Search

Lung cancer (LC) is a leading cause of cancer-related mortality worldwide. Lung Cancer Screening (LCS) programs that use low-dose computed tomography (LDCT) have been shown to reduce LC mortality by up to 25 % and are considered cost-effective. The European Health Union has encouraged its Member States to explore the feasibility of LCS implementation in their respective countries. The task force conducted a comprehensive literature review and engaged in extensive discussions to provide recommendations. These recommendations encompass the essential components required to initiate pilot LCS programs following the guidelines established by the World Health Organization. They were tailored to align with the specific context of the Portuguese healthcare system. The document addresses critical aspects, including the eligible population, methods for issuing invitations, radiological prerequisites, procedures for reporting results, referral processes, diagnostic strategies, program implementation, and ongoing monitoring. Furthermore, the task force emphasized that pairing LCS with evidence-based smoking cessation should be the standard of care for a high-quality screening program. This document also identifies areas for further research. These recommendations aim to guarantee that the implementation of a Portuguese LCS program ensures high-quality standards, consistency, and uniformity across centres., Competing Interests: Declaration of competing interest The authors have no conflicts of interest., (Copyright © 2024 Sociedade Portuguesa de Pneumologia. Published by Elsevier España, S.L.U. All rights reserved.)

Physiologically relevant human models that recapitulate the challenges of solid tumors and the tumor microenvironment (TME) are highly desired in the chimeric antigen receptor (CAR)-T cell field. We developed a breast cancer-on-chip model with an integrated endothelial barrier that enables the transmigration of perfused immune cells, their infiltration into the tumor, and concomitant monitoring of cytokine release during perfused culture over a period of up to 8 days. Here, we exemplified its use for investigating CAR-T cell efficacy and the ability to control the immune reaction with a pharmacological on/off switch. Additionally, we integrated primary breast cancer organoids to study patient-specific CAR-T cell efficacy. The modular architecture of our tumor-on-chip paves the way for studying the role of other cell types in the TME and thus provides the potential for broad application in bench-to-bedside translation as well as acceleration of the preclinical development of CAR-T cell products., Competing Interests: Declaration of interests M.H. is an inventor on patent applications and has been granted patents related to CAR technology, licensed in part to industry. M.H. is a cofounder and equity owner of T-CURX GmbH, Würzburg. M.H. receives speaker honoraria from BMS, Janssen, Kite/Gilead, and Novartis and research support from BMS., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

As the world's population grows older, vaccination is becoming a key strategy for promoting healthy aging. Despite scientific progress in adult vaccine development, obstacles such as immunosenescence and vaccine hesitancy remain. To unlock the potential of adult vaccines fully, we must enhance immunization programs, dispel misinformation, and invest in research that deepens our understanding of aging and immunity.

Research on the interaction between object and scene processing has a long history in the fields of perception and visual memory. Most databases have established norms for pictures where the object is embedded in the scene. In this study, we provide a diverse and controlled stimulus set comprising real-world pictures of 375 objects (e.g., suitcase), 245 scenes (e.g., airport), and 898 object-scene pairs (e.g., suitcase-airport), with object and scene presented separately. Our goal was twofold. First, to create a database of object and scene pictures, normed for the same variables to have comparable measures for both types of pictures. Second, to acquire normative data for the semantic relationships between objects and scenes presented separately, which offers more flexibility in the use of the pictures and allows disentangling the processing of the object and its context (the scene). Along three experiments, participants evaluated each object or scene picture on name agreement, familiarity, and visual complexity, and rated object-scene pairs on semantic congruency. A total of 125 septuplets of one scene and six objects (three congruent, three incongruent), and 120 triplets of one object and two scenes (in congruent and incongruent pairings) were built. In future studies, these objects and scenes can be used separately or combined, while controlling for their key features. Additionally, as object-scene pairs received semantic congruency ratings along the entire scale, researchers may select among a wide range of congruency values. ObScene is a comprehensive and ecologically valid database, useful for psychology and neuroscience studies of visual object and scene processing., (© 2023. The Author(s).)

Pancreatic in vitro research is of major importance to advance mechanistic understanding and development of treatment options for diseases such as diabetes mellitus. We present a thermoplastic-based microphysiological system aiming to model the complex microphysiological structure and function of the endocrine pancreas with concurrent real-time read-out capabilities. The specifically tailored platform enables self-guided trapping of single islets at defined locations: β-cells are assembled to pseudo-islets and injected into the tissue chamber using hydrostatic pressure-driven flow. The pseudo-islets can further be embedded in an ECM-like hydrogel mimicking the native microenvironment of pancreatic islets in vivo . Non-invasive real-time monitoring of the oxygen levels on-chip is realized by the integration of luminescence-based optical sensors to the platform. To monitor insulin secretion kinetics in response to glucose stimulation in a time-resolved manner, an automated cycling of different glucose conditions is implemented. The model's response to glucose stimulation can be monitored via offline analysis of insulin secretion and via specific changes in oxygen consumption due to higher metabolic activity of pseudo-islets at high glucose levels. To demonstrate applicability for drug testing, the effects of antidiabetic medications are assessed and changes in dynamic insulin secretion are observed in line with the respective mechanism of action. Finally, by integrating human pancreatic islet microtissues, we highlight the flexibility of the platform and demonstrate the preservation of long-term functionality of human endocrine pancreatic tissue.

The study of action observation and imagery, separately and combined, is expanding in diverse research areas (e.g., sports psychology, neurosciences), making clear the need for action-related stimuli (i.e., action statements, videos, and pictures). Although several databases of object and action pictures are available, norms on action videos are scarce. In this study, we validated a set of 60 object-related everyday actions in three different formats: action-statements, and corresponding dynamic (action videos) and static (object photos) stimuli. In Study 1, ratings of imageability, image agreement, action familiarity, action frequency, and action valence were collected from 161 participants. In Study 2, a different sample of 115 participants rated object familiarity, object valence, and object-action prototypicality. Most actions were rated as easy to imagine, familiar, and neutral or positive in valence. However, there was variation in the frequency with which participants perform these actions on a daily basis. High agreement between participants' mental image and action videos was also found, showing that the videos depict a conventional way of performing the actions. Objects were considered familiar and positive in valence. High ratings on object-action prototypicality indicate that the actions correspond to prototypical actions for most objects. 3ActStimuli is a comprehensive set of stimuli that can be useful in several research areas, allowing the combined study of action observation and imagery., (© 2022. The Psychonomic Society, Inc.)

Lactoferrin is a multifunctional protein that exhibits anti-inflammatory, immune regulating and anti-infective properties. One of its receptor sites is located on severe acute respiratory syndrome coronavirus 2. The binding of lactoferrin with heparin sulfate proteoglycans may prevent the first contact between the virus and host cells, thus preventing subsequent infection. Given that lactoferrin may act as a natural mucosal barrier, an intranasal treatment together with its oral intake can be hypothesized to prevent the spread, infection and inflammation caused by coronavirus disease 2019 (COVID-19). Moreover, the literature reports that vitamin D plays an essential role in promoting immune response. With its anti-inflammatory and immunoregulatory properties, vitamin D is critical for activating the immune system's defenses, improving immune cell function. Different studies also demonstrate that lactoferrin is a potential activator of the vitamin D receptor. In this sense, the combined use of lactoferrin (through an association of oral intake and a nasal spray formulation) and vitamin D could represent a valuable therapy for COVID-19 treatment and prevention. However, further randomized clinical trials are needed before recommending/prescribing them., Competing Interests: Conflict-of-interest statement: All authors declare no conflicts of interests., (©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.)

NEU-4438 is a lead for the development of drugs against Trypanosoma brucei , which causes human African trypanosomiasis. Optimized with phenotypic screening, targets of NEU-4438 are unknown. Herein, we present a cell perturbome workflow that compares NEU-4438's molecular modes of action to those of SCYX-7158 (acoziborole). Following a 6 h perturbation of trypanosomes, NEU-4438 and acoziborole reduced steady-state amounts of 68 and 92 unique proteins, respectively. After analysis of proteomes, hypotheses formulated for modes of action were tested: Acoziborole and NEU-4438 have different modes of action. Whereas NEU-4438 prevented DNA biosynthesis and basal body maturation, acoziborole destabilized CPSF3 and other proteins, inhibited polypeptide translation, and reduced endocytosis of haptoglobin-hemoglobin. These data point to CPSF3-independent modes of action for acoziborole. In case of polypharmacology, the cell-perturbome workflow elucidates modes of action because it is target-agnostic. Finally, the workflow can be used in any cell that is amenable to proteomic and molecular biology experiments., Competing Interests: The authors declare no competing interests., (© 2022 The Author(s).)

Obesity and associated diseases, such as diabetes, have reached epidemic proportions globally. In this era of "diabesity", white adipose tissue (WAT) has become a target of high interest for therapeutic strategies. To gain insights into mechanisms of adipose (patho-)physiology, researchers traditionally relied on animal models. Leveraging Organ-on-Chip technology, a microphysiological in vitro model of human WAT is introduced: a tailored microfluidic platform featuring vasculature-like perfusion that integrates 3D tissues comprising all major WAT-associated cellular components (mature adipocytes, organotypic endothelial barriers, stromovascular cells including adipose tissue macrophages) in an autologous manner and recapitulates pivotal WAT functions, such as energy storage and mobilization as well as endocrine and immunomodulatory activities. A precisely controllable bottom-up approach enables the generation of a multitude of replicates per donor circumventing inter-donor variability issues and paving the way for personalized medicine. Moreover, it allows to adjust the model's degree of complexity via a flexible mix-and-match approach. This WAT-on-Chip system constitutes the first human-based, autologous, and immunocompetent in vitro adipose tissue model that recapitulates almost full tissue heterogeneity and can become a powerful tool for human-relevant research in the field of metabolism and its associated diseases as well as for compound testing and personalized- and precision medicine applications., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Non-clinical models to study metabolism including animal models and cell assays are often limited in terms of species translatability and predictability of human biology. This field urgently requires a push towards more physiologically accurate recapitulations of drug interactions and disease progression in the body. Organ-on-chip systems, specifically multi-organ chips (MOCs), are an emerging technology that is well suited to providing a species-specific platform to study the various types of metabolism (glucose, lipid, protein and drug) by recreating organ-level function. This review provides a resource for scientists aiming to study human metabolism by providing an overview of MOCs recapitulating aspects of metabolism, by addressing the technical aspects of MOC development and by providing guidelines for correlation with in silico models. The current state and challenges are presented for two application areas: (i) disease modelling and (ii) pharmacokinetics/pharmacodynamics. Additionally, the guidelines to integrate the MOC data into in silico models could strengthen the predictive power of the technology. Finally, the translational aspects of metabolizing MOCs are addressed, including adoption for personalized medicine and prospects for the clinic. Predictive MOCs could enable a significantly reduced dependence on animal models and open doors towards economical non-clinical testing and understanding of disease mechanisms.

Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies., (© 2022. The Author(s).)

Gene therapies using adeno-associated viruses (AAVs) are among the most promising strategies to treat or even cure hereditary and acquired retinal diseases. However, the development of new efficient AAV vectors is slow and costly, largely because of the lack of suitable non-clinical models. By faithfully recreating structure and function of human tissues, human induced pluripotent stem cell (iPSC)-derived retinal organoids could become an essential part of the test cascade addressing translational aspects. Organ-on-chip (OoC) technology further provides the capability to recapitulate microphysiological tissue environments as well as a precise control over structural and temporal parameters. By employing our recently developed retina on chip that merges organoid and OoC technology, we analyzed the efficacy, kinetics, and cell tropism of seven first- and second-generation AAV vectors. The presented data demonstrate the potential of iPSC-based OoC models as the next generation of screening platforms for future gene therapeutic studies., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

African trypanosomes utilize glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) to evade the host immune system. VSG turnover is thought to be mediated via cleavage of the GPI anchor by endogenous GPI-specific phospholipase C (GPI-PLC). However, GPI-PLC is topologically sequestered from VSG substrates in intact cells. Recently, A. J. Szempruch, S. E. Sykes, R. Kieft, L. Dennison, et al. (Cell 164:246-257, 2016, https://doi.org/10.1016/j.cell.2015.11.051) demonstrated the release of nanotubes that septate to form free VSG + extracellular vesicles (EVs). Here, we evaluated the relative contributions of GPI hydrolysis and EV formation to VSG turnover in wild-type (WT) and GPI-PLC null cells. The turnover rate of VSG was consistent with prior measurements (half-life [ t 1/2 ] of ∼26 h) but dropped significantly in the absence of GPI-PLC ( t 1/2 of ∼36 h). Ectopic complementation restored normal turnover rates, confirming the role of GPI-PLC in turnover. However, physical characterization of shed VSG in WT cells indicated that at least 50% is released directly from cell membranes with intact GPI anchors. Shedding of EVs plays an insignificant role in total VSG turnover in both WT and null cells. In additional studies, GPI-PLC was found to have no role in biosynthetic and endocytic trafficking to the lysosome but did influence the rate of receptor-mediated endocytosis. These results indicate that VSG turnover is a bimodal process involving both direct shedding and GPI hydrolysis. IMPORTANCE African trypanosomes, the protozoan agent of human African trypanosomaisis, avoid the host immune system by switching expression of the variant surface glycoprotein (VSG). VSG is a long-lived protein that has long been thought to be turned over by hydrolysis of its glycolipid membrane anchor. Recent work demonstrating the shedding of VSG-containing extracellular vesicles has led us to reinvestigate the mode of VSG turnover. We found that VSG is shed in part by glycolipid hydrolysis but also in approximately equal part by direct shedding of protein with intact lipid anchors. Shedding of exocytic vesicles made a very minor contribution to overall VSG turnover. These results indicate that VSG turnover is a bimodal process and significantly alter our understanding of the "life cycle" of this critical virulence factor.

The advances in cancer immunotherapy come with several obstacles, limiting its widespread use and benefits so far only to a small subset of patients. One of the underlying challenges remains to be the lack of representative nonclinical models that translate to human immunity and are able to predict clinical efficacy and safety outcomes. In recent years, immunocompetent Cancer-on-Chip models emerge as an alternative human-based platform that enables the integration and manipulation of complex tumor microenvironment. In this review, we discuss novel opportunities offered by Cancer-on-Chip models to advance (mechanistic) immuno-oncology research, ranging from design flexibility to multimodal analysis approaches. We then exemplify their (potential) applications for the research and development of adoptive cell therapy, immune checkpoint therapy, cytokine therapy, oncolytic virus, and cancer vaccines., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cecile Zaupe is employee of Transgene S. A. Michael Hudecek is an inventor on granted patents and patent applications relating to CAR-T cell therapy. Birgit Fogal is an employee of Boehringer Ingelheim Pharmaceuticals, Inc., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

The most frequent form of dementia is Alzheimer's Disease (AD), a severe progressive neurological pathology in which the main cognitive functions of an individual are compromised. Recent studies have found that loneliness and living in isolation are likely to cause an acceleration in the cognitive decline associated with AD. Therefore, understanding social behaviours of AD patients is crucial to promote sociability, thus delaying cognitive decline, preserving independence, and providing a good quality of life. In this work, we analyze the localization data of AD patients living in assisted care homes to gather insights about the social dynamics among them. We use localization data collected by a system based on iBeacon technology comprising two components: a network of antennas scattered throughout the facility and a Bluetooth bracelet worn by the patients. We redefine the Relational Index to capture wandering and casual encounters, these being common phenomena among AD patients, and use the notions of Relational and Popularity Indexes to model, visualize and understand the social behaviour of AD patients. We leverage the data analyses to build predictive tools and applications to enhance social activities scheduling and sociability monitoring and promotion, with the ultimate aim of providing patients with a better quality of life. Predictions and visualizations act as a support for caregivers in activity planning to maximize treatment effects and, hence, slow down the progression of Alzheimer's disease. We present the Community Behaviour Prediction Table (CBPT), a tool to visualize the estimated values of sociability among patients and popularity of places within a facility. Finally, we show the potential of the system by analyzing the Coronavirus Disease 2019 (COVID-19) lockdown time-frame between February and June 2020 in a specific facility. Through the use of the indexes, we evaluate the effects of the pandemic on the behaviour of the residents, observing no particular impact on sociability even though social distancing was put in place.

The poor predictability of human liver toxicity is still causing high attrition rates of drug candidates in the pharmaceutical industry at the non-clinical, clinical, and post-marketing authorization stages. This is in part caused by animal models that fail to predict various human adverse drug reactions (ADRs), resulting in undetected hepatotoxicity at the non-clinical phase of drug development. In an effort to increase the prediction of human hepatotoxicity, different approaches to enhance the physiological relevance of hepatic in vitro systems are being pursued. Three-dimensional (3D) or microfluidic technologies allow to better recapitulate hepatocyte organization and cell-matrix contacts, to include additional cell types, to incorporate fluid flow and to create gradients of oxygen and nutrients, which have led to improved differentiated cell phenotype and functionality. This comprehensive review addresses the drug-induced hepatotoxicity mechanisms and the currently available 3D liver in vitro models, their characteristics, as well as their advantages and limitations for human hepatotoxicity assessment. In addition, since toxic responses are greatly dependent on the culture model, a comparative analysis of the toxicity studies performed using two-dimensional (2D) and 3D in vitro strategies with recognized hepatotoxic compounds, such as paracetamol, diclofenac, and troglitazone is performed, further highlighting the need for harmonization of the respective characterization methods. Finally, taking a step forward, we propose a roadmap for the assessment of drugs hepatotoxicity based on fully characterized fit-for-purpose in vitro models, taking advantage of the best of each model, which will ultimately contribute to more informed decision-making in the drug development and risk assessment fields., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Serras, Rodrigues, Cipriano, Rodrigues, Oliveira and Miranda.)

Background: -We aimed to identify the knowledge producers, the knowledge that they produce and the main areas of interest for health services research (HSR) in France, which is one of the priorities for public health research planning., Methods: -A two-phase approach comprising a bibliometric analysis and semi-structured interviews with 20 researchers and 13 staff members of public health bodies was used., Results: -In France today, interdisciplinary research teams are being organized in the main cities despite a lack of critical mass. The interviews showed that the term 'health services research' is vague with fuzzy boundaries between research, scientific expertise and evaluation. A keyword cluster analysis showed that French HSR is more hospital-centric compared to those countries that publish most frequently. The analysis also revealed a lack of research on methodological aspects., Conclusions: -We analyzed the structure of HSR in France and the specificity of the French publications in this field, compared with other countries. The results indicate the obstacles faced by researchers and decision-makers and offer insight into how these barriers may be removed., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Public Health Association. All rights reserved.)

Animal models are frequently used in drug discovery because they represent a mammalian in vivo model system, they are the closest approximation to the human brain, and experimentation in humans is not ethical. Working with postmortem human brain samples is challenging and developing human in vitro systems, which mimic the in vivo human brain, has been challenging. However, the use of animal models in drug discovery for human neurological diseases is currently under scrutiny because data from animal models has come with variations due to genetic differences. Evidence from the literature suggests that techniques to reconstruct multiple neurotransmission projections, which characterize neurological disease circuits in humans, in vitro, have not been demonstrated. This paper presents a multicompartment microdevice for patterning neurospheres and specification of neural stem cell fate toward networks of multiple neuronal phenotypes. We validated our design by specification of human neural stem cells to dopaminergic and GABAergic neurons in different compartments of the device, simultaneously. The neurospheres formed unrestricted robust neuronal circuits between arrays of neurospheres in all compartments of the device. Such a device design may provide a basis for formation of multineurotransmission circuits to model functional connectivity between specific human brain regions, in vitro, using human-derived neural stem cells. This work finds relevance in neurological disease modeling and drug screening using human cell-based assays and may provide the impetus for shifting from animal-based models.

The need for competent in vitro liver models for toxicological assessment persists. The differentiation of stem cells into hepatocyte-like cells (HLC) has been adopted due to its human origin and availability. Our aim was to study the usefulness of an in vitro 3D model of mesenchymal stem cell-derived HLCs. 3D spheroids (3D-HLC) or monolayer (2D-HLC) cultures of HLCs were treated with the hepatotoxic drug nevirapine (NVP) for 3 and 10 days followed by analyses of Phase I and II metabolites, biotransformation enzymes and drug transporters involved in NVP disposition. To ascertain the toxic effects of NVP and its major metabolites, the changes in the glutathione net flux were also investigated. Phase I enzymes were induced in both systems yielding all known correspondent NVP metabolites. However, 3D-HLCs showed higher biocompetence in producing Phase II NVP metabolites and upregulating Phase II enzymes and MRP7 . Accordingly, NVP-exposure led to decreased glutathione availability and alterations in the intracellular dynamics disfavoring free reduced glutathione and glutathionylated protein pools. Overall, these results demonstrate the adequacy of the 3D-HLC model for studying the bioactivation/metabolism of NVP representing a further step to unveil toxicity mechanisms associated with glutathione net flux changes.

It's not news to tell you that the coronavirus, known as COVID-19, is a worldwide pandemic. The initial outbreak of this novel virus in Wuhan in the Hubei province of China, first described in December 2019, has since moved on to being declared a pandemic by the World Health Organization. The classic description of COVID-19 is a respiratory illness that manifests with fever, dry cough, and dyspnea on exertion. However, gastrointestinal (GI) complication of COVID-19 is emerging as well. This was observed with similar viral respiratory illnesses, such as severe acute respiratory syndrome (SARS), which emerged in 2003, and the Middle East respiratory syndrome (MERS), which emerged in 2012. In a recently published, single-center case series of 138 consecutive hospitalized patients with confirmed COVID-19, investigators reported that approximately 10% of patients initially presented with GI symptoms, prior to the subsequent development of respiratory symptoms. Common and often very subtle symptoms included diarrhea, nausea, and abdominal pain, with a less common symptom being nonspecific GI illness. New studies are expanding our understanding of the possible fecal transmission of COVID-19. Assessment by polymerase chain reaction (PCR) has provided evidence of the virus in the stool and the oropharynx outside the nasopharynx and respiratory tract. Virus in the stool may be evident on presentation and last throughout the course of illness resolution for up to 12 days after the respiratory virus evidence is gone. In fact, in one of the most recent studies looking at 73 patients, approximately 24% remained positive in their stool for evidence of the virus, though not necessarily infection, after showing negative in respiratory samples. The Centers for Disease Control and Prevention (CDC) recommends that after two negative respiratory tests separated by ≥ 24 hours, patients can be dismissed from having transmissibility infection risk for COVID-19. The potential for fecal-oral transmission of COVID-19 needs to be strongly considered. Considering these cases and the lessons from SARS, many authors recommend that real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from feces should be performed routinely in SARS-CoV-2 patients., Competing Interests: The authors have declared that no competing interests exist., (Copyright © 2020, Cipriano et al.)

Fatty Acid Amide Hydrolase (FAAH) is a serine hydrolase that plays a key role in controlling endogenous levels of endocannabinoids. FAAH inhibition is considered a powerful approach to enhance the endocannabinoid signalling, and therefore it has been largely studied as a potential target for the treatment of neurological disorders such as anxiety or depression, or of inflammatory processes. We present two novel series of amide derivatives of ibuprofen designed as analogues of our reference FAAH inhibitor Ibu-AM5 to further explore its structure-activity relationships. In the new amides, the 2-methylpyridine moiety of Ibu-AM5 was substituted by benzylamino and piperazinoaryl moieties. The obtained benzylamides and piperazinoarylamides showed FAAH inhibition ranging from the low to high micromolar potency. The binding of the new amides in the active site of FAAH, estimated using the induced fit protocol, indicated arylpiperazinoamides binding the ACB channel and the cytosolic port, and benzylamides binding the ACB channel.

Members of the Apicomplexa phylum, including Plasmodium and Toxoplasma, have two types of secretory organelles (micronemes and rhoptries) whose sequential release is essential for invasion and the intracellular lifestyle of these eukaryotes. During invasion, rhoptries inject an array of invasion and virulence factors into the cytoplasm of the host cell, but the molecular mechanism mediating rhoptry exocytosis is unknown. Here we identify a set of parasite specific proteins, termed rhoptry apical surface proteins (RASP) that cap the extremity of the rhoptry. Depletion of RASP2 results in loss of rhoptry secretion and completely blocks parasite invasion and therefore parasite proliferation in both Toxoplasma and Plasmodium. Recombinant RASP2 binds charged lipids and likely contributes to assembling the machinery that docks/primes the rhoptry to the plasma membrane prior to fusion. This study provides important mechanistic insight into a parasite specific exocytic pathway, essential for the establishment of infection.

The deep peribiliary glands (DPBG) are a niche of progenitor cells in the wall of the biliary duct (BD) and are the second line of multiplication when severe lesion of the epithelium occurs. Previous studies have identified DPBG injury as a cause of post-liver transplant (LT) biliary stenosis; this complication is a major cause of post-LT morbidity. The incidence of biliary stenosis in our center is high (38.1%). This study evaluates the lesion of DPBG in response to ischemia. Graft BD was collected in adult LT between August 2016-July 2017, from donation after brain death. Samples of 45 grafts were collected at 2 moments: BD1-during graft preparation and BD2-before biliary anastomosis. Histological analysis of the samples was performed and then classified according to degree of lesion (0, ≤50%, and >50%). A comparison was made between the degree of lesion and graft ischemia, graft histology, donor, and procurement variables. The DPBG lesion was more frequent in BD2 (20.9% vs 7%, P = .079). BD2 lesions with DPBG lesions had higher medians and means at all times of ischemia. The difference was greater in the warm ischemia time (0: 43.3 ± 12.53 minutes vs ≤50%: 52.4 ± 14.38 minutes, P = .068). The group of BD1 with DPBG lesion presented superior median cold ischemia time (CIT). In the analysis of the remaining variables there were also no statistically significant differences. We concluded that during the period of CIT there is already lesion of the DPBG, which increases after reperfusion of the graft, in greater association with longer warm ischemia time., (Copyright © 2019. Published by Elsevier Inc.)

Introduction: A condition of high short-term mortality in the cirrhotic patient is Acute-on-Chronic Liver Failure (ACLF), characterized by organ failure (s) and usually preceded by acute decompensation (AD)., Objective: To determine the frequency, clinical profile, and mortality in cirrhotic patients hospitalized with ACLF., Materials and Methods: This is an observational analytical study conducted from July 2016 to June 2017. We established the ACLF condition through the criteria of the CANONIC study., Results: The study population was 118 patients, of whom 34 (28.8%) presented ACLF, 14 (41%) were Grade 1, 16 (47%) Grade 2 and 4 (11.9%) Grade 3. The average age was 61.5 years old, alcoholism being the most frequent etiology with 18 patients (53%) and mostly without episodes of AD (64.7%). The most frequent precipitating factors were: Digestive hemorrhage (41%) and infections (29.4%). The groups with and without ACLF were statistically significant in the Child-Turcot-Pugh score (CTP) (11.4 ± 1.8 vs. 8.69 ± 2.04; p < 0.0001 ). MELD score (26.4 ± 8.1 vs. 14.4 ± 4.6; p < 0.0001), leukocytes (11,809.7 +/- 6,906.3 per mm3 vs. 8,434.01 ± 5,434.9 per mm3; p: 0.006) and 28-day mortality (76.5% vs. 21.4%, p < 0.0001), with a relative risk (RR) of 3.5., Conclusions: The frecuency of ACLF was 28.8%, similar to that of the CANONIC study (30.9%). The digestive hemorrhage being the main precipitating factor. The CTP, MELD and leukocyte scores were highest in this group. Mortality in patients with ACLF was 3.5 times more frequent than in patients without ACLF.

Background: The predictors proposed by the American Society of Gastrointestinal Endoscopy (ASGE) are commonly used topredict the presence and management of choledocholithiasis., Objective: To evaluate the performance and precision of thepredictors of choledocholithiasis proposed by ASGE., Materials and Methods: Prospective and longitudinal study performed ata third level hospital during January 2015 to June 2017. All patients with high and intermediate probability of choledocholithiasiswho underwent endoscopic retrograde cholangiopancreatography (ERCP) were included according to the criteria proposedby the ASGE., Results: A total of 246 patients with suspected choledocholithiasis were analyzed. Of the 228 patients withhigh probability criteria 144 (63.2% = performance) had choledocholithiasis in ERCP with an accuracy of 62% (sensitivity:94.1% and specificity: 9.7%). Among the 18 patients with intermediate probability criteria, 9 (50% = performance) hadcholedocholithiasis with an accuracy of 38% (sensitivity: 5.9% and specificity: 90.3%). In the multivariate analysis, the presenceof stone in the bile duct by ultrasonography (OR: 1.937, 95% CI 1.048-3.580, p=0.035) and age 55 and over (OR: 2.121, 95%CI, 1.101-4.088, p=0.025) were the strongest predictors for choledocholithiasis., Conclusions: The application of the criteriaof the ASGE to predict the probability of choledocholithiasis, in our population has a performance greater than 50%, however,it is necessary to improve these parameters to avoid an unnecessary performance of ERCP.

Fatty acid amide hydrolase (FAAH) is a promising target for the development of drugs to treat neurological diseases. In search of new FAAH inhibitors, we identified 2-(4-cyclohexylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4g, with an IC 50 of 2.6 µM as a chemical starting point for the development of potent FAAH inhibitors. Preliminary hit-to-lead optimisation resulted in 2-(4-phenylphenoxy)-N-(3-(oxazolo[4,5-b]pyridin-2-yl)phenyl)acetamide, 4i, with an IC 50 of 0.35 µM.

Fatty acid amide hydrolase (FAAH) has a key role in the control of the cannabinoid signaling, through the hydrolysis of the endocannabinoids anandamide and in some tissues 2-arachidonoylglycerol. FAAH inhibition represents a promising strategy to activate the cannabinoid system, since it does not result in the psychotropic and peripheral side effects characterizing the agonists of the cannabinoid receptors. Here we present the discovery of a novel class of profen derivatives, the N-(heteroaryl)-2-(4-((2-(trifluoromethyl)pyridin-4-yl)amino)phenyl)propanamides, as FAAH inhibitors. Enzymatic assays showed potencies toward FAAH ranging from nanomolar to micromolar range, and the most compounds lack activity toward the two isoforms of cyclooxygenase. Extensive structure-activity studies and the definition of the binding mode for the lead compound of the series are also presented. Kinetic assays in rat and mouse FAAH on selected compounds of the series demonstrated that slight modifications of the chemical structure could influence the binding mode and give rise to competitive (TPA1) or non-competitive (TPA14) inhibition modes., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Efavirenz is an anti-HIV drug that presents relevant short- and long-term central nervous system adverse reactions. Its main metabolite (8-hydroxy-efavirenz) was demonstrated to be a more potent neurotoxin than efavirenz itself. This work was aimed to understand how efavirenz biotransformation to 8-hydroxy-efavirenz is related to its short- and long-term neuro-adverse reactions. To access those mechanisms, the expression and activity of Cyp2b enzymes as well as the thiolomic signature (low molecular weight thiols plus S-thiolated proteins) were longitudinally evaluated in the hepatic and brain tissues of rats exposed to efavirenz during 10 and 36days. Efavirenz and 8-hydroxy-efavirenz plasma concentrations were monitored at the same time points. Cyp2b induction had a delayed onset in liver (p<0.001), translating into increases in Cyp2b activity in liver and 8-hydroxy-efavirenz plasma concentration (p<0.001). Moreover, an increase in S-cysteinyl-glycinylated proteins (p<0.001) and in free low molecular weight thiols was also observed in liver. A distinct scenario was observed in hippocampus, which showed an underexpression of Cyp2b as well as a decrease in S-cysteinylated and S-glutathionylated proteins. Additionally, the observed changes in tissues were associated with a marked increase of S-glutathionylation in plasma. Our data suggest that the time course of efavirenz biotransformation results from different mechanisms for its short- and long-term neurotoxicity. The difference in the redox profile between liver and hippocampus might explain why, despite being mostly metabolized by the liver, this drug is neurotoxic. If translated to clinical practice, this evidence will have important implications in efavirenz short- and long-term neurotoxicity prevention and management., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The development of predictive in vitro stem cell-derived hepatic models for toxicological drug screening is an increasingly important topic. Herein, umbilical cord tissue-derived mesenchymal stem cells (hnMSCs) underwent hepatic differentiation using an optimized three-step core protocol of 24 days that mimicked liver embryogenesis with further exposure to epigenetic markers, namely the histone deacetylase inhibitor trichostatin A (TSA), the cytidine analogue 5-azacytidine (5-AZA) and dimethyl sulfoxide (DMSO). FGF-2 and FGF-4 were also tested to improve endoderm commitment and foregut induction during Step 1 of the differentiation protocol, being HHEX expression increased with FGF-2 (4 ng/mL). DMSO (1%, v/v) when added at day 10 enhanced cell morphology, glycogen storage ability, enzymatic activity and induction capacity. Moreover, the stability of the hepatic phenotype under the optimized differentiation conditions was examined up to day 34. Our findings showed that hepatocyte-like cells (HLCs) acquired the ability to metabolize glucose, produce albumin and detoxify ammonia. Global transcriptional analysis of the HLCs showed a partial hepatic differentiation degree. Global analysis of gene expression in the different cells revealed shared expression of gene groups between HLCs and human primary hepatocytes (hpHeps) that were not observed between HepG2 and hpHeps. In addition, bioinformatics analysis of gene expression data placed HLCs between the HepG2 cell line and hpHeps and distant from hnMSCs. The enhanced hepatic differentiation observed was supported by the presence of the hepatic drug transporters OATP-C and MRP-2 and gene expression of the hepatic markers CK18, TAT, AFP, ALB, HNF4A and CEBPA; and by their ability to display stable UGT-, EROD-, ECOD-, CYP1A1-, CYP2C9- and CYP3A4-dependent activities at levels either comparable with or even higher than those observed in primary hepatocytes and HepG2 cells. Overall, an improvement of the hepatocyte-like phenotype was achieved for an extended culture time suggesting a role of the epigenetic modifiers in hepatic differentiation and maturation and presenting hnMSC-HLCs as an advantageous alternative for drug discovery and in vitro toxicology testing.

3D cultures of human stem cell-derived hepatocyte-like cells (HLCs) have emerged as promising models for short- and long-term maintenance of hepatocyte phenotype in vitro cultures by better resembling the in vivo environment of the liver and consequently increase the translational value of the resulting data. In this study, the first stage of hepatic differentiation of human neonatal mesenchymal stem cells (hnMSCs) was performed in 2D monolayer cultures for 17 days. The second stage was performed by either maintaining cells in 2D cultures for an extra 10 days, as control, or alternatively cultured in 3D as self-assembled spheroids or in multicompartment membrane bioreactor system. All systems enabled hnMSC differentiation into HLCs as shown by positive immune staining of hepatic markers CK-18, HNF-4α, albumin, the hepatic transporters OATP-C and MRP-2 as well as drug-metabolizing enzymes like CYP1A2 and CYP3A4. Similarly, all models also displayed relevant glucose, phase I and phase II metabolism, the ability to produce albumin and to convert ammonia into urea. However, EROD activity and urea production were increased in both 3D systems. Moreover, the spheroids revealed higher bupropion conversion, whereas bioreactor showed increased albumin production and capacity to biotransform diclofenac. Additionally, diclofenac resulted in an IC 50 value of 1.51 ± 0.05 and 0.98 ± 0.03 in 2D and spheroid cultures, respectively. These data suggest that the 3D models tested improved HLC maturation showing a relevant biotransformation capacity and thus provide more appropriate reliable models for mechanistic studies and more predictive systems for in vitro toxicology applications.

The development of metabolically competent in vitro models is of utmost importance for predicting adverse drug reactions, thereby preventing attrition-related economical and clinical burdens. Using the antiretroviral drug nevirapine (NVP) as a model, this work aimed to validate rat hepatocyte 3D spheroid cultures as competent in vitro systems to assess drug metabolism and bioactivation. Hepatocyte spheroids were cultured for 12 days in a stirred tank system (3D cultures) and exposed to equimolar dosages of NVP and its two major Phase I metabolites, 12-OH-NVP and 2-OH-NVP. Phase I NVP metabolites were detected in the 3D cultures during the whole culture time in the same relative proportions reported in in vivo studies. Moreover, the modulation of SULT1A1 activity by NVP and 2-OH-NVP was observed for the first time, pointing their synergistic effect as a key factor in the formation of the toxic metabolite (12-sulfoxy-NVP). Covalent adducts formed by reactive NVP metabolites with N-acetyl-L-cysteine and bovine serum albumin were also detected by high-resolution mass spectrometry, providing new evidence on the relative role of the reactive NVP metabolites, 12-sulfoxy-NVP, and NVP quinone methide, in toxicity versus excretion pathways. In conclusion, these results demonstrate the validity of the 3D culture system to evaluate drug bioactivation, enabling the identification of potential biomarkers of bioactivation/toxicity, and providing new evidence to the mechanisms underlying NVP-induced toxic events. This model, integrated with the analytical strategies described herein, is of anticipated usefulness to the pharmaceutical industry, as an upstream methodology for flagging drug safety alerts in early stages of drug development.

This work investigated the relationships between room acoustics, background noise level, and vocal effort of a speaker in simulated classrooms of various volumes. Under simulated acoustic environments, talkers adjusted their vocal effort linearly with the voice support, i.e., the degree of amplification offered by the room to the voice of a speaker, at his own ears. The slope of this relationship, called the room effect, of -0.24 dB/dB was significant only in the case of the highest noise levels of 62 dB. The vocal comfort for the speaker, however, was found to be more closely related to noise annoyance than to room reverberance.

Introduction: Advances in neoadjuvant chemotherapy (NCT) have allowed surgical treatment in otherwise unresectable patients with colorectal liver metastases (CRLM). It is well known that NCT induces liver lesions such as sinusoidal obstruction syndrome (SOS) and steatohepatitis (SH). However, whether it affects postoperative morbidity remains controversial. The aim of this study was both to evaluate the impact of NCT on liver parenchyma and postoperative morbidity, and to identify preoperative predictive markers for liver injury., Patients and Methods: Among 140 patients undergoing liver resection for CRLM between 2010 and 2013, 70 underwent systemic NCT. Liver function tests, pathology, postoperative morbidity and mortality were compared between the two groups., Results: Univariate analysis revealed NCT as a cause of sinusoidal dilation (p = 0.09), peliosis (p = 0.028) and moderate and severe SOS (p = 0.004) and bevacizumab as a protective agent against moderate and severe SOS (p = 0.045). Diabetic patients were identified as having a lower incidence of sinusoidal dilation (p = 0.034) and a higher incidence of steatosis (p = 0.003). Multivariate analysis confirmed sinusoidal dilation as an independent cause for morbidity (p = 0.02) and liver-specific complications (p = 0.016). Preoperative level of GGT was identified as predictive factor for moderate and severe SOS and peliosis (p < 0.001 and p = 0.004, respectively)., Conclusion: The administration of NCT induces SOS-lesions, but can be partially prevented by bevacizumab and diabetes. Sinusoidal dilation is associated with increased postoperative morbidity. Preoperative GGT levels can be useful to predict the presence of SOS., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Multiple mechanisms related to metastases undergo redox regulation. Cu[15]pyN5 is a redox-active copper(II) complex previously studied as a chemotherapy sensitizer in mammary cells. The effects of a cotreatment with Cu[15]pyN5 and doxorubicin (dox) were evaluated in two human breast cancer cell lines: MCF7 (low aggressiveness) and MDA-MB-231 (highly aggressive). Cu[15]pyN5 decreased MCF7-directed cell migration. In addition, a cotreatment with dox and Cu[15]pyN5 reduced the proteolytic invasion of MDA-MB-231 cells. Cell detachment was not affected by exposure to these agents. Cu[15]pyN5 and dox significantly increased intracellular ROS in both cell lines. This increase could be at least partially due to H2 O2 accumulation. The combination of Cu[15]pyN5 with dox may be beneficial in breast cancer treatment as it could help reduce cancer cell migration and invasion. Moreover, the ligand [15]pyN5 has a high affinity for copper(II) and displays potential anti-angiogenic properties. Overall, we present a potential drug that might arrest the progression of breast cancer by different and complementary mechanisms., (© 2015 John Wiley & Sons A/S.)

Background: Obesity is a major risk factor for several chronic diseases. The BioEnterics(®) Intragastric Balloon (BIB(®)) is used worldwide as a short-term (6 months) treatment in morbid obese patients. However, removal of BIB(®) past 6 months is a common occurrence in clinical practice often as a result of patient factors. The aim of the present Italian multicentre cohort study was to evaluate the safety and efficacy of the BIB(®) left in situ for more than 6 months., Materials and Methods: A total of eight Italian centres participated in the study. Participating centres received a standardised questionnaire designed to capture safety and efficacy data. Weight loss data, as well as date, and reason for removal of the BIB(®) after 6 months were recorded. Adverse events, including mortality and complications, operators' subjective technical notes and findings, and difficulties during removal procedure were also collected., Results: Six hundred and eleven patients were included. In the majority of cases, patient extended BIB(®) treatment due to satisfactory weight loss (44 %). At 6 months, all patients achieved a BMI statistically lower than the initial BMI (p < 0.001). There was a non-significant trend towards greater BMIL % in patients who underwent removal up to 15 months versus the results achieved up to 6 months. No major complications were recorded., Conclusion: Extending the duration of BIB(®) use up to 14 months safely maintains weight loss and satiety with greater results than that up to 6 months, without complications.

Introduction: The secretion of trophic factors by mesenchymal stromal cells has gained increased interest given the benefits it may bring to the treatment of a variety of traumatic injuries such as skin wounds. Herein, we report on a three-dimensional culture-based method to improve the paracrine activity of a specific population of umbilical cord tissue-derived mesenchymal stromal cells (UCX®) towards the application of conditioned medium for the treatment of cutaneous wounds., Methods: A UCX® three-dimensional culture model was developed and characterized with respect to spheroid formation, cell phenotype and cell viability. The secretion by UCX® spheroids of extracellular matrix proteins and trophic factors involved in the wound-healing process was analysed. The skin regenerative potential of UCX® three-dimensional culture-derived conditioned medium (CM3D) was also assessed in vitro and in vivo against UCX® two-dimensional culture-derived conditioned medium (CM2D) using scratch and tubulogenesis assays and a rat wound splinting model, respectively., Results: UCX® spheroids kept in our three-dimensional system remained viable and multipotent and secreted considerable amounts of vascular endothelial growth factor A, which was undetected in two-dimensional cultures, and higher amounts of matrix metalloproteinase-2, matrix metalloproteinase-9, hepatocyte growth factor, transforming growth factor β1, granulocyte-colony stimulating factor, fibroblast growth factor 2 and interleukin-6, when compared to CM2D. Furthermore, CM3D significantly enhanced elastin production and migration of keratinocytes and fibroblasts in vitro. In turn, tubulogenesis assays revealed increased capillary maturation in the presence of CM3D, as seen by a significant increase in capillary thickness and length when compared to CM2D, and increased branching points and capillary number when compared to basal medium. Finally, CM3D-treated wounds presented signs of faster and better resolution when compared to untreated and CM2D-treated wounds in vivo. Although CM2D proved to be beneficial, CM3D-treated wounds revealed a completely regenerated tissue by day 14 after excisions, with a more mature vascular system already showing glands and hair follicles., Conclusions: This work unravels an important alternative to the use of cells in the final formulation of advanced therapy medicinal products by providing a proof of concept that a reproducible system for the production of UCX®-conditioned medium can be used to prime a secretome for eventual clinical applications.

Aβ-induced astrogliosis can worsen the eziopathogenesis of Alzheimer disease (AD) by the release of proinflammatory and pro-oxidant mediators. Activated glial cells may release also pro-angiogenic molecules. The role of angiogenesis in AD is still controversial: although angiogenesis brings oxygen and nutrients to injured tissue, it may also exacerbate reactive gliosis. Moreover, by altering blood-brain barrier permeability pro-angiogenic mediators promote passage of inflammatory/immune-competent cells into the brain, thereby exacerbating gliosis. The release of proangiogenic factors during astrogliosis may thus be a key-step in controlling AD progression. The endogenous fatty acid amide, palmitoylethanolamide (PEA), is a pleiotropic mediator exerting anti-inflammatory, antinociceptive and antiangiogenic effects in several in vitro and in vivo models of chronic-degenerative disease. In this study, we investigated the effects of PEA in AD angiogenesis and neuroinflammation by using conditioned medium from untreated and Aβ-treated C6 rat astroglioma cells and HUVEC human endothelial cells. PEA (10 -8 -10 -6 M) concentration-dependently reduced expression of pro-inflammatory and pro-angiogenic markers in Aβ (1 μg/mL)-stimulated C6 cells. Moreover, culture medium from PEA-treated C6 cells reduced HUVEC cell proliferation as compared to cells treated with conditioned medium from Aβ-treated C6 cells. Immunocytochemical analysis revealed that PEA treatment inhibited nuclear levels of mitogen-activated protein kinase 1 (the main pro-angiogenic pathway) and cytoplasmic vascular endothelial growth factor in HUVEC cells receiving C6 conditioned medium. Finally, the peroxisome proliferator-activated receptor alpha inhibitor GW6471, added to Aβ-treated C6 cells blocked all PEA effects in this model, suggesting that PEA acts through a proliferator-activated receptor alpha-dependent mechanism on astroglial cells. Collectively, these data support the potential therapeutic utility of PEA in AD.

Monoacylglycerol lipase (MAGL) is the enzyme responsible for the inactivation of the endocannabinoid 2-arachidonoylglycerol (2-AG). MAGL inhibitors show analgesic and tissue-protecting effects in several disease models. However, the few efficient and selective MAGL inhibitors described to date block the enzyme irreversibly, and this can lead to pharmacological tolerance. Hence, additional classes of MAGL inhibitors are needed to validate this enzyme as a therapeutic target. Here we report a potent, selective, and reversible MAGL inhibitor (IC50=0.18 μM) which is active in vivo and ameliorates the clinical progression of a multiple sclerosis (MS) mouse model without inducing undesirable CB1 -mediated side effects. These results support the interest in MAGL as a target for the treatment of MS., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cannabidiol (CBD) decreases insulitis, inflammation, neuropathic pain, and myocardial dysfunction in preclinical models of diabetes. We recently showed that CBD also improves vasorelaxation in the Zucker diabetic fatty (ZDF) rat, and the objective of the present study was to establish the mechanisms underlying this effect. Femoral arteries from ZDF rats and ZDF lean controls were isolated, mounted on a myograph, and incubated with CBD (10 μM) or vehicle for 2 hours. Subsequent vasorelaxant responses were measured in combination with various interventions. Prostaglandin metabolites were detected using enzyme immunoassay. Direct effects of CBD on cyclooxygenase (COX) enzyme activity were measured by oxygraph assay. CBD enhanced the maximum vasorelaxation to acetylcholine (ACh) in femoral arteries from ZDF lean rats (P < 0.01) and especially ZDF rats (P < 0.0001). In ZDF arteries, this enhancement persisted after cannabinoid receptor (CB) type 1, endothelial CB, or peroxisome proliferator-activated receptor-γ antagonism but was inhibited by CB2 receptor antagonism. CBD also uncovered a vasorelaxant response to a CB2 agonist not previously observed. The CBD-enhanced ACh response was endothelium-, nitric oxide-, and hydrogen peroxide-independent. It was, however, COX-1/2- and superoxide dismutase-dependent, and CBD enhanced the activity of both purified COX-1 and COX-2. The CBD-enhanced ACh response in the arteries was inhibited by a prostanoid EP4 receptor antagonist. Prostaglandin E2 metabolite levels were below the limits of detection, but 6-keto prostaglandin F1 α was decreased after CBD incubation. These data show that CBD exposure enhances the ability of arteries to relax via enhanced production of vasodilator COX-1/2-derived products acting at EP4 receptors., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Helicobacter pylori resistance to antimicrobial agents is steadily increasing. It is extremely important to be aware of the local prevalence of antibiotic resistance so as to adjust treatment strategies. During this single-centre, prospective study, we aimed to determine primary and secondary resistance rates of H. pylori to antibiotics as well as host and bacterial factors associated with this problem. Overall, 180 patients (131 female; mean age 43.4±13.5 years; primary resistance 103; secondary resistance 77) with positive (13) C-urea breath test were submitted to upper endoscopy with gastric biopsies. Helicobacter pylori was cultured and antimicrobial susceptibility was determined by Etest and molecular methods. Clinical and microbiological characteristics associated with resistance were evaluated by logistic regression analysis. Among the 180 isolates 50% were resistant to clarithromycin (primary 21.4%; secondary 88.3%), 34.4% to metronidazole (primary 29.1%; secondary 41.6%), 33.9% to levofloxacin (primary 26.2%; secondary 44.2%), 0.6% to tetracycline and 0.6% to amoxicillin. Being female was an independent predictor of resistance to clarithromycin and metronidazole. Previous, failed, eradication treatments were also associated with a decrease in susceptibility to clarithromycin. History of frequent infections, first-degree relatives with gastric carcinoma and low education levels determined increased resistance to levofloxacin. Mutations in the 23S rRNA and gyrA genes were frequently found in isolates with resistance to clarithromycin and levofloxacin, respectively. This study revealed that resistance rates to clarithromycin, metronidazole and levofloxacin are very high and may compromise H. pylori eradication with first-line and second-line empiric triple treatments in Portugal., (© 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.)

Background: The endocannabinoid system regulates cancer cell proliferation, and in prostate cancer a high cannabinoid CB1 receptor expression is associated with a poor prognosis. Down-stream mediators of CB1 receptor signaling in prostate cancer are known, but information on potential upstream regulators is lacking., Results: Data from a well-characterized tumor tissue microarray were used for a Bayesian network analysis using the max-min hill-climbing method. In non-malignant tissue samples, a directionality of pEGFR (the phosphorylated form of the epidermal growth factor receptor) → CB1 receptors were found regardless as to whether the endocannabinoid metabolizing enzyme fatty acid amide hydrolase (FAAH) was included as a parameter. A similar result was found in the tumor tissue, but only when FAAH was included in the analysis. A second regulatory pathway, from the growth factor receptor ErbB2 → FAAH was also identified in the tumor samples. Transfection of AT1 prostate cancer cells with CB1 receptors induced a sensitivity to the growth-inhibiting effects of the CB receptor agonist CP55,940. The sensitivity was not dependent upon the level of receptor expression. Thus a high CB1 receptor expression alone does not drive the cells towards a survival phenotype in the presence of a CB receptor agonist., Conclusions: The data identify two potential regulators of the endocannabinoid system in prostate cancer and allow the construction of a model of a dysregulated endocannabinoid signaling network in this tumor. Further studies should be designed to test the veracity of the predictions of the network analysis in prostate cancer and other solid tumors., (© 2014 The Authors. The Prostate published by Wiley Periodicals, Inc.)

Aims: A high consumption of fructose leads not only to peripheral changes in insulin sensitivity and vascular function, but also to central changes in several brain regions. Given the role of the endogenous cannabinoid system in the control of energy intake, we undertook a pilot study to determine whether a high fructose diet produced changes in brain CB1 receptor functionality., Main Methods: Male rats given access ad libitum to normal chow were given either water, glucose or fructose solutions to drink. CB1 receptor functionality was measured autoradiographically as the increase in [(35)S]GTPγS binding produced by the agonist CP55,940., Key Findings: Seven regions were investigated: the prefrontal cortex, caudate-putamen, hippocampal CA1-CA3, dentate gyrus, amygdala, and dorsomedial and ventromedial hypothalami. Two-way robust Wilcoxon analyses for each brain region indicated that the dietary treatment did not produce significant main effects upon agonist-stimulated [(35)S]GTPγS binding in any of the regions, in contrast to a significant main effect upon both leptin and adiponectin levels in the blood. However, a MANCOVA of the data controlling for leptin and adiponectin as co-variables identified a significant effect of glucose and fructose treatment for five weeks upon the [(35)S]GTPγS response in the ventromedial hypothalamus, a region of importance for regulation of appetite., Significance: It is concluded from this pilot study that palatable solutions do not produce overt changes in brain CB1 receptor functionality, although subtle changes in discrete brain regions may occur., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: It has been reported that direct activation of the cannabinoid CB1 receptor in epidermal growth factor (EGR)-stimulated PC-3 prostate cancer cells results in an anti-proliferative effect accompanied by a down-regulation of EGF receptors (EGFR). In the present study, we investigated whether similar effects are seen following inhibition of the endocannabinoid hydrolytic enzyme monoacylglycerol lipase (MGL)., Results: CB1 receptor expression levels were found to differ greatly between two experimental series conducted using PC-3 cells. The monoacylglycerol lipase inhibitor JZL184 increased levels of 2-arachidonoylglycerol in the PC-3 cells without producing changes in the levels of anandamide and related N-acylethanolamines. In the first series of experiments, JZL184 produced a small mitogenic effect for cells that had not been treated with EGF, whereas an anti-proliferative effect was seen for EGF-treated cells. An anti-proliferative effect for the EGF-treated cells was also seen with the CB receptor agonist CP55,940. In the second batch of cells, there was an interaction between JZL184 and CB1 receptor expression densities in linear regression analyses with EGFR expression as the dependent variable., Conclusions: Inhibition of MGL by JZL184 can affect EGFR expression. However, the use in our hands of PC-3 cells as a model to investigate the therapeutic potential of MGL inhibitors and related compounds is compromised by their variability of CB1 receptor expression.

Chronic inflammation, a condition frequently associated with several pathologies, is characterized by angiogenic and fibrogenic responses that may account for the development of granulomatous tissue. We previously demonstrated that the chymase, rat mast cell protease-5 (rMCP-5), exhibits pro-inflammatory and pro-angiogenic properties in a model of chronic inflammation sustained by mast cells (MCs), granuloma induced by the subcutaneous carrageenan-soaked sponge implant in rat. In this study, we investigated the effects of palmitoylethanolamide (PEA), an anti-inflammatory and analgesic endogenous compound, on rMCP-5 mRNA expression and Microphtalmia-associated Transcription Factor (MITF) activation in the same model of chronic inflammation. The levels of rMCP-5 mRNA were detected using semi-quantitative RT-PCR; the protein expression of chymase and extracellular signal-regulated kinases (ERK) were analyzed by western blot; MITF/DNA binding activity and MITF phosphorylation were assessed by electrophoretic mobility shift assay (EMSA) and immunoprecipitation, respectively. The administration of PEA (200, 400 and 800 µg/ml) significantly decreased rMCP-5 mRNA and chymase protein expression induced by λ-carrageenan. These effects were associated with a significant decrease of MITF/DNA binding activity and phosphorylated MITF as well as phosphorylated ERK levels. In conclusion, our results, showing the ability of PEA to inhibit MITF activation and chymase expression in granulomatous tissue, may yield new insights into the understanding of the signaling pathways leading to MITF activation controlled by PEA., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Geranium is one of the most popular ornamental plants in México. In December 2012, rust symptoms were observed on leaves of common geranium (Pelargonium × hortorum L. H. Bailey) growing in pots in garden landscapes in Morelia, Michoacán. Dark brown pustules with chlorotic halos appeared on the lower leaf surface. A center pustule surrounded by one or more partial-to-complete concentric circles of smaller pustules was observed in each lesion. Urediniospores were globose or subglobose to ovoid, light brown, echinulated, thin-walled with two more or less conspicuous subequatorial pores, and 21 to 29 × 18 to 24 μm (25.5 × 22.8 μm average). Teliospores were not observed. Based on these characters, the rust was identified as Puccinia pelargonii-zonalis Doidge (1,2). Pathogenicity tests were conducted on three healthy P. × hortorum plants that were sprayed with water droplets containing urediniospores. The inoculated plants were covered with a plastic bag and placed in a screened house. The bags were removed after 24 h. Afterwards, the plants were maintained outside the screened house in full sun at ambient temperature (24 to 30°C in the day and 5 to 10°C at night). Initial symptoms were observed 15 to 17 days post inoculation. Symptoms appeared as small light yellow spots on the upper surface of mature leaves. Urediniospores production on the lower surface of the leaves was evident 22 to 25 days post inoculation. To our knowledge, this is the first report of P. pelargonii-zonalis in the state of Michoacán, México. Geranium rust has been previously reported only in the state of Guanajuato (2). References: (1) E. M. Doidge. Bothalia 2:1, 1926. (2) H. L. Gallegos and G. B. Cummings. Uredinales (royas) de México. Vol. 1. Culiacán, Sinaloa, México, SARH, 1981.