Non-targeted analytical comparison of a heated tobacco product aerosol against mainstream cigarette smoke: does heating tobacco produce an inherently different set of aerosol constituents?

Smoking-related diseases remain a significant public health concern, and heated tobacco products (HTPs) have emerged as a potential alternative to cigarettes. While several studies have confirmed that HTP aerosols contain lower levels of harmful and potentially harmful constituents (HPHCs) than cigarette smoke, less is known about constituents that are intrinsically higher in HTP aerosols. This study provides a comprehensive comparative assessment of an HTP aerosol produced with Tobacco Heating System 2.2 (THS) and comparator cigarette (CC) smoke aiming at identifying all unique or increased compounds in THS aerosol by applying a broad set of LC-MS and GC × GC-MS methods. To focus on differences due to heating versus burning tobacco, confounding factors were minimized by using the same tobacco in both test items and not adding flavorants. Of all analytical features, only 3.5%-corresponding to 31 distinctive compounds-were significantly more abundant in THS aerosol than in CC smoke. A notable subset of these compounds was identified as reaction products of glycerol. The only compound unique to THS aerosol was traced back to its presence in a non-tobacco material in the test item and not a direct product of heating tobacco. Our results demonstrate that heating a glycerol-containing tobacco substrate to the temperatures applied in THS does not introduce new compounds in the resulting aerosol compared to CC smoke which are detectable with the method portfolio applied in this study. Overall, this study contributes to a better understanding of the chemical composition of HTP aerosols and their potential impact on human health.

Suppression of pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine in leaves of tobacco (N. tabacum L.).

BACKGROUND: Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis. RESULTS: The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X. CONCLUSION: Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine.

Optical Coherence Tomography Angiography Findings in Proliferative Diabetic Retinopathy.

PURPOSE: To characterize preretinal neovascularizations (NV) and their corresponding branching routes in proliferative diabetic retinopathy (PDR) with optical coherence tomography angiography (OCTA) and compare the findings with fluorescein angiography (FA). METHODS: In patients with PDR, angiograms were acquired with spectral-domain OCTA (CIRRUS 5000, OCTA AngioPlexTMCarl Zeiss Meditec, Inc.) and FA (Zeiss FF450PlusIR fundus camera or Spectralis HRA-OCT SLO, Heidelberg Engineering Inc.) and were consecutively evaluated. Neovascularization of the disc (NVD) and neovascularization elsewhere (NVE) were analyzed with 6 × 6 and 8 × 8 mm OCTA flow images and B-scans with flow registration. Segmentations of the vitreoretinal interface (VRI) and superficial retina were performed for analysis. Two independent investigators examined OCTA findings and compared them to corresponding FA. RESULTS: Forty-two eyes of 30 patients with PDR were analyzed. A total of 76 NV with their corresponding proliferation routes were visualized and characterized, with 55 (72.4%) proliferating along the posterior hyaloid membrane (PHM), 14 (18.4%) along the epiretinal membrane, and 7 (9.2%) along the fibrovascular membrane. The posterior vitreous was partially detached in 37 of 42 eyes (88.1%), completely detached in 1 of 42 eyes (2.4%), and adherent in 1 of 42 eyes (2.4%). In 38 of 42 cases, OCTA was superior (n = 23) or equivalent (n = 15) to FA in detecting NV and provided a more detailed information of the neovascular vessels. In 4 of 42 study eyes, OCTA was inferior to FA. CONCLUSIONS: OCTA is a useful tool to detect NV in PDR. In comparison to FA, OCTA has the advantages that it is noninvasive and the image capture takes only seconds. We were able to identify all NV and characterize their corresponding proliferation routes in the VRI, the superficial retina slab, or the B-scan with flow registration. Through evading the masking effect of dye leakage in FA, OCTA is capable of better visualization of NV. FA, however, remains essential for the detection of all NV, since OCTA supplies a smaller detection field. Additionally, we identified the PHM as the main proliferating route of diabetic NV (72.4%), marking it as an important structure for sprouting vessels in neoangiogenesis in PDR.

[Patient's Knowledge after Informed Consent for Intravitreal Injections]. / Intravitreale Injektionen ­ welche Informationen aus dem Aufklärungsgespräch bleiben Patienten im Gedächtnis?

BACKGROUND: Intravitreal treatment (IVT) is one of the most common ophthalmological procedures. Therapeutic effectiveness is however dependent on patient compliance. Unwanted treatment cessation rates are high though. The authors therefore decided to analyse the patient's knowledge and treatment expectations, as discrepancies are known to negatively affect compliance and thus treatment outcomes. PATIENTS AND METHODS: The study was designed as an exploratory survey. In total, 100 patients presenting to an outpatient clinic of a tertiary care centre from October to December 2016 were included. A structured, anonymised questionnaire was handed out, consisting mainly of question items with closed code lists as response domains. Solely descriptive analysis of results was performed. RESULTS: The median age of patients was 73 years. 70% had received more than 3 IVTs in at least one eye. Age-related macula degeneration was the most common underlying cause (52%). 64% expected improvement of visual acuity after IVT. 42% could not name one medication used in their IVT. 55% felt that the information provided during informed consent had been adequate. 69% did not know the post-surgical occurrence of endophthalmitis. Three patients were confident of being able to drive a car directly after IVT. CONCLUSION: Patient's knowledge of their underlying disease, treatment goals and complications rates exhibited some deficiencies. Standardised patient information sheets could be of significant use and were actively suggested by patients to improve the informed consent process.

Transport and fate of aflibercept in VEGF-A165-challenged retinal endothelial cells.

Retinal vessels are at least in part involved in clearing of Fc terminus-containing proteins from the vitreous. In vitro, the Fc fusion protein aflibercept is transported through a monolayer of unchallenged immortalized bovine retinal endothelial cells (iBREC), mediated by the neonatal Fc receptor (FcRn), but part of the Fc fusion protein is also degraded. Aflibercept's target VEGF-A not only enhances the permeability of REC by destabilization of tight junctions (TJs) thereby allowing for paracellular flow, it may also lower the intracellular stability of the Fc fusion protein by changing its binding properties to the FcRn. Therefore, we investigated the transport and fate of aflibercept in VEGF-A165-challenged iBREC. All cell culture media were supplemented with 5% fetal bovine serum (FBS) as its absence results in accumulation of aflibercept in iBREC due to deregulated expression of transport proteins. Early after exposure of a confluent iBREC monolayer cultivated on gold electrodes to 5% FBS, the cell index (CI) - assessed as a measure of barrier function, cell viability and cell adhesion - transiently declined but recovered again within a few hours to high values. These values remained stable for several days associated with a strong expression of the TJ-protein claudin-1, indicative of a functional barrier formed by the iBREC monolayer. Transient changes of the plasma membrane localizations of claudin-5 and vascular endothelial cadherin - both important for regulation of paracellular flow - accompanied the transient reduction of the CI not prevented by VEGF-binding proteins. Treatment of iBREC with 50 ng/ml VEGF-A165 for one day resulted in a strong and persistent decline of the CI associated with a low expression level of the TJ-protein claudin-1; reversion to normal values was complete one day after aflibercept's addition at a final concentration of 250 µg/ml. Expressions of other proteins involved in regulation of paracellular flow or transcellular transport were not significantly changed. More aflibercept passed through the monolayer of iBREC cultivated on permeable membrane inserts pretreated with VEGF-A for one day, but this was not affected by a FcRn-inhibiting antibody. Subcellular localization of aflibercept was hardly changed in VEGF-A-exposed iBREC 3 h after its addition to the cells; inhibition of (non)-lysosomal or proteasomal proteases then only weakly affected the amount of internalized aflibercept. iBREC also internalized VEGF-A which was barely detectable as early as 2 h after addition of aflibercept. In contrast, blocking the tyrosine kinase activity of VEGF receptor(s) did not prevent VEGF-A's uptake. Inhibition of cellular proteases strongly increased the amount of internalized VEGF-A in the absence and presence of the Fc fusion protein. We therefore conclude that a FcRn-mediated transport plays a minor role in aflibercept's passage through a leaky barrier of REC. Even early after addition of aflibercept to VEGF-A-exposed iBREC, the levels of free intracellular VEGF-A are low, as aflibercept likely prevents binding of VEGF-A to its receptor. Interestingly, the growth factor's detrimental effects still persist for nearly one day.

VEGF receptor 2 inhibitor nintedanib completely reverts VEGF-A165-induced disturbances of barriers formed by retinal endothelial cells or long-term cultivated ARPE-19 cells.

Various severe ocular diseases are associated with an elevated intravitreal expression of VEGF-A which increases the permeability of retinal endothelial cells (REC) or retinal pigment epithelial (RPE) cells in vivo and in vitro. Inhibition of VEGF receptor 2 (VEGFR2) is sufficient to completely prevent VEGF-A165-induced dysfunctions of barriers formed by long-term cultivated, immortal human ARPE-19 cells or immortalized bovine retinal endothelial cells (iBREC). Extended exposure to VEGF-A could result in additional activation of other growth factor receptors, potentially promoting synergistic effects of corresponding factors on various cellular processes including angiogenesis. Based on these observations, we investigated whether blocking of VEGFR2 is also sufficient to revert VEGF-A-induced changes of the barriers consisting of iBREC (i.e. inner blood-retina barrier) or ARPE-19 cells (i.e. outer blood-retina barrier) in vitro. Alterations of confluent monolayers' properties induced by treatment with VEGF-A165 for one day followed by addition of small molecule inhibitors of the VEGFR2 were determined by continuous cell index (CI) measurements using the microelectronic biosensor system for cell-based assays xCELLigence. VEGF-A165 induced a long-lasting drop of the otherwise high CI of iBREC accompanied by reduced expression of the tight junction (TJ) protein claudin-1 and subtle changes of the plasma membrane localizations of TJ-protein claudin-5 and of vascular endothelial cadherin. Blocking mainly VEGFR2 with 10 nM nintedanib, 10 nM tivozanib or 500 nM ZM323881 efficiently reverted these changes within one day; higher concentrations of nintedanib or additional inhibition of neuropilin-1 were not superior. Interestingly, the CI of short-term cultivated, confluent ARPE-19 cells slightly increased in the presence of VEGF-A165, but was not changed by nintedanib. In contrast, VEGF-A165 markedly reduced the transepithelial electrical resistance of ARPE-19 cells cultivated on porous membrane inserts for three weeks, which was also accompanied by a significant loss of the then strongly plasma membrane-expressed TJ-protein ZO-1. These alterations were completely reverted within one day by 10 nM nintedanib of which higher concentrations were not superior. None of the inhibitors tested diminished the strong barrier properties of iBREC or long-term cultivated ARPE-19 cells. Taken together, inhibition of VEGFR2 efficiently reverts VEGF-A165-induced barrier disturbances of both cell types forming and regulating the inner and outer blood-retina barrier. As synergistic actions of growth factors seem to play only a minor role in inducing a barrier dysfunction, specific inhibition of VEGFR2 could be an interesting option to treat VEGF-A-induced macular edema without obvious effects on vitality and functions of REC and RPE cells.

Comparison between Findings in Optical Coherence Tomography Angiography and in Fluorescein Angiography in Patients with Diabetic Retinopathy.

OBJECTIVE: To compare typical findings of diabetic retinopathy in optical coherence tomography angiography (OCTA) and fluorescein angiography (FA). SUBJECTS/METHODS: 42 patients were enrolled in this study. We performed FA and obtained en face 3 × 3 mm OCTA images of the macular region. The count of microaneurysms (MAs) and the size of the foveal avascular zone (FAZ) were compared. The assessability of the imaging modalities was graded in each eye. RESULTS: 53 eyes of 42 patients with a mean age of 61 years were included. 36/53 eyes revealed nonproliferative diabetic retinopathy, 17/53 eyes had proliferative diabetic retinopathy. The mean size of the FAZ was 0.39 mm2 in FA and 0.42 mm2 in OCTA. The mean MA count was 14 in FA and 13 in OCTA. The assessability was favorable to OCTA in 38-41/53 eyes regarding the FAZ and favorable to FA in 45-49/53 eyes regarding MAs. CONCLUSION: We found a good agreement for the size of the FAZ and a weak agreement regarding the count of MAs in both imaging modalities. The readers favored OCTA for the assessment of the FAZ and FA for the assessment of MAs. Complementary use of FA and OCTA ensures the best diagnostic approach in patients with diabetic retinopathy.

[Recanalisation by Probing with a Bicanalicular Silicone Tube (Oggel Silicone Intubation) in Patients with Acquired Lacrimal Stenosis]. / Rekanalisierung durch Sondierung mit Oggel-Sonden-Implantation bei erworbener Tränenwegsstenose.

OBJECTIVES: Recanalisation and bicanalicular splinting with silicone tube is one therapeutic option for acquired lacrimal duct stenosis, and restenosis is frequently achieved. The aim was to determine the therapeutic success of Oggel silicone intubation after lacrimal syringing and probing. We also analysed other characteristics of the complaint and compared the therapeutic success with published results. METHODS: Therapeutic success was defined as subjective absence of complaints. Other complaint features - directly after explantation of the Oggel tube, one month after explantation and one year after explantation - were retrospectively collected by a patient questionnaire, and a questionnaire for the attending ophthalmologist. These were completed with information from the patient's file. We included 82 procedures performed at the Dept. of Ophthalmology, University of Ulm, between 2006 and 2015. Statistical testing of the impact of risk factors on therapeutic success was performed, using logistic regression analysis and the Mann-Whitney U test. Subgroups were compared using Pearson-Chi-square and Fisher's exact tests and a Kaplan-Meier analysis was also performed. RESULTS: Therapeutic success could be documented in 80.8% of patients after explantation of the Oggel tube, in 61.6% of patients after one month and in 42.5% of patients at one year (n = 82). There were no significant differences between the subgroups of stenosis localisation, or the patients' age or sex. There was also no significant influence of the patient's age, the preoperative duration of complaints or risk factors predisposing for lacrimal duct stenosis. For the observation period of one year, the Kaplan-Meier analysis gave a probability of complaint-freeness of 52.2% for the patient group, as well as a mean duration of the complaint-free period of 34.2 weeks. CONCLUSION: The results allow a prognostic assessment of the therapeutic success in the absence of significant differences in the subgroups. The therapeutic success of bicanalicular silicone intubation with the Oggel tube was in the lower third of comparable studies. The results conform to the observation that there is a progressive decline of therapeutic success. The comparison of the literature turned out to be methodically more difficult due to differences in study characteristics. Standardisation would make sense for future studies.

[The Importance of the Preoperative Information from a Patient's Perspective - a Structured Interview of 100 Ophthalmological University-Based Outpatients]. / Die Erwartungen an das Aufklärungsgespräch aus Patientensicht ­ strukturierte Befragung von 100 Patienten einer ophthalmologischen Universitätsambulanz.

BACKGROUND: The patient's knowledge about their illness, as well as their expectations regarding pre-intervention, consultation and treatment, may differ from the physician's assumptions. Therefore, it is of great importance that the physician can identify misconceptions and missing knowledge and to focus on those points in the preoperative consultation, as well as meeting patient expectations as to the consultation itself. The aim of this study was to identify such expectations and the knowledge gaps of patients scheduled for ophthalmologic treatment. METHOD: An anonymous questionnaire containing predominantly closed questions was handed out to 100 patients in an ophthalmological outpatient clinic of a tertiary care center. Answers were mostly single choice items on a rating scale. RESULTS: 55% of patients had received ophthalmological interventions prior to receiving the questionnaire; 36% received more than two. More than half had not informed themselves about the planned procedure prior to their appointment. They were worried the most about complications (59%) and least about the anaesthesia (29%). When asked, patients attributed the highest priority to provision of information regarding complications and most often requested information on implications of the planned surgery on daily activities. CONCLUSION: Roughly half of the patients came without having informed themselves prior to the consultation. A comprehensive explanation with regard to success rates and possible post-surgical impairments appears to be essential. Possibilities of new media, such as the internet, surprisingly do not seem to be of importance to patients in this context.

[Augmented Reality in Ophthalmology: Technical Innovation Complements Education for Medical Students]. / Augmented Reality in der Augenheilkunde: Technische Innovation ergänzt studentische Lehre.

BACKGROUND: At the Medical Faculty of the University of Ulm, the learning objectives of ophthalmology are taught to students in a one-week block course in the fourth year of study. According to the National Competency-based Learning Objective Catalogue Medicine (NKLM), one of these learning objectives is the clinical examination of the eye, including the eye fundus examination. In order to best train this expertise as defined in the NKLM, a novel, augmented reality-based training simulator (Eyesi Indirect, VRmagic Holding AG, Mannheim, Germany) was integrated into the teaching at the University Eye Hospital Ulm. During the block week, two key questions were examined: Does the one week block lesson increase interest in ophthalmology? How do students assess the use of an innovative e-learning technology compared to three other classical teaching methods for learning ophthalmoscopy? MATERIAL/METHODS: As part of the student teaching (block weeks July and October 2016), 292 students were questioned by questionnaire anonymously and on a voluntary basis regarding their assessment of the block week and the training simulator at the beginning and end of the one week event. The ophthalmoscopies were classically practiced on the ophthalmoscope trainer, head model and on fellow students and then performed on the new training simulator. RESULTS: Overall, there was little interest among the students in ophthalmology. However, the block week helped to increase interest (p < 0.001). The assessments of attractiveness before and after the block week of ophthalmology as a later professional goal and as an optional subject in the practical year were also significantly increased (p < 0.001 or p = 0.031). With regard to the use of e-learning technologies, it was shown that the handling of the simulator for learning ophthalmoscopy was rated more attractive by the students than the classical technology-based teaching methods (all p < 0.01). The joy of learning could only be further increased by practicing on a fellow student (p = 0.051). CONCLUSION: The results of the present study show a positive impact of the block week on the attractiveness assessment of the specialty by the participating students. Innovative e-learning methods can increase the enjoyment of learning. However, the training simulator cannot provide a complete replacement of the training on a real person.

[Invasive Eyelid Infections by ß-Hemolytic Streptococci of Serogroup A]. / Invasive Lidinfektionen durch ß-hämolysierende Streptokokken der Serogruppe A.

BACKGROUND: Invasive soft tissue infections by Streptococcus pyogenes are rapidly progressive and potentially life-threatening infectious diseases. These can also affect the eyelid. Aggressive virulence factors and the synthesis of exotoxins can lead to complications, such as periorbital necrotizing fasciitis (PONF) and streptococcal toxic shock syndrome (STSS). The clinical picture is characterized by four patients with invasive eyelid infections. MATERIALS AND METHODS: Photographic documentation, radiological imaging, laboratory and smear diagnostics and intravenous antibiotic therapy were performed on all patients according to the recommendations of the German Robert Koch Institute and the local infectiology board. RESULTS: In all patients, Streptococcus pyogenes was culturally detected in a direct swab. The antibiogram showed sensitivity to the common intravenous antibiotics. The time interval between symptom onset and presentation at the clinic was between two days and one week. All patients had high systemic inflammatory parameters on admission: Pat. 1: CRP 259 mg/l, leukocytes 20.1 giga/l; Pat. 2: CRP 375 mg/l, leukocytes 15.6 giga/l; Pat. 3: CRP 378 mg/l, leukocytes 38.7 giga/l; Pat. 4: CRP 483 mg/l, leukocytes 1.7 giga/l; normal values: CRP < 5 mg/l, leucocytes 4.4 - 11.3 giga/l. In Pat. 2 and 3, a periorbital necrotizing fasciitis was diagnosed due to rapidly progressing necrosis in the area of cutis and subcutis and systemic toxicity. Pat. 3 and 4 met the diagnostic criteria of STSS. Pat. 2, 3 and 4 had to be relocated to an intermediate or intensive care unit with sepsis, despite immediate intravenous antibiotic therapy. Patient 3 underwent surgical debridement during the stay in the intensive care unit. Thanks to interdisciplinary management (ophthalmology, infectiology, ear, nose and throat medicine, internal medicine and intensive care medicine), all patients were finally discharged from our inpatient treatment in a significantly improved general condition. CONCLUSION: Invasive streptococcal infections represent a challenge in the daily routine of an ophthalmologist. Interdisciplinary management and immediate onset of high-dose intravenous antibiotic therapy are crucial for successful treatment.

Fate of the Fc fusion protein aflibercept in retinal endothelial cells: competition of recycling and degradation.

PURPOSE: Intravitreal injection of the VEGF-binding protein aflibercept is widely used to treat various ocular diseases. In vitro, immortalized bovine retinal endothelial cells (iBREC) take up and transport aflibercept through the cell layer in a serum-dependent manner, likely mediated through the neonatal Fc receptor (FcRn), but degradation of the Fc domain-containing protein might be a competing intracellular process. Therefore, aflibercept's associations with proteins either involved in FcRn-mediated transport or in the lysosomal pathway were studied. METHODS: Confluent iBREC pre-cultivated with or without FBS were exposed for 4 h to in vivo achievable 250 µg/ml aflibercept, before cells were harvested for immunofluorescence staining or preparation of protein extracts. Intracellular localization of aflibercept and putative co-localizations with proteins involved in transport of IgG/FcRn complexes, i.e., endosomal Rab4 and Rab11, components of the cytoskeleton, motor proteins, or with marker proteins characteristic of multivesicular bodies or lysosomes were assessed by co-immunofluorescence stainings. Amounts of expressed endogenous proteins and of internalized aflibercept were determined by Western blot analyses. RESULTS: Aflibercept-specific perinuclear staining overlapped with that of the motor protein dynein whereas double staining with an anti-kinesin antibody resulted in a patchy pattern. In addition, aflibercept was typically present close to microtubules and often co-localized with α-tubulin. Rab4 and Rab11 stainings partly overlapped with the perinuclear staining of aflibercept whereas co-localization with Rab7 (in late endosomes/lysosomes) was only rarely seen. Interestingly, aflibercept but not the IgG bevacizumab broadly co-localized with the cation-independent mannose 6-phosphate receptor characteristic of multivesicular endosomes. In accordance with partial degradation beside transcytosis, the amount of intracellular aflibercept increased when cells were treated with protease inhibitors MG-132 or MG-101. Serum-deprived iBREC expressed less Rab11 and dynein but slightly more Rab4. CONCLUSION: After uptake by iBREC, aflibercept is present in organelles associated with FcRn-mediated transport, but part of the protein is subject to degradation. Transport inhibition of aflibercept during cultivation without FBS is likely a consequence of an attenuated exocytosis due to decreased expression of Rab11.

[In Vitro Study of Subablative Er : YAG Laser and Diode Laser Therapy vs. Thermal Cautery in Microbial Keratitis]. / In-vitro-Studie mit subablativem Er:YAG-Laser und Diodenlaser vs. Thermokauter zur Therapie der mikrobiellen Keratitis.

BACKGROUND: In microbial keratitis, thermal cautery leads to thermal necrosis associated with germ reduction. Subablative heating of the cornea using an Er : YAG laser or diode laser is an alternative option. Thermal effects and reproducibility of both procedures were investigated. MATERIAL AND METHODS: Thermal effects were monitored with an infrared imaging system. Temperature of stromal tissue of enucleated porcine eyes was measured. Effects of thermal injury were rated by microscopic photography and histopathologic examination. Reduction of living bacteria was visualized by laser scanning microscopy and fluorescent stain on stromal tissue models containing E. coli bacteria. RESULTS: Corneal samples showed thermal damage depths of 110 - 250 µm after thermal cautery at contact times of 1 - 3 s. Superficial temperature ranged from 70 to 80 °C. Thickness of thermal necrosis after laser irradiation was between 50 and 270 µm. Temperature ranged from 60 to 120 °C. Laser scanning microscopy of a stromal tissue model, comprising Escherichia coli bacteria in soft agar, showed a mortification of bacteria over 40% up to 100 µm depth after treatment for 25 s. Treatment with diode laser showed an even better effect: more than 60% of bacteria were dead up to 250 µm depth and more than 50% up to 450 µm depth in the stromal tissue model. CONCLUSIONS: All procedures are able to induce high temperatures in tissue to achieve effective germ reduction in corneal stroma. Effect and reproducibility of the thermal cautery procedure depends on the surgeon's experience. Energy dose and thermal effect can be calculated for the Er : YAG and diode laser procedure to affect the infected stromal layer selectively.

[Innovative Teaching Methods: Wet Lab-based Ophthalmosurgical Education for Medical Students]. / Innovative Lehrmethoden: Einsatz eines ophthalmochirurgischen Wetlabs im Studentenunterricht.

BACKGROUND: An increased demand for young physicians in Germany is causing increased competition to attract the best minds, even during their undergraduate studies. Therefore, it is the task of the university hospitals to offer their students the best possible courses in order to arouse interest in the relevant subject area. We therefore examined the impact of an ophthalmosurgical wet lab on teaching undergraduate medical students. MATERIAL/METHODS: Undergraduate medical students were offered voluntary participation in the ophthalmosurgical wet lab during an ophthalmology block week. At the beginning and at the end of the week, as well as at the end of the wet lab, the students answered questionnaires with questions about the interest in the field of ophthalmology and the specific assessment of the wet lab. RESULTS: The interest of the students in ophthalmology was increased by the block week and additionally by the wet lab. The wet lab enriched the block week and was rated as very good by the participants. The overall rating of the block week was significantly better among participants in the wet lab than in the group without wet lab participation. The wet lab gave the students great pleasure, enabled them to apply the acquired knowledge, gain valuable insights into ophthalmology, and was easily implemented. CONCLUSION: The integration of a wet lab into the block week was evaluated very positively by the students and gives them a valuable insight into the field of ophthalmology.

[Comparison Between Automated and Manual Measurement of Foveal Avascular Zone Retinopathy in Optical Coherence Tomography]. / Evaluation der automatisierten Messung von ischämischen Veränderungen der Makula bei diabetischer Makulopathie.

OBJECTIVE: A comparison between automated and manual measurements of a foveal avascular zone in optical coherence tomography angiography (OCTA) in patients with diabetic retinopathy (DR). MATERIAL AND METHODS: Consecutive patients with non-proliferative DR were included in this study. All patients received an OCTA, with a 3 × 3 mm scan of the macular region taken with the Zeiss OCT CIRRUS 5000 with the AngioPlex module. The size of the foveal avascular zone (FAZ) was determined both manually and with the help of the automated measurement metrics. Next, the measurements obtained using manual and automated methods were compared. In addition, the circularity index determined in metrics was examined for correlations with the size and area of the FAZ. RESULTS: Thirty-four eyes from 28 patients with non-proliferative diabetic retinopathy with a mean age of 63 years were included. The mean size of the foveal avascular zone was 0.34 ± 0.12 mm2 (0.08 - 0.65 mm2) for manual evaluation and 0.23 ± 0.11 mm2 (range 0.03 - 0.49 mm2) in metrics. The circularity index in metrics averaged 0.58 and showed a statistically significant correlation with the size of the manually measured FAZ. CONCLUSION: There was a comparable result for the size of the FAZ in both measurement methods. Automated measurements with metrics can reliably represent changes in the FAZ for most patients, based on the calculated area, as well as on the circumference and the circularity index.

[Visualization of Retinal Neovascularization with Optical Coherence Tomography Angiography in Comparison with Fluorescein Angiography]. / Darstellung von retinalen Neovaskularisationen mit der optischen Kohärenztomografie-Angiografie im Vergleich mit der Fluoreszeinangiografie.

OBJECTIVE: Comparison of retinal neovascularizations of the disc (NVD) and elsewhere (NVE) in optical coherence tomography angiography (OCTA) and fluorescein angiography (FA) in patients with proliferative diabetic retinopathy. MATERIALS AND METHODS: 15 consecutive patients were included in this study. All patients received an OCTA with a 3 × 3 mm scan of the region of interest with the ZEISS OCT Cirrus 5000 with the AngioPlex module. The size of the neovascularization (NV) was determined manually in OCTA and FA and compared between the two methods. RESULTS: 20 eyes of 15 patients with proliferative diabetic retinopathy with an average age of 57 years were included. The mean size of NVDs was 3.44 mm2 in OCTA and 3.75 mm2 in FA, the mean size of NVDs was 1.06 mm2 in OCTA and 1.54 mm2 in FA. Taking into account a generally larger area measured in the FA, the two methods showed good overall agreement. CONCLUSION: There was a good agreement for the size of the NVs in both methods. OCTA can be used as a simple and non-invasive method to visualize retinal neovascularizations.

[Free-Floating Intraocular Cysts]. / Frei fluktuierende intraokulare Zysten.

BACKGROUND: Free-floating intraocular cysts may be found in the anterior chamber (FZV) and the vitreous (FZG). The first description of a cyst was 150 years ago, and they are considered to be ocular rarities. MATERIALS AND METHODS: The actual knowledge about FZV and FZG is shown on the basis of two exemplary patients. RESULTS AND DISCUSSION: Patient 1 had a FZV as an incidental finding which had a smooth surface, a slight pigmentation and was translucent. The ultrasound biomicroscopy revealed an echo-free interior space. Without the patient's discomfort and missing treatment indication, a watch-and-wait strategy was chosen. Cysts of the iris can be classified as primary and secondary cysts. Primary cysts of the iris can arise from the stroma as the pigment epithelium wherein it is believed that FZV descend from the pigment epithelium. Secondary cysts and FZV can be generated by tumors, inflammation, epithelial ingrowth, the use of eye-drops or intraocular foreign bodies. Patient 2 showed marked myopic fundus changes and an FZG with a yellowish-greenish surface; the transparency was reduced and the surface was not pigmented. The ultrasound examination also revealed an echo-free interior space. Clinical controls were advised. Congenital and acquired causes are discussed for the formation of FZG. FZG could originate from the pigment epithelium of the iris, but there are conflicting study results. Trauma, inflammation and chorioretinal diseases are considered as a reason for acquired causes of FZG. The genesis, especially of FZG, is still unclear. For the treatment of patients with FZV and FZG, it is important to know the potential causes to be able to make a therapeutic decision. High quality photographic and sonographic documentation is needed in the watch-and-wait strategy.

Inhibition of Single Routes of Intracellular Signaling is Not Sufficient to Neutralize the Biphasic Disturbance of a Retinal Endothelial Cell Barrier Induced by VEGF-A165.

BACKGROUND/AIMS: Hallmark of diabetic macular edema is the enhanced permeability of retinal endothelial cells (REC) induced by vascular endothelial growth factor (VEGF-A165), which acts through activating specific receptors. To improve the predictability of inhibitors' potentials to block harmful effects of VEGF-A165, we investigated if its signaling pathways triggered in REC are redundant. METHODS: Immortalized bovine REC monolayers were treated with inhibitors specific for various protein kinases in combination with VEGF-A165. Permeability was monitored continuously by measurements of the cell index (CI) to reveal even subtle and transient changes. Expression of tight junction (TJ) proteins was determined as additional indicator of barrier stability. RESULTS: After a sharp but transient CI drop caused by VEGF-A165 early after its addition, further exposure resulted in a continuous CI decline over several days associated with loss of TJ protein claudin-1. Both phases were blocked by inhibition of VEGF receptor 2. Tested inhibitors of intracellular kinases had a limited or no effect, or were efficient only in certain phases of exposure to VEGF-A165, e.g. inhibiting protein kinase C only prevented the early response. High concentrations of some inhibitors even resulted in VEGF-independent barrier destabilization. CONCLUSIONS: Specific kinase inhibitors differently affect VEGF-A165-triggered processes in distinct phases of its action. VEGF-A165-initiated signaling is redundant and blocking of key proteins of single pathways is not sufficient to suppress REC barrier breakdown.

Neonatal Fc receptor FcRn is involved in intracellular transport of the Fc fusion protein aflibercept and its transition through retinal endothelial cells.

Retinal endothelial cells (REC) likely contribute to the clearance of intravitreally injected IgG. Because this is of high relevance to the pharmacokinetic assessment of the widely used therapeutic Fc fusion protein aflibercept, we studied its transport through immortalized bovine REC (iBREC) in detail. For shuttling of IgG or Fc fusion proteins like aflibercept, endothelial cells use the highly conserved neonatal Fc receptor (FcRn) also expressed in iBREC where it is down regulated by serum depletion. Therefore, we focused on studying intracellular localization and transport of aflibercept under conditions affecting its interaction with the FcRn. Intracellular localization of aflibercept was assessed by Western-blot analyses of subcellular protein fractions or by immunofluorescence staining. After uptake in a temperature-dependent process, aflibercept co-localized with early endosomes, which harbor FcRn. Similar amounts of aflibercept were co-extracted with proteins from membranes/organelles irrespectively of the amount of FBS in the culture medium. Lowering the concentration of FBS resulted in a strong, but reversible association with cytoskeletal proteins suggesting a block in intracellular transport. In accordance with this finding, aflibercept's transport through an iBREC monolayer grown on porous membrane inserts was markedly delayed in the absence of FBS in the culture medium indicating that aflibercept is taken up but not exocytosed under these conditions. Transcytosis of aflibercept was also strongly delayed by inhibition of phosphatidylinositol 3-kinase with LY294002, which affects FcRn-mediated IgG transport. A similar inhibition of aflibercept's transport was observed with IgG-binding proteins (i.e. protein A or protein G) that block interaction between FcRn and aflibercept. Interfering with aflibercept's binding to the FcRn with protein A (or protein G) or the inhibitory FcRn-specific monoclonal antibody 1G3 resulted in a reduced amount of intracellular aflibercept. Taken together, our results strongly suggest that FcRn is involved in transport of aflibercept through REC in vitro.

[ISO 9001:2015 Certification in Quality Management]. / Zertifizierung des Qualitätsmanagements nach DIN EN ISO 9001:2015.

Quality management improves the structures, processes and results of organizations of all kinds. Many practices and clinics have their existing quality management system certified according to ISO 9001, (e.g., to check their own quality management system or to obtain a testimonial against third parties). The latest version ISO 9001:2015 contains some changes, both structurally and in terms of content. These changes can be met with reasonable efforts. An ISO 9001:2015 certification represents a value for your organization, but these advantages are often not directly measurable.