evaluated with optical coherence tomography (TD-OCT and SD-OCT). RESULTS: Mean follow-up was 8.3 years (5-12); mean age was 67 years (46-84). In the observation group, median BCVA (logMAR) at first exam was 0.2 (LH), 0.1 (PH), 0.2 (cleavedPH) and at last exam 0.3 (LH, p = 0.02), 0.2 (PH), 0.15 (cleavedPH). In the vitrectomy group, median BCVA at first exam was 0.4 (LH), 0.3 (PH), 0.25 (cleavedPH); before vitrectomy BCVA was 0.5 (LH), 0.35 (PH), 0.35 (cleavedPH); and at last exam BCVA increased to 0.3 (LH), 0.2 (PH, p < 0.05), 0.1 (cleavedPH, p < 0.05). At last exam, BCVA of LH was significantly worse compared to PH and cleavedPH. In the observation group, 6 of 29 eyes with PH or cleavedPH showed a spontaneous resolution of the epiretinal membrane with improvement of the foveal contour. Nine of 16 eyes with LH and 2/20 eyes with PH presented lamellar hole-associated epiretinal proliferation (LHEP) in SD-OCT. CONCLUSIONS: LH, PH, and cleavedPH are often stable over a very long time. LH tends to worse visual function compared to PH and cleavedPH. A spontaneous separation of epiretinal membranes in the long-term is not uncommon. Vitreoretinal intervention should be considered in cases with significant visual loss or functional and morphological progression.

[Vision Loss in Association with Silicone Oil]. / Die Visusminderung in Zusammenhang mit Silikonöltamponaden.

Silicone oil tamponades have been a key feature of vitreoretinal surgery for more than 40 years. The use of these durable vitreous substitutes has not only been limited to tractive retinal detachments, inflammatory chorioretinopathies or other complicated vitreoretinal diseases, but also to macular holes and rhegmatogenous retinal detachment. During the last 12 years, an increasing number of cases of vision loss during or after the removal of silicone oil have been reported in the literature. In up to one third of patients with uncomplicated macula-attached retinal detachment with good preoperative vision, continuing vision loss is seen. In most of these cases, thinning of the inner retinal layers can be found. The underlying pathological mechanism is unknown. A direct connection to silicone oil is likely, but has not been proven. However, the increasing number of reports raises evidence that silicone oil itself might be the reason for this phenomenon, which is not observed in gas tamponades. Vision loss during or after the removal of silicone oil is an underestimated problem. The safety and toxicity of silicone oil should be reexamined systematically. The search for novel vitreous replacements is indispensable. The use of silicone oil should be strictly evaluated and balanced against other possible tamponades in every individual case.

The role of pigment epithelial detachment in AMD with submacular hemorrhage treated with vitrectomy and subretinal co-application of rtPA and anti-VEGF.

PURPOSE: To assess the incidence of pigment epithelial detachment (PED) in age-related macular degeneration (AMD) with submacular hemorrhage (SMH) and its response to treatment with pars plana vitrectomy (ppV), subretinal co-application of recombinant tissue plasminogen activator (rtPA) and anti-VEGF, and an intravitreal gas tamponade. METHODS: Consecutive interventional case series of 132 eyes of 129 patients with neovascular AMD with SMH. All eyes underwent ppV with subretinal co-application of rtPA and bevacizumab followed by a gas tamponade. Postoperatively, two additional intravitreal anti-VEGF injections were applied monthly, followed by intravitreal anti-VEGF injections applied PRN thereafter. PEDs and SMHs were evaluated with SD-OCT pre- and postoperatively. RESULTS: Preoperatively, 88 of 132 (67%) eyes were examined by OCT, and in 81 of these eyes the RPE could be visualised. A PED was found in 74 (91%) eyes, and no PED was found in five (6%) eyes. Median height of preoperative PED was 503 µm (range 150-1242, n = 65) and reduced to 344 (n = 62) and 306 µm (n = 27) after 3 and 12 months respectively. Two eyes showed a pre-existing rip of the RPE. Postoperatively, a rip was documented in 12 of 128 (9%) eyes. Median height of SMH was 762 µm (range 217-1840), median diameter was 4.3 (1.5-15) disc diameter. A complete displacement of the SMH from the fovea was achieved in 112 of 129 (87%) eyes. Overall, median best-corrected logMAR visual acuity (BCVA) improved significantly from preoperative 1.6 (0.5-2.0, n = 132) to 1.0 (0.2-2.0) 3 (n = 132) and 12 months (n = 74) postoperatively. Excluding eyes with pre-existing macular scars (n = 22), BCVA 3 months postoperatively was 0.8. Height of PED or SMH did not correlate with postoperatively BCVA, while size of SMH showed a mild correlation (rho = 0.25, p = 0.005). CONCLUSION: PpV with subretinal co-application of rtPA and bevacizumab and an intravitreal gas tamponade effectively displaces SMH and improves BCVA. Preoperatively, PED is found in the majority of eyes. Height of PED or SMH did not correlate with postoperatively BCVA. Tears of the RPE occur as frequently as in exudative AMD without SMH.

Paediatric non-progression following grandmother-to-child HIV transmission.

BACKGROUND: In contrast to adult HIV infection, where slow disease progression is strongly linked to immune control of HIV mediated by protective HLA class I molecules such as HLA-B*81:01, the mechanisms by which a minority of HIV-infected children maintain normal-for-age CD4 counts and remain clinically healthy appear to be HLA class I-independent and are largely unknown. To better understand these mechanisms, we here studied a HIV-infected South African female, who remained a non-progressor throughout childhood. RESULTS: Phylogenetic analysis of viral sequences in the HIV-infected family members, together with the history of grand-maternal breast-feeding, indicated that, unusually, the non-progressor child had been infected via grandmother-to-child transmission. Although HLA-B*81:01 was expressed by both grandmother and grand-daughter, autologous virus in each subject encoded an escape mutation L188F within the immunodominant HLA-B*81:01-restricted Gag-specific epitope TL9 (TPQDLNTML, Gag 180-188). Since the transmitted virus can influence paediatric and adult HIV disease progression, we investigated the impact of the L188F mutant on replicative capacity. When this variant was introduced into three distinct HIV clones in vitro, viral replicative capacity was abrogated altogether. However, a virus constructed using the gag sequence of the non-progressor child replicated as efficiently as wildtype virus. CONCLUSION: These findings suggest alternative sequences of events: the transmission of the uncompensated low fitness L188F to both children, potentially contributing to slow progression in both, consistent with previous studies indicating that disease progression in children can be influenced by the replicative capacity of the transmitted virus; or the transmission of fully compensated virus, and slow progression here principally the result of HLA-independent host-specific factors, yet to be defined.

A new cytokine release assay: a simple approach to monitor the immune status of HIV-infected patients.

PURPOSE: To test a new assay based on an ex vivo cytokine release from whole blood for the monitoring of immune changes in human immunodeficiency virus (HIV)-infected patients. METHODS: A pilot study of outpatients with HIV infection (n = 9) at a large academic hospital who were divided into three groups: HIV-infected patients on highly active antiretroviral therapy (HAART) with a CD4(+) cell count >350/µL (group I) or a CD4(+) cell count <350/µL (group II) and HIV-infected HAART-naïve subjects with a CD4(+) cell count >350/µL (group III). All groups were compared with healthy volunteers (n = 3). The ex vivo cytokine release assay was performed in a three-step process: (1) blood collection, (2) whole-blood ex vivo incubation over 48 h without or with a standard set of well-defined recall antigens as comparable to those used formerly in the skin delayed-type hypersensitivity (DTH) test, (3) cytokine determination from the assay supernatant. RESULTS: Under stimulated conditions, untreated HIV-infected patients with a CD4(+) count >350/µL had similar interleukin-2 (IL-2) levels in the supernatant of the whole-blood incubation to HIV-infected patients on HAART with a low CD4(+) count. Both groups revealed lower IL-2 levels in the supernatant than HIV-infected patients on HAART and with a CD4(+) count >350/µL or healthy volunteers. The determination of interferon-γ and tumour necrosis factor-α in the supernatant showed a similar arrangement of cytokines between groups. CONCLUSIONS: Our results suggest that this cytokine release assay could be a suitable tool to mirror the immunological responsiveness of patients with HIV infection in a gradual manner; further studies are required in order to assess its value in HAART monitoring.

Vectorial release of matrix metalloproteinases (MMPs) from porcine RPE-choroid explants following selective retina therapy (SRT): towards slowing the macular ageing process.

The purpose of this study was to investigate release of matrix metalloproteinases (MMP) 2 and 9 during retinal pigment epithelium (RPE) wound healing after Selective Retina Therapy (SRT) with laser energy levels below and above the threshold of RPE cell death. Following exposure to SRT using a prototype pulsed Nd:YLF laser with energies of 80-180 mJ/cm(2) fresh porcine RPE-monolayers with Bruch's membrane and choroid were cultured in modified Ussing chambers which separate the apical (RPE-facing) and basal (choroid facing) sides of the RPE monolayer. Threshold energy for RPE cell death and wound healing were determined with calcein-AM viability test. Inactive and active forms of MMP 2 and 9 were quantified within tissue samples and in the culture medium of the apical and basal compartments of the Ussing chamber using gelatine zymography. Laser energies of 160-180 mJ/cm(2) resulted in cell death within 1 h while 120-140 mJ/cm(2) resulted in delayed death of exposed RPE cells. All cells survived 80 and 100 mJ/cm(2). Laser spots healed within 6 days after SRT accompanied by a transient vectorial increase of MMPs. SRT with 180 mJ/cm(2) increased active MMP 2 by 1.9 (p < 0.05) and 1.6 (p < 0.05) fold in tissue and basal compartments, respectively, without alterations in the apical compartment. Pro-MMP 2 levels were also significantly increased in all compartments (p < 0.05). Release of MMP 9 was not altered. Laser energy below the threshold of RPE cell death did not alter the release of MMP 2 or 9. The findings suggest that the release of active MMP 2 on the basal side of the RPE during wound healing following SRT may address age-related pathological changes of Bruch's membrane with a potential to slow degenerative macular ageing processes before irreversible functional loss has occurred.

[Screening for glaucoma by blue light-pattern ERG and OCT--are the methods comparable?]. / Glaukomscreening mit dem blaulicht-muster-ERG und dem OCT--sind beide verfahren gleichwertig?

BACKGROUND: Up to now, several examinations are necessary to identify early glaucoma. It is also possible to analyse the peripapillary retinal nerve fibre layer thickness by means of OCT. The loss of retinal ganglion cells can be measured by pattern ERG, therefore this method could be used for the detection of early glaucoma. This study compares the results of the blue light-pattern ERG with the Stratus OCT for glaucoma early recognition. PATIENTS AND METHODS: We studied 30 healthy test persons, 20 patients with glaucoma and 20 patients with suspected glaucoma. Each group was examined using eye pressure measurements, funduscopy, perimetry, pachymetry und analysing the retinal nerve fibre layer by the Stratus OCT. The diagnostic work-up was completed by the pattern ERG and an upgrade with a blue light filtering glass. The results of the blue light-pattern ERG and the OCT ("healthy" or "pathological") were compared in a cross table and the agreement between these two raters was measured by the Cohen's kappa coefficient. RESULTS: Pattern ERG and blue light-pattern ERG decrease with advancing age. After developing an age-matched calibration graph we defined standard values up to the age of 56 years. All included subjects were classified by Stratus OCT and blue light-pattern ERG as healthy. We recognised a significant reduction of the retinal nerve fibre layer thickness and the amplitude in the pattern ERG in the cluster of glaucoma patients. For most of them, a blue light-pattern ERG could not be recorded. Nine out of 40 glaucoma suspect eyes were judged as "pathological". Regarding the amplitudes of the blue light-pattern ERG 25 of 28 eyes under suspicion of glaucoma were assigned as "healthy". With the aid of the cross table we estimated a Cohen's kappa of 0.4. The sensitivity of the blue light-pattern ERG was computed to be 70 % and the specificity to be 97.7 %. For the OCT we calculated the sensitivity to be 50 % and the specificity to be 100 %. CONCLUSION: The blue light-pattern ERG correlates in 87 % of our cases with the OCT. Healthy test persons are certainly identified just as well as definitely glaucomatous patients. To distinguish people suspected of early glaucoma the blue light-pattern ERG can be used as an additional, fast and economic diagnostic procedure.

Distinct Immunoglobulin Fc Glycosylation Patterns Are Associated with Disease Nonprogression and Broadly Neutralizing Antibody Responses in Children with HIV Infection.

A prophylactic HIV vaccine would ideally induce protective immunity prior to sexual debut. Children develop broadly neutralizing antibody (bnAb) responses faster and at higher frequencies than adults, but little is known about the underlying mechanisms or the potential role of Fc-mediated effector functions in disease progression. We therefore performed systems immunology, with immunoglobulin profiling, on HIV-infected children with progressive and nonprogressive disease. Pediatric nonprogressors (PNPs) showed distinct immunoglobulin profiles with an increased ability to elicit potent Fc-mediated natural killer (NK)-cell effector functions. In contrast to previous reports in adults, both groups of children showed high levels of gp120-specific IgG Fc glycan sialylation compared to bulk IgG. Importantly, higher levels of Fc glycan sialylation were associated with increased bnAb breadth, providing the first evidence that Fc sialylation may drive affinity maturation of HIV-specific antibodies in children, a mechanism that could be exploited for vaccination strategies.IMPORTANCE To protect future generations against HIV, a vaccine will need to induce immunity by the time of sexual debut and hence requires immunization during childhood. Current strategies for a prophylactic HIV vaccine include the induction of a broadly neutralizing antibody response and the recruitment of potent effector functions of immune cells via the constant antibody Fc region. In this study, we show that nonprogressing HIV-infected children mounted antibody responses against HIV that were able to mediate potent Fc effector functions, which may contribute to the control of HIV replication. Children who had specific glycan structures on the Fc portion of antibodies against HIV were able to neutralize a broader range of HIV variants, providing evidence of a potential role of Fc glycovariation in the development of bnAbs against HIV. These findings complement our knowledge of the distinct immune landscape in early life that could be exploited in the development of vaccine strategies.

Treating age-related macular degeneration - interaction of VEGF-antagonists with their target.

The neutralization of VEGF is the current treatment of choice for age-related macular degeneration. Current approaches include anti-VEGF-antibodies and -Fab Fragments, aptamers, soluble receptors (Traps) and siRNA. The molecular properties of VEGF and its antagonists are reviewed and the pathways of action of these substances are discussed.

[A new haploscopic test device excludes blinking induced monocular perception]. / Ein neues haploskopisches Testverfahren verhindert monokulare Perzeption durch Blinzeln.

BACKGROUND: Haploscopic tests like the pola-test are used to detect functional loss of vision, aggravation or simulation. They aim at confusing the tested person about the relation of disparate monocular visual perceptions to the right and left eyes. The haploscopic confusion can be overcome easily by single sided blinking. Other investigators have faced this problem by presenting optotypes very briefly, thus excluding blinking artefacts. We introduce a new device for shutting off the optotype projection whenever blinking occurs. MATERIALS AND METHODS: A newly developed mechanism slightly presses small lever-switches onto each upper eyelid of a test person while performing the pola test. The levers move up and down following eyelid movements. Lid closure triggers a shut off of the optotype projector. The device was tested on 30 persons. RESULTS: The device worked properly on 59/60 eyes. 24/30 persons did not realize the haploscopic confusion at the beginning, but 6/30 did right away. 18/30 would not realize the haploscopic confusion even after some experience. CONCLUSIONS: The new device worked on nearly all patients and effectively kept most of them from overcoming the haploscopic confusion. It is a useful upgrading for haploscopic tests, making them applicable even in repeated examinations. It should be helpful to detect single-sided functional loss of vision.

[YAG laser vitreolysis for treatment of symptomatic vitreous opacities]. / YAG-Laser-Vitreolyse zur Behandlung von störenden Glaskörpertrübungen.

YAG laser vitreolysis is an innovative procedure to treat symptomatic vitreous floaters. The treatment decision is based on taking a comprehensive patient history, thorough clinical examination, realistic expectations, and detailed patient information including risk of the procedure. Manifestation of vitreous opacities and possible subjective impairments are considerably variable. Dynamic changes over time are possible. Therefore, not all vitreous opacities are suitable for laser treatment. A classification according to histoanatomical origin can aid the treatment decision. In comparison to other ophthalmic YAG laser applications, YAG laser vitreolysis differs predominantly by its specific technical equipment requirements. In addition, treatment is more complex and time consuming, requires appropriate experience and knowledge of the surgeon, and should be carried out with utmost care and accuracy.

[Intravitreal administration of triamcinolone and bevacizumab for pigment epithelial detachment in conjunction with AMD]. / Intravitreales Triamcinolon und Bevacizumab bei Pigmentepithelabhebung im Rahmen der AMD.

BACKGROUND: The effect of the vascular endothelial growth factor (VEGF) inhibitors triamcinolone and bevacizumab (Avastin) on serous pigment epithelial detachment (PED) related to age-dependent macular degeneration (AMD) was analysed retrospectively. PATIENTS AND METHODS: Data of 45 patients (45 eyes) were evaluated: 11 patients received an intravitreal injection with triamcinolone and 16 with bevacizumab. The remaining 18 patients received no therapy. Visual acuity (VA), height of the PED, retinal thickness and the cystoid component of the PED were compared after 6 months follow-up. RESULTS: Over 90% showed a stabilisation or improvement in VA after bevacizumab therapy; 63% of those who received triamcinolone showed VA stabilisation. Patients who received no therapy had a significant decrease in VA. CONCLUSIONS: Our data seem to implicate that VA outcome is much better after bevacizumab treatment. VA outcome seems to correlate with the cystoid component of the foveal oedema rather than with the height of PED. A greater number of patients with a longer period of follow-up are necessary to confirm these results.

[Retinal laser coagulation with the pattern scanning laser--report of first clinical experience]. / Retinale Laserkoagulation mit dem Pattern Scanning Laser--erster klinischer Erfahrungsbericht.

BACKGROUND: The PASCAL pattern scanning laser can be applied to induce multiple lesions in retinal laser coagulation. First clinical experience is evaluated. PATIENTS/MATERIALS UND METHODS: The PASCAL pattern scanning laser is a frequency doubled Nd:YAG laser (532 nm). Exposition times are 10-30 msec and power is up to 2000 mW per single lesion. Clinical experience was gained in 33 patients and the subjective perception of pain was compared in 27 patients. The pain of 14 patients in group 1 (pattern) and the pain of 13 patients in group 2 (single lesion) were compared to the pain of previous conventional single spot laser coagulation. RESULTS: No complications occurred. In group 1 (pattern), 10 patients had less severe pain than in conventional laser coagulation, 4 patients had similar pain. In group 2 (single lesion), 6 patients had less severe pain, 7 patients had similar pain. Differences were not significant in Fisher's exact test (p = 0.17). CONCLUSIONS: In the pattern technique, pain also occurs. Time saving seems to be possible. A wide understanding of the interaction between laser and tissue is necessary to avoid over-coagulation. For macular grid laser coagulation, the single spot technique seems to be safer.