Human Echolocators Have Better Localization Off Axis.

Here, we report novel empirical results from a psychophysical experiment in which we tested the echolocation abilities of nine blind adult human experts in click-based echolocation. We found that they had better acuity in localizing a target and used lower intensity emissions (i.e., mouth clicks) when a target was placed 45° off to the side compared with when it was placed at 0° (straight ahead). We provide a possible explanation of the behavioral result in terms of binaural-intensity signals, which appear to change more rapidly around 45°. The finding that echolocators have better echo-localization off axis is surprising, because for human source localization (i.e., regular spatial hearing), it is well known that performance is best when targets are straight ahead (0°) and decreases as targets move farther to the side. This may suggest that human echolocation and source hearing rely on different acoustic cues and that human spatial hearing has more facets than previously thought.

The potential of gene therapy for recessive dystrophic epidermolysis bullosa.

Epidermolysis bullosa (EB) encompasses a heterogeneous group of inherited skin fragility disorders, with mutations in genes encoding the basement membrane zone (BMZ) proteins that normally ensure dermal-epidermal integrity. Of the four main EB types, recessive dystrophic EB (RDEB), especially the severe variant, represents one of the most debilitating clinical entities, with recurrent mucocutaneous blistering and ulceration leading to chronic wounds, infections, inflammation, scarring and ultimately cutaneous squamous cell carcinoma, which leads to premature death. Improved understanding of the molecular genetics of EB over the past three decades and advances in biotechnology have led to rapid progress in developing gene and cell-based regenerative therapies for EB. In particular, RDEB is at the vanguard of advances in human clinical trials of advanced therapeutics. Furthermore, the past decade has witnessed the emergence of a real collective, global effort involving academia and industry, supported by international EB patient organizations such as the Dystrophic Epidermolysis Bullosa Research Association (DEBRA), among others, to develop clinically relevant and marketable targeted therapeutics for EB. Thus, there is an increasing need for the practising dermatologist to become familiar with the concept of gene therapy, fundamental differences between various approaches, and their human applications. This review explains the principles of different approaches of gene therapy, summarizes its journey, and discusses its current and future impact in RDEB.

Intrapatient comparisons of efficacy in a single-arm trial of entrectinib in tumour-agnostic indications.

BACKGROUND: Entrectinib is a tropomyosin receptor kinase inhibitor approved for the treatment of neurotrophic tyrosine receptor kinase (NTRK) fusion-positive solid tumours based on single-arm trials. Traditional randomised clinical trials in rare cancers are not feasible; we conducted an intrapatient analysis to evaluate the clinical benefit of entrectinib versus prior standard-of-care systemic therapies. METHODS: Patients with locally advanced/metastatic NTRK fusion-positive tumours enrolled in the global phase II, single-arm STARTRK-2 trial were grouped according to prior systemic therapy and response. The key analysis used growth modulation index [GMI; ratio of progression-free survival (PFS) on entrectinib to time to discontinuation (TTD) on the most recent prior therapy]; ratio ≥1.3 indicated clinically meaningful efficacy. Additional analyses investigated TTD and objective response rate (ORR) for entrectinib and prior therapies. RESULTS: Seventy-one patients were included; 51 received prior systemic therapy. In 38 patients who progressed on prior therapy, ORR was 60.5% (23/38) with entrectinib and 15.8% (6/38) with the most recent prior therapy. Median PFS [11.2 months; 95% confidence interval (CI) 6.7-not estimable] for entrectinib exceeded median TTD (2.9 months; 95% CI 2.0-4.9) for most recent prior therapy. From the intrapatient analysis of GMI, 65.8% had a ratio ≥1.3 and median GMI was 2.53. Consistent results were observed at more stringent GMI thresholds; 60.5% of patients had GMI ≥1.5 or ≥1.8 and 57.9% had GMI ≥2.0. CONCLUSIONS: ORR was high and PFS was longer on entrectinib versus TTD on prior therapy. Furthermore, 65.8% of patients experienced clinically meaningful benefit based on GMI. This intrapatient analysis demonstrates comparative effectiveness of entrectinib in a rare, heterogeneous adult population.

Biomarker-guided trials: Challenges in practice.

Biomarker-guided trials have drawn considerable attention as they promise to lead to improvements in the benefit-risk ratio of treatments and enhanced opportunities for drug development. A variety of such designs have been proposed in the literature, many of which have been adopted in practice. Implementing such trial designs in practice can be challenging, and identifying those challenges was the main objective of a workshop organised by the MRC Hubs for Trials Methodology Research Network's Stratified Medicine Working Group in March 2017. Participants reflected on completed and ongoing biomarker-guided trials to identify the practical challenges encountered. Here, the key challenges identified during the workshop including those related to funding, ethical and regulatory issues, recruitment, monitoring of samples and laboratories, biomarker assessment, and data sharing and resources, are discussed. Despite the complexities often associated with biomarker-guided trials, the workshop concluded that they can play an important role in advancing the field of personalised medicine. Therefore, it is important that the practical challenges surrounding their implementation are acknowledged and addressed.

Human Click-Based Echolocation of Distance: Superfine Acuity and Dynamic Clicking Behaviour.

Some people who are blind have trained themselves in echolocation using mouth clicks. Here, we provide the first report of psychophysical and clicking data during echolocation of distance from a group of 8 blind people with experience in mouth click-based echolocation (daily use for > 3 years). We found that experienced echolocators can detect changes in distance of 3 cm at a reference distance of 50 cm, and a change of 7 cm at a reference distance of 150 cm, regardless of object size (i.e. 28.5 cm vs. 80 cm diameter disk). Participants made mouth clicks that were more intense and they made more clicks for weaker reflectors (i.e. same object at farther distance, or smaller object at same distance), but number and intensity of clicks were adjusted independently from one another. The acuity we found is better than previous estimates based on samples of sighted participants without experience in echolocation or individual experienced participants (i.e. single blind echolocators tested) and highlights adaptation of the perceptual system in blind human echolocators. Further, the dynamic adaptive clicking behaviour we observed suggests that number and intensity of emissions serve separate functions to increase SNR. The data may serve as an inspiration for low-cost (i.e. non-array based) artificial 'cognitive' sonar and radar systems, i.e. signal design, adaptive pulse repetition rate and intensity. It will also be useful for instruction and guidance for new users of echolocation.

Enhancing photocatalytic degradation of the cyanotoxin microcystin-LR with the addition of sulfate-radical generating oxidants.

This study investigated the coupling of sulfate radical generating oxidants, (persulfate, PS and peroxymonosulfate, PMS) with TiO2 photocatalysis for the degradation of microcystin-LR (MC-LR). Treatment efficiency was evaluated by estimating the electrical energy per order (EEO). Oxidant addition at 4 mg/L reduced the energy requirements of the treatment by 60% and 12% for PMS and PS, respectively compared with conventional photocatalysis. Quenching studies indicated that both sulfate and hydroxyl radicals contributed towards the degradation of MC-LR for both oxidants, while Electron Paramagnetic Resonance (EPR) studies confirmed that the oxidants prolonged that lifetime of both radicals (concentration maxima shifted from 10 to 20 min), allowing for bulk diffusion and enhancing cyanotoxin removal. Structural identification of transformation products (TPs) formed during all treatments, indicated that early stage degradation of MC-LR occurred mainly on the aromatic ring and conjugated carbon double bonds of the ADDA amino acid. In addition, simultaneous hydroxyl substitution of the aromatic ring and the conjugated double carbon bonds of ADDA (m/z = 1027.5) are reported for the first time. Oxidant addition also increased the rates of formation/degradation of TPs and affected the overall toxicity of the treated samples. The detoxification and degradation order of the treatments was UVA/TiO2/PMS > UVA/TiO2/PS>> UVA/TiO2.

Human echolocators adjust loudness and number of clicks for detection of reflectors at various azimuth angles.

In bats it has been shown that they adjust their emissions to situational demands. Here we report similar findings for human echolocation. We asked eight blind expert echolocators to detect reflectors positioned at various azimuth angles. The same 17.5 cm diameter circular reflector placed at 100 cm distance at 0°, 45° or 90° with respect to straight ahead was detected with 100% accuracy, but performance dropped to approximately 80% when it was placed at 135° (i.e. somewhat behind) and to chance levels (50%) when placed at 180° (i.e. right behind). This can be explained based on poorer target ensonification owing to the beam pattern of human mouth clicks. Importantly, analyses of sound recordings show that echolocators increased loudness and numbers of clicks for reflectors at farther angles. Echolocators were able to reliably detect reflectors when level differences between echo and emission were as low as -27 dB, which is much lower than expected based on previous work. Increasing intensity and numbers of clicks improves signal-to-noise ratio and in this way compensates for weaker target reflections. Our results are, to our knowledge, the first to show that human echolocation experts adjust their emissions to improve sensory sampling. An implication from our findings is that human echolocators accumulate information from multiple samples.

Measurement of lentiviral vector titre and copy number by cross-species duplex quantitative PCR.

Lentiviruses are the vectors of choice for many preclinical studies and clinical applications of gene therapy. Accurate measurement of biological vector titre before treatment is a prerequisite for vector dosing, and the calculation of vector integration sites per cell after treatment is as critical to the characterisation of modified cell products as it is to long-term follow-up and the assessment of risk and therapeutic efficiency in patients. These analyses are typically based on quantitative real-time PCR (qPCR), but as yet compromise accuracy and comparability between laboratories and experimental systems, the former by using separate simplex reactions for the detection of endogene and lentiviral sequences and the latter by designing different PCR assays for analyses in human cells and animal disease models. In this study, we validate in human and murine cells a qPCR system for the single-tube assessment of lentiviral vector copy numbers that is suitable for analyses in at least 33 different mammalian species, including human and other primates, mouse, pig, cat and domestic ruminants. The established assay combines the accuracy of single-tube quantitation by duplex qPCR with the convenience of one-off assay optimisation for cross-species analyses and with the direct comparability of lentiviral transduction efficiencies in different species.

Sampling strategies for phlebotomine sand flies (Diptera: Psychodidae) in Europe.

The distribution of phlebotomine sand flies is widely reported to be changing in Europe. This can be attributed to either the discovery of sand flies in areas where they were previously overlooked (generally following an outbreak of leishmaniasis or other sand fly-related disease) or to true expansion of their range as a result of climatic or environmental changes. Routine surveillance for phlebotomines in Europe is localized, and often one of the challenges for entomologists working in non-leishmaniasis endemic countries is the lack of knowledge on how to conduct, plan and execute sampling for phlebotomines, or how to adapt on-going sampling strategies for other haematophagous diptera. This review brings together published and unpublished expert knowledge on sampling strategies for European phlebotomines of public health concern in order to provide practical advice on: how to conduct surveys; the collection and interpretation of field data; suitable techniques for the preservation of specimens obtained by different sampling methods; molecular techniques used for species identification; and the pathogens associated with sand flies and their detection methods.

Systemic gene transfer of binding immunoglobulin protein (BiP) prevents disease progression in murine collagen-induced arthritis.

Summary Recombinant human binding immunoglobulin protein (BiP) has previously demonstrated anti-inflammatory properties in multiple models of inflammatory arthritis. We investigated whether these immunoregulatory properties could be exploited using gene therapy techniques. A single intraperitoneal injection of lentiviral vector containing the murine BiP (Lenti-mBiP) or green fluorescent protein (Lenti-GFP) transgene was administered in low- or high-dose studies during early arthritis. Disease activity was assessed by visual scoring, histology, serum cytokine and antibody production measured by cell enzyme-linked immunosorbent assay (ELISA) and ELISA, respectively. Lentiviral vector treatment caused significant induction of interferon (IFN)-γ responses regardless of the transgene; however, further specific effects were directly attributable to the BiP transgene. In both studies Lenti-mBiP suppressed clinical arthritis significantly. Histological examination showed that low-dose Lenti-mBiP suppressed inflammatory cell infiltration, cartilage destruction and significantly reduced pathogenic anti-type II collagen (CII) antibodies. Lenti-mBiP treatment caused significant up-regulation of soluble cytotoxic T lymphocyte antigen-4 (sCTLA-4) serum levels and down-regulation of interleukin (IL)-17A production in response to CII cell restimulation. In-vitro studies confirmed that Lenti-mBiP spleen cells could significantly suppress the release of IL-17A from CII primed responder cells following CII restimulation in vitro, and this suppression was associated with increased IL-10 production. Neutralization of CTLA-4 in further co-culture experiments demonstrated inverse regulation of IL-17A production. In conclusion, these data demonstrate proof of principle for the therapeutic potential of systemic lentiviral vector delivery of the BiP transgene leading to immunoregulation of arthritis by induction of soluble CTLA-4 and suppression of IL-17A production.

The role of indigenous phlebotomine sandflies and mammals in the spreading of leishmaniasis agents in the Mediterranean region.

An updated view of the establishment and spread of the leishmaniases in Europe is presented, mostly with respect to newly emerging and re-emerging foci and the incrimination of neglected as well as new reservoir hosts. At the same time, a concept of specific versus permissive vectors reassesses the potential role of various sandfly species in Leishmania transmission and considers the risk of introduction of exotic Leishmania species in Europe. The leishmaniases are dynamic diseases and the circumstances of transmission are continually changing in relation to environmental, demographic and human behavioural factors. Changes in the habitat of the natural hosts and vectors, immunosuppressive conditions (like infection with human immunodeficiency virus (HIV) or organ transplantation-associated therapies in humans) and the consequences of war, all contribute to the transformation of the epidemiology of leishmaniasis. Such changes should be considered when studying the spread of the disease throughout Europe for targeted control measures to safeguard public health.

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter.

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site.

Therapeutic pneumoperitoneum in a patient with pulmonary tuberculosis and persistent fever.

Artificial pneumoperitoneum represents a therapeutic technique first applied in the treatment of pulmonary tuberculosis (TB) in prechemotherapy antimycobacterial era. A 25-year-old patient presented with pulmonary TB diagnosed during the 8th month of her pregnancy. She was febrile and in severe clinical condition. An antituberculous regimen of four primary drugs was initiated immediately after the caesarean section. There was no clinical improvement after 3 months despite full drug sensitivity of Mycobacterium tuberculosis isolates. An artificial pneumoperitoneum was applied along with the drug treatment for 6 months. Soon the patient became afebrile, her body weight increased and sputum smears gave negative results. The combination of the old technique of therapeutic pneumoperitoneum along with the current antituberculosis treatment proved to be effective in this advanced case of pulmonary TB initially unresponsive to drug therapy alone.

Leishmania donovani s.l.: evaluation of the proliferation potential of promastigotes using CFSE staining and flow cytometry.

Leishmania infantum causes visceral leishmaniasis in all countries in the Mediterranean basin. It uses Phlebotomine sandflies as vectors where the promastigote stage develops, reproduces and becomes infective. Therefore the reproductive power of the promastigotes determines the inoculum size of the isolate. Ten Leishmania strains from Cyprus: two Leishmania donovani and eight L. infantum were used to study the proliferation capacity of the promastigotes. Population increase during a 6-day culture period was assessed quantitatively, by haematocytometer enumeration, and qualitatively by following the division history of each population during the same period by CFSE staining and flow cytometry. The strains exhibited different proliferation rates with L. infantum showing higher multiplication rates than L. donovani. These differences may represent their fitness capabilities and their ability to synchronize the multiplication activity of individual members in the population for the production of a sizeable inoculum in time for the vector's blood meal.

Toxoplasmosis in pregnant women in Crete.

In Greece there are no official recommendations concerning the management of pregnant women for the prevention of congenital toxoplasmosis. A protocol for monitoring pregnant women was designed in order to differentiate between acute and latent toxoplasmosis and was tested successfully for 7 years. The maternofetal transmission rate in Crete was assessed and a map showing seroprevalence of pregnant women in all prefectures of Greece was prepared. The high seroprevalence of Toxoplasma gondii in Greece (up to 46% in some areas) may be explained by: (a) the presence of a great number of stray cats; (b) the Greek diet consisting of large amounts of raw, wild vegetables and salads that could easily be contaminated with oocysts; (c) the high consumption of meat, smoked pork and sausages, well-documented sources of T. gondii infection. T. gondii genotypes were characterized, directly from clinical samples, after PCR-RFLP on the SAG2 gene and sequence analysis at the restriction sites. They belonged to all 3 clonal lineages.

Evolving radiological features of hypothalamo-pituitary lesions in adult patients with Langerhans cell histiocytosis (LCH).

Langerhans cell histiocytosis (LCH) is a rare, systemic disease caused by monoclonal expansion of dendritic cells that shows a particular predilection for the hypothalamic-pituitary system (HPS). We studied the function (anterior and posterior pituitary hormonal secretion) and morphology using magnetic resonance imaging (MRI) of the HPS in 17 adult patients (seven males, median age 35 years, range 18-59 years) with multisystem LCH. We also evaluated the evolution of structural HPS abnormalities in relation to pituitary function and response to treatment in 12 of these patients during a median follow-up period of 3.75 years (range 1.5-10 years). Of the 17 patients, 14 (82%) had abnormal HPS imaging, and 12 (70%) had more than one area involved. Lack of the bright spot of the posterior pituitary lobe was typically found in all patients with the diagnosis of diabetes insipidus (DI). Eight patients (47%) had infundibular enlargement, six (35%) pituitary infiltration, four (24%) partially or completely empty sella, three (18%) hypothalamic involvement, and two (12%) infundibular atrophy. DI was found in 16 patients (94%) and anterior pituitary hormonal deficiency (APHD) in 10 patients (59%); two patients had single (12%) and 8 (47%) multiple APHD. During the follow-up period there was improvement of the initially demonstrated HPS pathology in seven (47%) patients, and five (33%) of them had received at least one form of treatment. APHD and DI persisted in all patients except in one in whom established gonadotrophin deficiency recovered. In summary, DI and APHD are very common in patients with multisystem LCH and are almost always associated with abnormal HPS imaging.

Binge eating disorder versus overeating: a failure to replicate and common factors in severely obese treatment seeking women.

The disordered eating symptoms, general psychopathology and dieting history among obese women diagnosed with Binge Eating Disorder (BED) and obese women who overeat (OE) are examined. One hundred and thirty women (n=83 with BED and n=47 who overeat) seeking treatment for an eating disorder were diagnosed using the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV) criteria and the Eating Disorders Examination (EDE). They also completed a battery of psychometric tests. Despite adequate statistical power to detect differences, MANOVAs revealed very few significant differences between the groups. Loss of control of eating does not adequately differentiate these two groups within an eating disorders treatment-seeking context. It is likely that only the most acutely distressed from each group is seeking treatment, so that differences found in a community sample would not be found. Using a principal components analysis, factors within each group that may underlie the common psychopathology of both groups (i.e. disordered eating symptoms, general psychopathology and dieting history) were found.

LCR-mediated, long-term tissue-specific gene expression within replicating episomal plasmid and cosmid vectors.

Locus control regions (LCRs) are transcriptional regulatory elements, which possess a dominant chromatin remodelling and transcriptional activating capability conferring full physiological levels of expression on a gene linked in cis, when integrated into the host cell genome. Using the human beta-globin LCR (betaLCR) as a model, we show that this class of control element can drive high levels of tissue-specific gene expression in stably transfected cultured cells from within an Epstein-Barr virus-based plasmid REV. Furthermore, a 38-kb betaLCR minilocus-REV cosmid vector was efficiently retained and maintained therapeutic levels of beta-globin transgene expression in the absence of drug selective pressure over a 2-month period of continuous culture equivalent to at least 60 generations. This demonstrates for the first time the feasibility of using REVs for gene therapy of the haemoglobinopathies. Importantly, our results demonstrate that as in the case of integrated transgenes, expression from within REVs is prone to silencing but that the inclusion of the betaLCR prevented this repression of gene function. Therefore, appropriate control elements to provide and maintain tissue-specific gene expression, as well as the episomal status of REVs is a crucial feature in vector design. Our data suggest that LCRs can contribute to this vital function.