Uptake of Ranibizumab but Not Bevacizumab into Uveal Melanoma Cells Correlates with a Sustained Decline in VEGF-A Levels and Metastatic Activities.

Despite the implication of vascular endothelial growth factor-A (VEGF-A) in the pathophysiology of uveal melanoma (UM), the anti-VEGF-A antibody bevacizumab yielded conflicting results on UM growth. Here, we evaluated whether bevacizumab and ranibizumab, a humanized Fab-fragment against VEGF-A, can enter UM cells and induce a sustained physiological response. The primary and metastatic UM cell lines Mel-270 and OMM-2.5 were exposed to bevacizumab or ranibizumab for one day and were maintained further in untreated medium for a total of three days. Both antibodies significantly reduced the levels of extracellular VEGF-A and the angiogenic potential of the conditioned medium after one day. These inhibitory effects of bevacizumab diminished by day three. Ranibizumab suppressed the metabolic activity, proliferation, and intracellular VEGF-A levels in a cell-type and concentration-dependent manner, whereas bevacizumab exerted no effect. Both drugs were detected inside early endosomes within the UM cells, with the stronger and sustained colocalization of ranibizumab. Our results therefore demonstrated the more potent and persistent suppressive activity of ranibizumab on the UM cells, possibly due to its higher level of uptake and prolonged intracellular retention. Further research on the endosome dynamics in UM cells might provide valuable insight into the response of these heterogenous tumors to therapeutic antibodies.

Rift Valley fever virus minigenome system for investigating the role of L protein residues in viral transcription and replication.

Replicon systems are important tools for investigating viral RNA synthesis. We have developed an ambisense minigenome system for Rift Valley fever virus (RVFV) with the aim to analyse the effects of L gene mutations on viral transcription versus replication. The overall activity of the replication complex was assessed by expression of a luciferase reporter gene. Northern blot analysis enabled differentiation between synthesis of viral mRNA and replication intermediates. The functionality of the system was demonstrated by probing residues predictably involved in the cap-snatching endonuclease active site in the L protein. Corresponding mutations led to a selective defect in the viral mRNA synthesis as described for other bunyaviruses. The analysis of further L gene mutants revealed an essential role of a C-terminal region in the RVFV L protein in viral transcription. In summary, the established minigenome system is suitable for functional testing of the relevance of residues for viral transcription and replication.

Laboratory Findings, Compassionate Use of Favipiravir, and Outcome in Patients With Ebola Virus Disease, Guinea, 2015-A Retrospective Observational Study.

BACKGROUND: In 2015, the laboratory at the Ebola treatment center in Coyah, Guinea, confirmed Ebola virus disease (EVD) in 286 patients. The cycle threshold (Ct) of an Ebola virus-specific reverse transcription-polymerase chain reaction assay and 13 blood chemistry parameters were measured on admission and during hospitalization. Favipiravir treatment was offered to patients with EVD on a compassionate-use basis. METHODS: To reduce biases in the raw field data, we carefully selected 163 of 286 patients with EVD for a retrospective study to assess associations between potential risk factors, alterations in blood chemistry findings, favipiravir treatment, and outcome. RESULTS: The case-fatality rate in favipiravir-treated patients was lower than in untreated patients (42.5% [31 of 73] vs 57.8% [52 of 90]; P = .053 by univariate analysis). In multivariate regression analysis, a higher Ct and a younger age were associated with survival (P < .001), while favipiravir treatment showed no statistically significant effect (P = .11). However, Kaplan-Meier analysis indicated a longer survival time in the favipiravir-treated group (P = .015). The study also showed characteristic changes in blood chemistry findings in patients who died, compared with survivors. CONCLUSIONS: Consistent with the JIKI trial, this retrospective study revealed a trend toward improved survival in favipiravir- treated patients; however, the effect of treatment was not statistically significant, except for its influence on survival time.

Apolipoprotein A-I Mimetic Peptide L-4F Removes Bruch's Membrane Lipids in Aged Nonhuman Primates.

Purpose: Multiple evidence lines support Bruch's membrane lipid deposition as a major precursor of soft drusen and age-related macular degeneration as including a potentially treatable atherosclerosis-like progression in the subretinal pigment epithelium (RPE)-basal lamina space. We evaluated the effect of anti-inflammatory, antiatherogenic peptide L-4F on Bruch's membrane of aged nonhuman primates in a dose-escalating study. Methods: Macaca fascicularis ≥20 years of age evaluated by color fundus photography and optical coherence tomography received monocular intravitreal injections of L-4F (n = 7) or a placebo-scrambled peptide (n = 2) in 6 doses of 25 to 175 µg over 6 months. Eyes were processed for detection and masked semiquantitative assessment of macular Bruch's membrane neutral lipid (oil red O staining), esterified cholesterol (filipin histochemistry), membrane attack complex (immunofluorescence), and paramacular thickness (transmission electron microscopy). Results: Bruch's membrane neutral lipid, esterified cholesterol, and membrane attack complex were cleared and ultrastructure was improved in L-4F-injected eyes, compared to placebo-injected eyes. Fellow eyes were also affected to the same degree as the injected eyes. Punctate yellow fundus lesions without corresponding RPE elevations on optical coherence tomography correlated to RPE lipoidal degeneration (engorgement with lipid droplets), which was unchanged by this treatment. Conclusions: Clinical-stage apolipoprotein A-I mimetic peptide L-4F, delivered intravitreally in repeated doses, produced a substantial pharmacologic reduction of Bruch's membrane lipid and restoration of ultrastructure in a nonhuman primate model that exhibits an important precursor of soft drusen, if not soft drusen themselves.

Catastrophic antiphospholipid syndrome causing ST-segment elevation myocardial infarction with non-obstructive coronary arteries.

A 51-year-old woman with known primary antiphospholipid syndrome presented with a 4-day history of chest and abdominal pain, inferior ST-segment elevation on a 12-lead ECG and a subtherapeutic international normalised ratio. In view of a significantly raised high-sensitivity troponin I assay, inferior wall hypokinesis on transthoracic echocardiography and despite unobstructed epicardial vessels on emergency coronary angiography, a diagnosis of myocardial infarction was made. Furthermore, the patient also developed both bilateral adrenal haemorrhages leading to acute adrenal insufficiency and microvascular thrombotic renal disease concurrently. The patient therefore fulfilled the diagnostic criteria for catastrophic antiphospholipid syndrome presenting with cardiac, endocrine and renal involvement. Early diagnosis permitted appropriate treatment with anticoagulation, dual antiplatelet therapy, secondary prevention and corticosteroid replacement therapy and led to a full recovery. This case highlights first the importance of adequate anticoagulation in antiphospholipid syndrome and, second, the potentially fatal, multiorgan complication of failure to do so.

[Patient-Reported Treatment Satisfaction with Stereotactic Radiotherapy in Neovascular Age-Related Macular Degeneration]. / Patientenzufriedenheit nach stereotaktischer Radiotherapie bei exsudativer altersbedingter Makuladegeneration.

BACKGROUND: Stereotactic radiotherapy (SRT) in conjunction with the common intravitreal injections (IVI) is a new adjuvant approach in neovascular age-related macular degeneration (AMD) patients. The aim of our study was to investigate factors influencing patient satisfaction one year after SRT. METHODS: A questionnaire was administered to 35 AMD patients who had consecutively undergone SRT using the IRay®-device at the Department of Ophthalmology, University of Lübeck. In addition to descriptive statistics, responses were evaluated by correlation analysis. Moreover, subgroup analyses were performed, using a classification of IVI responders (annual injection rate after SRT ≤ 3), visual acuity (VA) responders (VA improvement ≥ 0.2 logMAR) and double responders (annual injection rate after SRT ≤ 3 as well as VA improvement ≥ 0.2 logMAR). RESULTS: The response rate was 86%. With respect to their treatment expectations, twice as many patients hoped to receive less injections instead of a better vision. Those hoping for less injections were significantly more satisfied with their clinical outcome. In addition, IVI-responders were significantly more satisfied than IVI-non-responders, while VA-responders were not, compared to VA-non-responders. CONCLUSIONS: Patient satisfaction seems to depend on patients' comprehension of how SRT affects their disease and what kinds of expectations were set. It is of utmost importance to provide the patients with adequate and comprehensible education and to define realistic goals prior to SRT.

Impact of correct anatomical slab segmentation on foveal avascular zone measurements by optical coherence tomography angiography in healthy adults.

PURPOSE: To evaluate the impact of correct anatomical slab segmentation on foveal avascular zone (FAZ) dimensions in the superficial capillary plexus (SCP) and deep capillary plexus (DCP) using optical coherence tomography angiography (OCTA). METHODS: Participants with healthy retinas were recruited, and 5 × 5 mm OCTA images were acquired using the Canon HS-100 Angio eXpert module. FAZ size was measured in automatically (AS, manufacturer-based) and manually (MS, anatomical-based) segmented OCTA slabs by two experienced graders. FAZ dimensions, inter-rater agreement, and correlation to demographic and retinal parameters were evaluated. RESULTS: A total of 38 eyes from 20 healthy adult subjects were included in this cross-sectional study. While in AS slabs, the FAZ in the SCP was smaller than in the DCP, in MS images, it was the opposite. MS had a relevant impact on inter-rater agreement of FAZ measurements in the SCP. The FAZ area in both plexus correlated inversely with the central retinal thickness (CRT), irrespective of the segmentation applied. Furthermore, an enlargement of FAZ size in the DCP with increasing age was found. Finally, the FAZ in female participants was significantly larger than in their male counterparts, regardless of the evaluated plexus and chosen segmentation. CONCLUSIONS: Correct anatomical slab segmentation has a significant impact on FAZ size measurements. Not adjusting the segmentation boundaries represents a significant source of error for measuring FAZ area and confounds comparisons across studies as well as OCTA devices.

ApoA-I Mimetic Peptide 4F Reduces Age-Related Lipid Deposition in Murine Bruch's Membrane and Causes Its Structural Remodeling.

PURPOSE: Accumulation of lipoprotein-derived lipids including esterified and unesterified cholesterol in Bruch's membrane of human eyes is a major age-related change involved in initiating and sustaining soft drusen in age-related macular degeneration (AMD). The apolipoprotein (apo) A-I mimetic peptide 4F is a small anti-inflammatory and anti-atherogenic agent, and potent modifier of plasma membranes. We evaluated the effect of intravitreally-injected 4F on murine Bruch's membrane. METHODS: We tested single intravitreal injections of 4F doses (0.6 µg, 1.2 µg, 2.4 µg, and placebo scrambled peptide) in ApoEnull mice ≥10 months of age. After 30 days, mice were euthanized. Eyes were processed for either direct immunofluorescence detection of esterified cholesterol (EC) in Bruch's membrane whole mounts via a perfringolysin O-based marker linked to green fluorescent protein or by transmission electron microscopic visualization of Bruch's membrane integrity. Fluorescein isothiocyanate-conjugated 4F was traced after injection. RESULTS: All injected eyes showed a dose-dependent reduction of Bruch's membrane EC with a concomitant ultrastructural improvement compared to placebo treated eyes. At a 2.4 µg dose of 4F, EC was reduced on average by ~60% and Bruch's membrane returned to a regular pentalaminar structure and thickness. Tracer studies confirmed that injected 4F reached intraocular targets. CONCLUSION: We demonstrated a highly effective pharmacological reduction of EC and restoration of Bruch's membrane ultrastructure. The apoA-I mimetic peptide 4F is a novel way to treat a critical AMD disease process and thus represents a new candidate for treating the underlying cause of AMD.

Tramadol for neuropathic pain in adults.

BACKGROUND: This review is an update of a review of tramadol for neuropathic pain, published in 2006; updating was to bring the review in line with current standards. Neuropathic pain, which is caused by a lesion or disease affecting the somatosensory system, may be central or peripheral in origin. Peripheral neuropathic pain often includes symptoms such as burning or shooting sensations, abnormal sensitivity to normally painless stimuli, or an increased sensitivity to normally painful stimuli. Neuropathic pain is a common symptom in many diseases of the peripheral nervous system. OBJECTIVES: To assess the analgesic efficacy of tramadol compared with placebo or other active interventions for chronic neuropathic pain in adults, and the adverse events associated with its use in clinical trials. SEARCH METHODS: We searched CENTRAL, MEDLINE, and Embase for randomised controlled trials from inception to January 2017. We also searched the reference lists of retrieved studies and reviews, and online clinical trial registries. SELECTION CRITERIA: We included randomised, double-blind trials of two weeks' duration or longer, comparing tramadol (any route of administration) with placebo or another active treatment for neuropathic pain, with subjective pain assessment by the participant. DATA COLLECTION AND ANALYSIS: Two review authors independently extracted data and assessed trial quality and potential bias. Primary outcomes were participants with substantial pain relief (at least 50% pain relief over baseline or very much improved on Patient Global Impression of Change scale (PGIC)), or moderate pain relief (at least 30% pain relief over baseline or much or very much improved on PGIC). Where pooled analysis was possible, we used dichotomous data to calculate risk ratio (RR) and number needed to treat for an additional beneficial outcome (NNT) or harmful outcome (NNH), using standard methods. We assessed the quality of the evidence using GRADE and created 'Summary of findings' tables. MAIN RESULTS: We identified six randomised, double-blind studies involving 438 participants with suitably characterised neuropathic pain. In each, tramadol was started at a dose of about 100 mg daily and increased over one to two weeks to a maximum of 400 mg daily or the maximum tolerated dose, and then maintained for the remainder of the study. Participants had experienced moderate or severe neuropathic pain for at least three months due to cancer, cancer treatment, postherpetic neuralgia, peripheral diabetic neuropathy, spinal cord injury, or polyneuropathy. The mean age was 50 to 67 years with approximately equal numbers of men and women. Exclusions were typically people with other significant comorbidity or pain from other causes. Study duration for treatments was four to six weeks, and two studies had a cross-over design.Not all studies reported all the outcomes of interest, and there were limited data for pain outcomes. At least 50% pain intensity reduction was reported in three studies (265 participants, 110 events). Using a random-effects analysis, 70/132 (53%) had at least 50% pain relief with tramadol, and 40/133 (30%) with placebo; the risk ratio (RR) was 2.2 (95% confidence interval (CI) 1.02 to 4.6). The NNT calculated from these data was 4.4 (95% CI 2.9 to 8.8). We downgraded the evidence for this outcome by two levels to low quality because of the small size of studies and of the pooled data set, because there were only 110 actual events, the analysis included different types of neuropathic pain, the studies all had at least one high risk of potential bias, and because of the limited duration of the studies.Participants experienced more adverse events with tramadol than placebo. Report of any adverse event was higher with tramadol (58%) than placebo (34%) (4 studies, 266 participants, 123 events; RR 1.6 (95% CI 1.2 to 2.1); NNH 4.2 (95% CI 2.8 to 8.3)). Adverse event withdrawal was higher with tramadol (16%) than placebo (3%) (6 studies, 485 participants, 45 events; RR 4.1 (95% CI 2.0 to 8.4); NNH 8.2 (95% CI 5.8 to 14)). Only four serious adverse events were reported, without obvious attribution to treatment, and no deaths were reported. We downgraded the evidence for this outcome by two or three levels to low or very low quality because of small study size, because there were few actual events, and because of the limited duration of the studies. AUTHORS' CONCLUSIONS: There is only modest information about the use of tramadol in neuropathic pain, coming from small, largely inadequate studies with potential risk of bias. That bias would normally increase the apparent benefits of tramadol. The evidence of benefit from tramadol was of low or very low quality, meaning that it does not provide a reliable indication of the likely effect, and the likelihood is very high that the effect will be substantially different from the estimate in this systematic review.

RNA Interference Restricts Rift Valley Fever Virus in Multiple Insect Systems.

The emerging bunyavirus Rift Valley fever virus (RVFV) is transmitted to humans and livestock by a large number of mosquito species. RNA interference (RNAi) has been characterized as an important innate immune defense mechanism used by mosquitoes to limit replication of positive-sense RNA flaviviruses and togaviruses; however, little is known about its role against negative-strand RNA viruses such as RVFV. We show that virus-specific small RNAs are produced in infected mosquito cells, in Drosophila melanogaster cells, and, most importantly, also in RVFV vector mosquitoes. By addressing the production of small RNAs in adult Aedes sp. and Culex quinquefasciatus mosquitoes, we showed the presence of virus-derived Piwi-interacting RNAs (piRNAs) not only in Aedes sp. but also in C. quinquefasciatus mosquitoes, indicating that antiviral RNA interference in C. quinquefasciatus mosquitoes is similar to the described activities of RNAi in Aedes sp. mosquitoes. We also show that these have antiviral activity, since silencing of RNAi pathway effectors enhances viral replication. Moreover, our data suggest that RVFV does not encode a suppressor of RNAi. These findings point toward a significant role of RNAi in the control of RVFV in mosquitoes. IMPORTANCE Rift Valley fever virus (RVFV; Phlebovirus, Bunyaviridae) is an emerging zoonotic mosquito-borne pathogen of high relevance for human and animal health. Successful strategies of intervention in RVFV transmission by its mosquito vectors and the prevention of human and veterinary disease rely on a better understanding of the mechanisms that govern RVFV-vector interactions. Despite its medical importance, little is known about the factors that govern RVFV replication, dissemination, and transmission in the invertebrate host. Here we studied the role of the antiviral RNA interference immune pathways in the defense against RVFV in natural vector mosquitoes and mosquito cells and draw comparisons to the model insect Drosophila melanogaster. We found that RVFV infection induces both the exogenous small interfering RNA (siRNA) and piRNA pathways, which contribute to the control of viral replication in insects. Furthermore, we demonstrate the production of virus-derived piRNAs in Culex quinquefasciatus mosquitoes. Understanding these pathways and the targets within them offers the potential of the development of novel RVFV control measures in vector-based strategies.

Persistence and clearance of Ebola virus RNA from seminal fluid of Ebola virus disease survivors: a longitudinal analysis and modelling study.

BACKGROUND: By January, 2016, all known transmission chains of the Ebola virus disease (EVD) outbreak in west Africa had been stopped. However, there is concern about persistence of Ebola virus in the reproductive tract of men who have survived EVD. We aimed to use biostatistical modelling to describe the dynamics of Ebola virus RNA load in seminal fluid, including clearance parameters. METHODS: In this longitudinal study, we recruited men who had been discharged from three Ebola treatment units in Guinea between January and July, 2015. Participants provided samples of seminal fluid at follow-up every 3-6 weeks, which we tested for Ebola virus RNA using quantitative real-time RT-PCR. Representative specimens from eight participants were then inoculated into immunodeficient mice to test for infectivity. We used a linear mixed-effect model to analyse the dynamics of virus persistence in seminal fluid over time. FINDINGS: We enrolled 26 participants and tested 130 seminal fluid specimens; median follow up was 197 days (IQR 187-209 days) after enrolment, which corresponded to 255 days (228-287) after disease onset. Ebola virus RNA was detected in 86 semen specimens from 19 (73%) participants. Median duration of Ebola virus RNA detection was 158 days after onset (73-181; maximum 407 days at end of follow-up). Mathematical modelling of the quantitative time-series data showed a mean clearance rate of Ebola virus RNA from seminal fluid of -0·58 log units per month, although the clearance kinetic varied greatly between participants. Using our biostatistical model, we predict that 50% and 90% of male survivors clear Ebola virus RNA from seminal fluid at 115 days (90% prediction interval 72-160) and 294 days (212-399) after disease onset, respectively. We also predicted that the number of men positive for Ebola virus RNA in affected countries would decrease from about 50 in January 2016, to fewer than 1 person by July, 2016. Infectious virus was detected in 15 of 26 (58%) specimens tested in mice. INTERPRETATION: Time to clearance of Ebola virus RNA from seminal fluid varies greatly between individuals and could be more than 13 months. Our predictions will assist in decision-making about surveillance and preventive measures in EVD outbreaks. FUNDING: This study was funded by European Union's Horizon 2020 research and innovation programme, Directorate-General for International Cooperation and Development of the European Commission, Institut national de la santé et de la recherche médicale (INSERM), German Research Foundation (DFG), and Innovative Medicines Initiative 2 Joint Undertaking.

Introducing MARCo: Histoserological Findings of a Multi-Organic Paraneoplastic Syndrome in Cutaneous Melanoma Patients.

INTRODUCTION: Cutaneous melanoma (CM) is an aggressive skin cancer entity, causing most skin cancer-related deaths. Autoimmune disorders have been described as potential paraneoplastic complications. The purpose of this study was to elucidate the possibility of a combinatory paraneoplastic affection of the retina and cochlea in patients with CM. METHODS: Sera samples from CM patients were used for indirect immunofluorescence on histological retinal and cochlear sections. Furthermore, the serum specimen of a patient with symptomatic affection of both organs was analyzed by multiplex ELISA (enzyme-linked immunosorbent assay) for various cytokines including CD163 (cluster of differentiation 163). RESULTS: Eleven patients were diagnosed with CM. Autoantibodies against structures of the inner ear were confirmed in all patients who were tested positive for antiretinal antibodies as well. CD163 was significantly elevated in the double-symptomatic patient, who developed metastatic disease. CONCLUSION: Paraneoplastic disease of CM can affect more than one organ and this affect may be correlated with the individual prognosis. Therefore, a thorough anamnesis is needed to avoid missing potential symptoms.

Analysis of Diagnostic Findings From the European Mobile Laboratory in Guéckédou, Guinea, March 2014 Through March 2015.

BACKGROUND: A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. METHODS: The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcription-polymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. RESULTS: The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged <15 years had the highest proportion of Ebola virus-malaria parasite coinfections. The case-fatality ratio was high in patients aged <5 years (80%) and those aged >74 years (90%) and low in patients aged 10-19 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of <5 and ≥45 years, and, among children aged 5-14 years, malaria parasite coinfection were independent determinants of a poor EVD outcome. CONCLUSIONS: Virus load, age, and malaria parasite coinfection play a role in the outcome of EVD.

Ranibizumab interacts with the VEGF-A/VEGFR-2 signaling pathway in human RPE cells at different levels.

Vascular endothelial growth factor (VEGF) secreted by the retinal pigment epithelium (RPE) plays an important role in ocular homeostasis, but also in diseases, most notably age-related macular degeneration (AMD). To date, anti-VEGF drugs like ranibizumab have been shown to be most effective in treating these pathologic conditions. However, clinical trials suggest that the RPE could degenerate and perish through anti-VEGF treatment. Herein, we evaluated possible pathways and outcomes of the interaction between ranibizumab and human RPE cells (ARPE-19). Results indicate that ranibizumab affects the VEGF-A metabolism in RPE cells from an extra- as well as intracellular site. The drug is taken up into the cells, with the VEGF receptor 2 (VEGFR-2) being involved, and decreases VEGF-A protein levels within the cells as well as extracellularly. Oxidative stress plays a key role in various inflammatory disorders of the eye. Our results suggest that oxidative stress inhibits RPE cell proliferation. This anti-proliferative effect on RPE cells is significantly enhanced through ranibizumab, which does not inhibit RPE cell proliferation substantially in absence of relevant oxidative stress. Therefore, we emphasize that anti-VEGF treatment should be selected carefully in AMD patients with preexistent extensive RPE atrophy.

Fc Receptor Inhibition Reduces Susceptibility to Oxidative Stress in Human RPE Cells Treated with Bevacizumab, but not Aflibercept.

BACKGROUND/AIMS: VEGF-A is induced by oxidative stress, and functions as a survival factor for various cell types, including retinal pigment epithelial (RPE) cells. Anti-vascular endothelial growth factor (VEGF) drugs like aflibercept and bevacizumab have shown to be most effective in treating neovascular age-related macular degeneration (AMD), however uptake of the drugs might lead to interference with cell physiology. Herein, we evaluated the significance of the Fc receptor (FcR) within this context and moreover explored the impact of VEGF inhibition under normal conditions as well as under oxidative stress, in terms of potential adverse effects. METHODS: ARPE-19 (human RPE) cells were treated with aflibercept and bevacizumab in presence or absence of H2O2 as oxidative stress stimulus. After 24h cells were evaluated for drug uptake, VEGF-A expression and secretion, levels of intracellular reactive oxygen species (ROS) as well as cell proliferation. Experiments were repeated with cells being pre-incubated with an FcR inhibitor prior to drug application. RESULTS: Both drugs inhibited extracellular levels of VEGF-A and were taken up into the RPE, resulting in significantly reduced intracellular levels of VEGF-A. When oxidative stress was applied, intracellular ROS levels in cells treated with both drugs rose, and cell proliferation was reduced. Prior incubation with the FcR inhibitor lessened the uptake of bevacizumab, but not aflibercept into RPE cells, and simultaneously enhanced cell survival under oxidative stress conditions. CONCLUSIONS: Our results indicate that uptake and accumulation of aflibercept and bevacizumab within RPE cells affect the intracellular VEGF-A metabolism negatively, leading to a biologically relevant reduced cell survival under oxidative stress. The FcR plays a substantial role in the uptake of bevacizumab, but not aflibercept, which allows an enhanced RPE cell survival through FcR blockage in an environment dominated by oxidative stress, as clinically significant for various inflammatory retinal disorders.

Culex pipiens and Culex torrentium populations from Central Europe are susceptible to West Nile virus infection.

West Nile virus (WNV), a Flavivirus with an avian primary host, is already widespread in Europe and might also pose an infection risk to Germany, should competent mosquito vectors be present. Therefore, we analysed the ability of WNV to infect German Culex mosquitoes with special emphasis on field collected specimens of Culex torrentium and Culex pipiens biotype pipiens. We collected egg rafts of Culex mosquitoes over two subsequent seasons at two geographically distinct sampling areas in Germany and differentiated the samples by molecular methods. Adult females, reared from the various egg rafts, were challenged with WNV by feeding of artificial blood meals. WNV infection was confirmed by real-time RT-PCR and virus titration. The results showed that field collected C. pipiens biotype pipiens and C. torrentium mosquitoes native to Germany are susceptible to WNV infection at 25 °C as well as 18 °C incubation temperature. C. torrentium mosquitoes, which have not been established as WNV vector so far, were the most permissive species tested with maximum infection rates of 96% at 25 °C. Furthermore, a disseminating infection was found in up to 94% of tested C. pipiens biotype pipiens and 100% of C. torrentium. Considering geographical variation of susceptibility, C. pipiens biotype pipiens mosquitoes from Southern Germany were more susceptible to WNV infection than corresponding populations from Northern Germany. All in all, we observed high infection and dissemination rates even at a low average ambient temperature of 18 °C. The high susceptibility of German Culex populations for WNV indicates that an enzootic transmission cycle in Germany could be possible.

Distribution and genetic structure of Aedes japonicus japonicus populations (Diptera: Culicidae) in Germany.

In recent years, the number of imported cases of arthropod-borne diseases in Europe, such as dengue fever, has increased steadily, as did the emergence and distribution of invasive insect vectors. Consequently, the risk of disease spreading into previously unaffected regions through invasive mosquitoes is also increasing. One example of an invasive mosquito is Aedes japonicus japonicus (A. j. japonicus), which spread from its original habitat in Japan to North America and Europe. This species has been shown to act as a vector for Japanese encephalitis and West Nile viruses. In Europe, A. j. japonicus has been detected in Switzerland, Belgium, Slovenia, and Germany, where it has become a resident species. Here, we describe the recent spread and genetic structure of A. j. japonicus populations in Germany. By monitoring the species in Baden-Württemberg in 2011 and 2012, we observed a considerable enlargement of the infested area from 54 municipalities in 2011 to 124 municipalities in 2012. To elucidate the colonization of Europe by A. j. japonicus, seven microsatellite loci were studied in 106 individuals sampled in Germany and Switzerland in 2012. The same markers were genotyped in 31 North American and 26 Japanese specimens. Population genetic analyses indicated that A. j. japonicus in Baden-Württemberg and North Rhine-Westphalia represented two genetically distinct populations with FST-values of 0.073-0.152, suggesting that they originated from two independent introduction events in the past. These results are of particular interest in light of vectorial variability for the transmission of viruses and other pathogens in Europe.

Detection of esterified cholesterol in murine Bruch's membrane wholemounts with a perfringolysin O-based cholesterol marker.

PURPOSE: To investigate the effects of Bruch's membrane (BrM) neutral lipid deposition in mouse models and its significance to aging and age-related macular degeneration, it is essential to reliably detect small quantities of neutral lipids including esterified cholesterol (EC). In chorioretinal sections and BrM wholemounts, we tested a novel fluorescent cholesterol marker based on the bacterial toxin perfringolysin O (PFO) and compared results with those obtained with the classic cholesterol dye filipin. METHODS: An engineered plasmid containing the specific cholesterol binding domain (D4) of PFO fused to green fluorescent protein (GFP) was expressed in cultured E. coli, isolated, purified, and concentrated. A total of 150 BrM-choroid wholemounts and chorioretinal sections of 11- to 13-month-old ApoE(null) mice were prepared and stained with PFO/D4-GFP or filipin for EC. Samples were examined by epifluorescence microscopy. RESULTS: The fluorescence intensity of PFO/D4-GFP was strong, stable, and, if small quantities of EC were present, superior to filipin. In all specimens, we could sharply locate the PFO/D4-GFP signal to BrM. A semiquantitative evaluation of BrM lipid deposition is possible by measuring PFO/D4-GFP fluorescence intensity. CONCLUSIONS: The use of PFO/D4-GFP allowed a robust and direct detection of EC in aged murine BrM. In wholemount samples, its strong and stable fluorescence facilitated a semiquantitative evaluation of BrM-EC content over a large area. The patterns of EC deposition in murine BrM wholemounts are comparable with findings in human BrM wholemounts. Perfringolysin O/D4-GFP could be an important tool for investigating the effects of BrM lipid deposition in mouse models.

Sleeping with the fishes: electromagnetic interference causing an inappropriate implantable cardioverter defibrillator shock.

A 60-year-old man with a cardiac defibrillator implanted due to previous ventricular fibrillation arrest and ischaemic cardiomyopathy received a shock while cleaning his fish pond. At the time, his immersed arm was close to a submersed water pump, but the patient was asymptomatic. As a result of the shock he lost consciousness, but collapsed backwards, away from the pond. Interrogation of the device revealed a high-frequency artefact that was sensed by the device and triggered a shock. Device parameters were otherwise normal. Subsequently, the submersed water pump was found to be the source of an external alternating current leak and was identified as the likely cause of the inappropriate shock due to electromagnetic interference (EMI). Awareness of potential sources of EMI along with evaluation of data with a detailed clinical history is warranted in all cases.

The use of morphometric wing characters to discriminate female Culex pipiens and Culex torrentium.

The reliability of the length of wing radial vein r(2/3) as a character for the morphological discrimination of the two potential arbovirus vectors Culex pipiens s.s. and Cx. torrentium from Germany was reassessed, after this character had been neglected for more than 40 years. Additionally, multivariate morphometric analyses were applied to evaluate wing shape variation between both species. Although high-throughput molecular tools are now available to differentiate the two species, a simple, low-cost routine alternative may be useful in the absence of a molecular laboratory, such as under semi-field conditions. A thin-plate splines transformation confirmed that primarily the shrinkage of vein r(2/3) is responsible for the wing differences between the two species. In the bivariate analysis, the r(2/3)/r3 indices of Cx. pipiens s.s. and Cx. torrentium were 0.185 and 0.289, respectively, resulting in a correct classification of more than 91% of all tested specimens. Using the absolute length of vein r(2/3) alone still allowed for more than 90% accurate discrimination. Furthermore, classification accuracy of linear discriminant analysis exceeded 97%.