Ex Vivo Perfusion Culture of Large Blood Vessels in a 3D Printed Bioreactor.

Vascular disease forms the basis of most cardiovascular diseases (CVDs), which remain the primary cause of mortality and morbidity worldwide. Efficacious surgical and pharmacological interventions to prevent and treat vascular disease are urgently needed. In part, the shortage of translational models limits the understanding of the cellular and molecular processes involved in vascular disease. Ex vivo perfusion culture bioreactors provide an ideal platform for the study of large animal vessels (including humans) in a controlled dynamic environment, combining the ease of in vitro culture and the complexity of the live tissue. Most bioreactors are, however, custom manufactured and therefore difficult to adopt, limiting the reproducibility of the results. This paper presents a 3D printed system that can be easily produced and applied in any biological lab, and provides a detailed protocol for its setup, enabling users' operation. This innovative and reproducible ex vivo perfusion culture system enables the culture of blood vessels for up to 7 days in physiological conditions. We expect that adopting a standardized perfusion bioreactor will support a better understanding of physiological and pathological processes in large animal blood vessels and accelerate the discovery of new therapeutics.

A new TCAD simulation method for direct CMOS electron detectors optimization.

A custom CMOS image sensor hardened by design is characterized in a transmission electron microscope, with the aim to extract basic parameters such as the quantum efficiency, the modulation transfer function and finally the detective quantum efficiency. In parallel, a new methodology based on the combination of Monte Carlo simulation of electron distributions and TCAD simulations is proposed and performed on the same detector, and for the first time the basic parameters of a direct CMOS electron detector are extracted thanks to the TCAD. The methodology is validated by means of the comparison between experimental and simulation results. This simulation method may be used for the development of future electron detectors.

Porcine Organotypic Epicardial Slice Protocol: A Tool for the Study of Epicardium in Cardiovascular Research.

The epicardium has recently gained interest in the cardiovascular field due to its capacity to support heart regeneration after ischemic injury. Models to study the epicardium of large animals in vitro are limited and mainly based on epicardial cell isolation/differentiation from stem cells, followed by 2D cells culture. In this method paper, we describe the procedure to obtain and culture 3D organotypic heart slices presenting an intact epicardium, as a novel model to study the epicardial physiology and activation. Epicardial slices are obtained from porcine hearts using a high-precision vibratome and retain a healthy epicardial layer embedded in its native extracellular environment and connected with other cardiac cells (cardiomyocytes, fibroblasts, vascular cells etc.). Epicardial slices can be cultured for 72 h, providing an ideal model for studying the epicardium physiology or perform pharmacological interventions/gene therapy approaches. We also report on methods to assesses the viability and composition of the epicardial slices, and evaluate their architecture in 3D through tissue decoloration. Finally, we present a potential application for a nanomaterial-based gene transfer method for tracking of epicardial cells within the slice. Crucially, given the similarity in morphology and physiology of porcine heart with its human counterpart, our system provides a platform for translational research while providing a clinically relevant and ethical alternative to the use of small animals in this type of research.

3D Printed Bioreactor Enabling the Pulsatile Culture of Native and Angioplastied Large Arteries.

Routine interventions such as balloon angioplasty, result in vascular activation and remodeling, often requiring re-intervention. 2D in vitro models and small animal experiments have enabled the discovery of important mechanisms involved in this process, however the clinical translation is often underwhelming. There is a critical need for an ex vivo model representative of the human vascular physiology and encompassing the complexity of the vascular wall and the physical forces regulating its function. Vascular bioreactors for ex vivo culture of large vessels are viable alternatives, but their custom-made design and insufficient characterization often hinders the reproducibility of the experiments. The objective of the study was to design and validate a novel 3D printed cost-efficient and versatile perfusion system, capable of sustaining the viability and functionality of large porcine arteries for 7 days and enabling early post-injury evaluations. MultiJet Fusion 3D printing was used to engineer the EasyFlow insert, converting a conventional 50 ml centrifuge tube into a mini bioreactor. Porcine carotid arteries either left untreated or injured with an angioplasty balloon, were cultured under pulsatile flow for up to 7 days. Pressure, heart rate, medium viscosity and shear conditions were adjusted to resemble arterial in vivo hemodynamics. Tissue viability, cell activation and matrix remodeling were analyzed by immunohistochemistry, and vascular function was monitored by duplex ultrasound. Culture conditions in the EasyFlow bioreactor preserved endothelial coverage and smooth muscle organization and extracellular matrix structure in the vessel wall, as compared to static culture. Injured arteries presented hallmarks of early remodeling, such as intimal denudation, smooth muscle cell disarray and media/adventitia activation in flow culture. Duplex ultrasound confirmed continuous pulsatile blood flow conditions, dose-dependent vasodilator response to nitroglycerin in untreated vessels and impaired dilator response in angioplastied vessels. The scope of this work is to validate a low-cost, robust and reproducible system to explore the culture of native and injured large arteries under pulsatile flow. While the study of vascular pathology is beyond the scope of the present paper, our system enables future investigations and provides a platform to test novel therapies and devices ex vivo, in a patient relevant system.

Pericytes' Circadian Clock Affects Endothelial Cells' Synchronization and Angiogenesis in a 3D Tissue Engineered Scaffold.

Angiogenesis, the formation of new capillaries from existing ones, is a fundamental process in regenerative medicine and tissue engineering. While it is known to be affected by circadian rhythms in vivo, its peripheral regulation within the vasculature and the role it performs in regulating the interplay between vascular cells have not yet been investigated. Peripheral clocks within the vasculature have been described in the endothelium and in smooth muscle cells. However, to date, scarce evidence has been presented regarding pericytes, a perivascular cell population deeply involved in the regulation of angiogenesis and vessel maturation, as well as endothelial function and homeostasis. More crucially, pericytes are also a promising source of cells for cell therapy and tissue engineering. Here, we established that human primary pericytes express key circadian genes and proteins in a rhythmic fashion upon synchronization. Conversely, we did not detect the same patterns in cultured endothelial cells. In line with these results, pericytes' viability was disproportionately affected by circadian cycle disruption, as compared to endothelial cells. Interestingly, endothelial cells' rhythm could be induced following exposure to synchronized pericytes in a contact co-culture. We propose that this mechanism could be linked to the altered release/uptake pattern of lactate, a known mediator of cell-cell interaction which was specifically altered in pericytes by the knockout of the key circadian regulator Bmal1. In an angiogenesis assay, the maturation of vessel-like structures was affected only when both endothelial cells and pericytes did not express Bmal1, indicating a compensation system. In a 3D tissue engineering scaffold, a synchronized clock supported a more structured organization of cells around the scaffold pores, and a maturation of vascular structures. Our results demonstrate that pericytes play a critical role in regulating the circadian rhythms in endothelial cells, and that silencing this system disproportionately affects their pro-angiogenic function. Particularly, in the context of tissue engineering and regenerative medicine, considering the effect of circadian rhythms may be critical for the development of mature vascular structures and to obtain the maximal reparative effect.

OCTAVA: An open-source toolbox for quantitative analysis of optical coherence tomography angiography images.

Optical coherence tomography angiography (OCTA) performs non-invasive visualization and characterization of microvasculature in research and clinical applications mainly in ophthalmology and dermatology. A wide variety of instruments, imaging protocols, processing methods and metrics have been used to describe the microvasculature, such that comparing different study outcomes is currently not feasible. With the goal of contributing to standardization of OCTA data analysis, we report a user-friendly, open-source toolbox, OCTAVA (OCTA Vascular Analyzer), to automate the pre-processing, segmentation, and quantitative analysis of en face OCTA maximum intensity projection images in a standardized workflow. We present each analysis step, including optimization of filtering and choice of segmentation algorithm, and definition of metrics. We perform quantitative analysis of OCTA images from different commercial and non-commercial instruments and samples and show OCTAVA can accurately and reproducibly determine metrics for characterization of microvasculature. Wide adoption could enable studies and aggregation of data on a scale sufficient to develop reliable microvascular biomarkers for early detection, and to guide treatment, of microvascular disease.

Migraine attacks in the pharmacy: a gender subanalysis on treatment preferences.

In 2014 our group published the results of a survey conducted in Piedmont, Italy, on the patterns of use and dispensing of drugs in patients requesting assistance from pharmacists for relief of a migraine attack. Epidemiological studies on migraine have consistently shown that migraine is far more common among women than men. This gender difference is also reflected in the higher percentage of women visiting a pharmacy to obtain treatment or advice for headache attacks. In this study, we further explored gender differences in healthcare-seeking behavior and use of migraine medications. The aim of the study was to determine whether women made better selective use of migraine medications and whether visiting a headache center for consultation and treatment reflected awareness of how best to manage their condition. Among the drugs usually taken for relieving head pain, there was no statistically significant difference between men and women in the routine use of NSAIDs (55.6 vs. 51.6 %) or ergot derivatives (8.7 vs. 9.3 %). Statistically significant differences emerged between men and women (27.9 vs. 35.4 %) in the use of triptans (p = 0.003; OR 1.41, 95 % CI 1.12-1.78) and in the use of combined medications (8.5 vs. 12.2 %) (p = 0.029; OR 1.49, 95 % CI 1.04-2.14) but not in the use of simple OTC non-NSAIDs. Less men than women sought professional medical care for managing migraine (65.7 vs. 72.4 %) (p = 0.003; OR 0.71, 95 % CI 0.57-0.89); more women than men sought treatment at a headache center (21.7 vs. 17.4 %) (p = 0.044; OR 1.31, 95 % CI 1.07-1.72).

[Eligibility to a Maastricht III - type organ donation according to the protocol proposed by the French organ procurement organization (Agence de la biomedicine) among patients in a medico surgical intensive care unit]. / Potentiel de patients éligibles à un prélèvement d'organes de type Maastricht III dans le cadre du protocole de l'Agence de la biomédecine dans un service de réanimation polyvalente.

OBJECTIVE: Because of graft shortages, an experimental programme of organ donation after Maastricht 3-type circulatory death (M3) has been proposed by the French organ procurement organization (Agence de la biomedicine: ABM). The aim of the study was to estimate how many potential patients were eligible for an M3-type organ donation, amongst deceased patients who have had life-support withdrawn. PATIENTS AND METHODS: We conducted a retrospective study looking at the notes of deceased patients in a French general intensive care unit (ICU), where organ donation is arranged in DBD donors. RESULTS: Over the year 2013, 1475 patients were admitted in ICU and 215 died. One hundred and one patients were brain-injured and 26 of them died following a decision to withdrawn life-support and without contraindication to organ donation. Among them, 2 patients (8%) met the criteria for the French M3-type organ donation protocol. A 12.5% increase in organ donation activity of our team and five organ transplantations could have been considered. CONCLUSION: If M3 organ donation is considered, a significant increase in transplantation would be expected.

Migraine attacks in the pharmacy: a survey in Piedmont, Italy.

Headache patients often consult a pharmacist in an attempt to obtain momentary pain relief without having been given any previous expert advice. A specific questionnaire was distributed to the pharmacies in order to assess the patterns of use and dispensing of analgesic medications to the headache patient who turns to the pharmacist for relief of a painful attack. This study aimed at identifying migraine patients who self-medicated, with further end points including whether these patients shared any particular clinical characteristics, the most common type of analgesic medications used, and what, if anything, was recommended by the pharmacist; lastly, which health care professional, if any, routinely managed the patient's headaches. A total of 9,100 questionnaires were distributed to the pharmacies and the complete 3,065 were included in the database. The ID Migraine Screener Test was used to classify subjects into 4 groups: "Definite migraine" (3/3 positive answers: n = 1,042; 34 %), "Probable migraine" (2/3: n = 969; 31.6 %), "Unlikely migraine" (1/3: n = 630; 20.5 %), and "Other headaches" (0/3: n = 424; 13.8 %). Only Definite and Probable migraines (n = 2,011) are considered in this paper. Amongst the drugs usually taken by the patients, NSAIDs were more common in the Probable migraine group (60.7 %) than in the Definite migraine (44.7 %) group (p < 0.001). On the contrary, triptans were more commonly used by the Definite migraine group (42.9 %) than the Probable migraine (23.7 %) group (p < 0.001), and combination drugs were preferentially (p < 0.001) chosen by the Definite (13.8 %) rather than the Probable migraine group (8.7 %). A total of 29.2 % of respondents reported that for the management of their headaches, they did not avail themselves of any type of professional healthcare, such as their general practitioner, a headache specialist, or a Headache Center.

Endonasal mucosal contact points in chronic migraine.

Some anatomo-functional alterations of the nose may be considered as possible causes of headache: deviations of the nasal septum, abnormal turbinates, especially middle or superior, with consequent areas of mucosal contact with the septum. This study was performed on 100 subjects, 27 chronic migraine (CM) sufferers and 73 subjects who never suffered from migraine as control group. In the CM group, a direct endoscopic assessment was carried out in order to search for mucosal points of contact. Following the endoscopy, the patients underwent a computerized tomography (CT) in order to confirm the mucosal contact and for a better evaluation of its localization. The control group (C group) consisted of subjects who underwent a CT of the skull for various reasons. In CM group, a mucosal contact was highlighted in 14 patients (51.8 %); it was unilateral in 50 % of cases. In C group, the contact was present in 27 cases (36.9 %); in 81.5 % of them (n = 22), it was unilateral. A single site of contact was present in 6 (22 %) patients in CM group and 20 (27.3 %) patients in C group; more sites, in 8 (29.6 %) CM group patients and in 7 (9.5 %) patients of the C group. The connection between subjects and the number of single or multiple contacts in the two groups was statistically significant (p = 0.049). Furthermore, the frequency of the septum-middle turbinate was significantly (p = 0.0013) more frequent in CM sufferers (13/14) compared with control subjects (11/27). This study suggests, although with extremely early data, the need to select carefully patients for a possible surgical approach, using various parameters: in particular, the site of the mucosal contact, favoring the cases with multiple areas of contact, mainly between septum-middle turbinate and septum-superior turbinate.

Effects of the acupoints PC 6 Neiguan and LR 3 Taichong on cerebral blood flow in normal subjects and in migraine patients.

Acupuncture has been proven to be effective in the treatment of various cardiovascular disorders; it acts both on the peripheral flow and on the cerebral flow. Our study aimed to evaluate the effects of the insertion of PC 6 Neiguan and LR 3 Taichong acupoints on the cerebral blood flow (CBF) in the middle cerebral artery (MCA). These effects were measured in a group of patients suffering from migraine without aura (Group M) and in a healthy control group (Group C). In the study, we included 16 patients suffering from migraine without aura, classified according to the criteria of the International Headache Society, and 14 healthy subjects as a control group. The subjects took part in the study on two different days, and on each day, the effect of a single acupoint was evaluated. Transcranial Doppler was used to measure the blood flow velocity (BFV) in the MCA. Our study showed that the stimulation of PC 6 Neiguan in both groups results in a significant and longlasting reduction in the average BFV in the MCA. After pricking LR 3 Taichong, instead, the average BFV undergoes a very sudden and marked increase; subsequently, it decreases and tends to stabilize at a slightly higher level compared with the baseline, recorded before needle insertion. Our data seem to suggest that these two acupoints have very different effects on CBF. The insertion of PC 6 Neiguan probably triggers a vasodilation in MCA, while the pricking of LR 3 Taichong determines a rapid and marked vasoconstriction.

Acupuncture in cluster headache: four cases and review of the literature.

Although cluster headache (CH) is the most disabling form of primary headache, little evidences regarding alternative and complementary therapies are available. Only few dated studies and some isolated cases are described. We describe four patients with CH treated with acupuncture as a preventive treatment, combined with verapamil or alone. All patients received acupuncture treatment twice/week for 2 weeks, then once/week for 8 weeks, and then once/alternate weeks for 2 weeks. According to Traditional Chinese Medicine the acupoints selected were: Ex HN-5 Taiyang, GB 14 Yangbai (both only on the affected side), GB 20 Fengchi (on both sides), LI 4 Hegu, LR 2 Xingjiang, SP 6 Sanyinjiao, ST 36 Zusanli (all on both sides). At each point, after the insertion of the needle, the feeling of "De Qi" was evoked; after obtaining this sensation the acupoints were not further stimulated for a period of 20 min, until their extraction. In all patients an interruption of cluster attacks was obtained. To our knowledge, this is the first report concerning acupuncture in CH patients which details the protocol approach, acupoints and duration of the treatment. Our results offer the opportunity to discuss the emerging role of acupuncture in the therapy of CH, assuming a possible influence on opioid system.

PulseNet USA: a five-year update.

PulseNet USA is the molecular surveillance network for foodborne infections in the United States. Since its inception in 1996, it has been instrumental in detection, investigation and control of numerous outbreaks caused by Shiga toxin-producing Escherichia coli O157:[H7] (STEC O157), Salmonella enterica, Listeria monocytogenes, Shigella spp., and Campylobacter. This paper describes the current status of the network, including the methodologies used and its future possibilities. The currently preferred subtyping method in the network is pulsed-field gel electrophoresis (PFGE), a proven highly discriminatory molecular subtyping method. New simpler sequencebased subtyping methods are under development and validation to complement and eventually replace PFGE. PulseNet is essentially a cluster detection network, but the data in the system will now also be used in attribution analyses of sporadic infections. The PulseNet platform will also be used as a primary tool in preparedness and response to acts of food bioterrorism.

Transferencia de genes plasmídicos de resistencia a antibióticos mediante conjugación entre cepas de Escherichia coli provenientes de procesos diarréicosde niños menores a 3 años de edad. Hospital Materno Infantil de la ciudad de La Paz / Transfer of plasmid genes of resistance to antibiotics through conjugation between strains of escherichia coli from processes diarréicos de smaller children than 3 years of edad. Hospital Maternal and Child of the city of La Paz

Escherichia coli es el agente etiológico enterobacteriano más común causante de enfermedades diarreicas en lapoblación infantil. Por este motivo se estudió la transferencia por conjugación de genes plasmídicos de resistencia a antibióticos de bacterias E. coli provenientes de heces de niños menores de 3 años con procesos diarreicos que asistieron al laboratorio clínico delHospital materno infantil durante abril y diciembre del 2003. Se analizó el antibiograma de 44 cepas de E. colique presentó altos porcentajes de resistencia (Amoxicilina 95.55%, cotrimoxazol 68.88%, cloranfenicol51.11%). Por otra parte, las asociaciones más comunes de antibióticos fueron amoxicilina-cotrimoxazol (65%), y amoxicilina-cotrimoxazol-cloranfenicol (47,73%). Los experimentos de conjugación presentaron frecuencias entre 10-5 a 1 0-7, siendo de mayor valor para latransferencia de amoxicilina y menor para la amikacina. Los porcentajes de resistencia y las frecuencias deconjugación demuestran que la mayoría de los genes de resistencia son capaces de ser transferidos por conjugación. Lo que implica probablemente que muchosde estos genes están localizados en elementos genéticos como plásmidos y transposones.

Busqueda de marcadores RAPD (Ramdom Amplified Polymorphic DNA) de dos especies Cripticas del complejo Sordida / Search of cripticas markers (Random Amplified Polymorphic DNA) of two species of the sordida complex

La erradicación de los insectos vectores Trypanosornci cruzt (agente etiológico del mal de Chagas) ha causado cambios inesperados en el comportamiento poblacional de éstos, reemplazando los vectores primarios por otros secundarios, como Triatoma sordida y T. guasayana (en Bolivia). Estos vectores han mostrado una alta capacidad de adaptación al hábitat humano. Por eso es importante identificar y estudiar la dinámica de poblaciones de estos insectos, sobre la base de marcadores de especie, una de las herramientas moleculares para este efecto propuesta en este trabajo es el RAPD. A partir de ADN extraido de especimenes de Triatoma guasayana, 7'. sordida Ql. T. sordida G2 y T. infestans, clasificadas anteriormente por isoenzimas; se amplificó fragmentos con 10 cebadores decaméricos del MT A (OPERON), 9 de los 10 cebadores utilizados presentaron perfiles RAPD, obteniéndose 170 perfiles de 1 a 8 fragmentos. Se pudo evidenciar la existencia de dos marcadores RAPD específicos: (1) un fragmento de 995 pb con el cebador OPAO1 para 7'. sordida G2, (2) un fragmento de 986 pb con el cebador OPAO2 para 7' sordida Gi

[Tic disorders in children and adolescents. Clinical and genetic features, comorbidity]. / Disturbo da tic in età evolutiva. Aspetti clinici e genetici, comorbidità.

UNLABELLED: Tic disorders in children and adolescents. Clinical and genetic features, comorbidity. BACKGROUND: Aim of the study is to evaluate the clinical and genetic characteristics of tic disorders, in view of individuating similarities or differences relevant to the prognosis among different nosological groups. METHODS: A retrospective study of 79 children and adolescents (average age 9.3 yrs) was performed. The cases were diagnosed according to DSM-IV as: transitory tics (TT) 13 cases; chronic tics (CT) 50 cases; Tourette disease (TD) 16 cases. They were compared to a control group of 18 school age children without any neurological or psychiatric disturbance. The study included: semi-structured interviews focused on natural history of the disturbances, familiarity, presence of perinatal pathology, comorbidity; neurological examination, EEG, psychodiagnostic tests and investigation. RESULTS: Mean age of onset and type of first symptoms are the same in the three groups. Compared to the control group there is a significant increase in: familiarity for tics disturbances in TD; presence of perinatal pathological factors in the three groups of patients; comorbidity for obsessive-compulsive disorder (OCD) in CT and TD, comorbidity with ADHD in CT group. Three clinical cases are reported to exemplify the mixed features in the families and the different responsivity to the pharmacological treatment.

[Continuous parascalene block for shoulder surgery]. / Bloqueo paraescalénico continuo en la cirugía del hombro.

OBJECTIVE: To evaluate the efficacy and incidence of side effects of parascalene brachial plexus block for shoulder surgery and for postoperative analgesia. PATIENTS AND METHODS: This prospective descriptive study enrolled 186 patients scheduled for conventional (open) and arthroscopic shoulder operations. The parascalene approach to the brachial plexus was used, with an 18-G x 4.5 cm cannula (30 degrees bevel), applying the technique of detecting perforation of the prevertebral fascia. Mepivacaine 1.5% was administered for anesthesia, beginning with a test dose of 5 ml followed by doses of 25 ml. Bupivacaine 0.125% with 1/400,000 adrenaline was given for postoperative analgesia. Two groups were established according to type of surgery. In group A were patients undergoing open surgery and those with articular stiffness undergoing arthroscopic surgery; analgesics were delivered in continuous perfusion of 5 to 8 ml/h. Group B patients, who underwent arthroscopic surgery, received the analgesic in boluses of 10 to 14 ml every 4 to 6 hours. We assessed perception of perforation of the fascia, provocation of paresthesia, number of puncture attempts, anesthetic efficacy, postsurgical analgesia (visual analogue scale from 0 to 10) and complications. RESULTS: Blockade was effective for 155 of the 186 patients (83.33%). In 118 (63.44%) the perception of perforation of the prevertebral fascia was ostensible, in 52 (27.95%) it was slight and in 16 (8.6%) it was undetectable. Paresthesia was triggered by the cannula in 18 cases (9.13%) and by the test dose in 181 cases (97.31%). The mean number of puncture attempts was 1.2 +/- 0.09 per patient. During the early postoperative period analgesia was adequate for 92 (84.40%) of the 109 patients in group A. In group B, 45 patients (97.82%) required 1 to 2 analgesic doses (mean 1.4 +/- 2 doses) and one patient (2.17%) needed 6 doses within the first 24 hours. Catheters remained inserted as long as 36 hours to 10 days (mean 3.5 days) in group A. In group B they had been removed by 24 hours. Complications were transitory and slight. No cases of pneumothorax, no intra- or epidural anesthesia was required and no instances of vascular puncture occurred. CONCLUSIONS: The parascalene approach is easy to use and entails few risks while providing excellent anesthetic conditions for open and arthroscopic shoulder operations. It is useful for ambulatory analgesia to facilitate early rehabilitation after surgery.