Efficient generation and transcriptomic profiling of human iPSC-derived pulmonary neuroendocrine cells

Expansion of pulmonary neuroendocrine cells (PNECs) is a pathological feature of many human lung diseases. Human PNECs are inherently difficult to study due to their rarity (\u3c1% of total lung cells) and a lack of established protocols for their isolation. We used induced pluripotent stem cells (iPSCs) to generate induced PNECs (iPNECs), which express core PNEC markers, including ROBO receptors, and secrete major neuropeptides, recapitulating known functions of primary PNECs. Furthermore, we demonstrate that differentiation efficiency is increased in the presence of an air-liquid interface and inhibition of Notch signaling. Single-cell RNA sequencing (scRNA-seq) revealed a PNEC-associated gene expression profile that is concordant between iPNECs and human fetal PNECs. In addition, pseudotime analysis of scRNA-seq results suggests a basal cell origin of human iPNECs. In conclusion, our model has the potential to provide an unlimited source of human iPNECs to explore PNEC pathophysiology associated with several lung diseases

ULTRASTRUCTURAL FEATURES OF RAT UTERINE BLOOD-TISSUE BARRIER DURING DIFFERENT FUNCTIONAL STATES

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Microanatomy of the respiratory system of Bufo arenarum and Melanophryniscus stelzneri stelzneri (Anura, Bufonidae)

En el presente estudio se ha determinado que la microanatomíadel sistema respiratorio de B. arenarum y M.s.stelzneri son enalgunos aspectos muy diferentes. Utilizando técnicas macroscópicas, de microscopía óptica y demicroscopía electrónica, se caracterizaron anatómicamente ehistológicamente el sistema respiratorio de ambas especies. Para ellose dividió el estudio en I) vías aéreas extrapulmonares, II) pulmón y III) cuerpos neuroepiteliales. Las vías extrapulmonares están reducidas a una laringotráqueaconstituida por un par de cartílagos aritenoides y el cricoides. En B.arenarum a diferencia de M. s. stelzneri la laringotráquea estádividida en dos cámaras, una anterior y otra posterior delimitadasambas por membranas. La mucosa que reviste el lumen de lalaringotráquea así como las membranas que tapizan las cámaraspresenta una diversidad celular importante. En B. arenarum la últimaporción de la laringotráquea se continúa con dos cámaras “bronquiales” que se abren cada una de ellas a un pulmón mientrasque en M.s. stelzneri la laringotráquea se comunica directamente conlos pulmones. Los pulmones son diferentes en ambas especies. En B.arenarum la estructura interna es más compleja por la presencia detres tipos de septos: primarios, secundarios y terciarios concaracterísticas histológicas propias, mientras que en M. s. stelzneri laorganización interna es simple por la presencia solamente de septosterciarios. El parénquima pulmor es del tipo faveolar para B.arenarum y edicular para M. s. stelzneri. El epitelio respiratorio estáconstituido por neumocitos los cuales emiten prolongacionescitoplasmáticas que cubren a los capilares sanguíneos. Estasprolongaciones junto con el endotelio de los capilares constituyen labarrera de intercambio gaseoso. En M.s. stelzneri se determinó la presencia sobre los septos decuerpos neuroepiteliales a los cuales se les adjudica una posiblefunción secretora y/o receptora.The present study has determined that the microanatomy of therespiratory system of B. arenarum and M.s.stelzneri are different insome aspects. Light and electron microscopy were used to study the anatomyand histology of the respiratory system of both species. For it wasdivided it the study in I) extrapulmonary airways, II) lung and III)neuroepithelial bodies. The extrapulmonary airways in these species are reduced to alaryngotrachea constituted by a couple of arytenoid cartilages and acricoid cartilage. In B. arenarum contrary to M.s.stelzneri thelaryngotrachea is divided in two laryngeal chambers, anterior andposterior. The epithelia of the chambers as well as the membranesthat limit this chambers present an important cellular diversity. In B.arenarum the last portion of the laryngotrachea connected with twobronchial chambers which give out onto the lung. The lungs in both species are different. In B. arenarum theinternal structure is more complex by the presence of three types ofsepta: first, second and third order. In M.s. stelzneri the parenchymaforms a polygonal network arrangement, therefore the parenchyma isedicular in both. These space are delimited by the interconnection ofsepta which are covered by respiratory epithelium. This epitheliumconsists of one type of pneumocytes, which emit a thin cytoplasmicprocess that cover the pulmonary capillaries to form the outer layerof the air-blood barrier. In M.s. stelzneri neuroepithelial bodies (NEB) appear randomlydistributed over the septa. Each NEB consists of special cells, thecorpuscular cells (cc) which contains numerous dense core vesicles (dcv). The NEB are associated with afferent and efferent terminalnerves. The location of the NEB, the presence of the dcv, theoccurrence of synaptic contacts between nerve endings and the ccand the occurrence of capillaries close to the NEB, suggest areceptosecretory function for the NEB in the lung of M.s. stelzneri.Fil: Hermida, Gladys N.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina

Pharmacological classification of muscarinic receptors involved in the nervous control of airway smooth muscle tone in the guinea-pig

The work in this thesis was carried out to investigate modulation of pulmonary sympathetic neurotransmission by endogenous neurotransmitters and autacoids. An in vitro preparation has been developed which allows selective stimulation of post-ganglionic noradrenergic sympathetic nerves innervating airway smooth muscle of the guinea-pig. When the pressure within the fluid-filled tracheal tube had been raised, stimulation of the sympathetic nerve trunk evoked a relaxation response. Sympathetic nerve-induced relaxations were reduced in the presence of muscarinic agonists in a dose-dependent manner, when compared to those obtained at the same intraluminal pressure in the presence of the stable thromboxane analogue U46619, prostaglandin F2a and histamine. These differences were not due to differences in postjunctional "physiological" antagonism, as noradrenaline attenuated the postjunctional contractile actions of U46619 and acetylcholine to a similar extent. These results suggest a prejunctional inhibitory action of muscarinic agonists on sympathetic neurotransmission. This suggestion was confirmed with muscarinic antagonists. The selective M2 antagonists did not alter the postjunctional action of acetylcholine, nor its inhibitory effect on sympathetic nerve-induced relaxations. In contrast, the inhibitory effect of acetylcholine could be blocked with atropine and with M3 muscarinic antagonists. Endogenous acetylcholine was also shown to inhibit sympathetic nerve- induced relaxations. Stimulation of the vagal nerve trunk, simultaneously with sympathetic nerve stimulation caused inhibition of sympathetic nerve-induced relaxations. In addition, the anticholinesterase physostigmine attenuated sympathetic nerve-induced relaxations probably via facilitation of spontaneous acetylcholine release. These results suggest that in the guinea-pig trachea, acetylcholine released from the adjacent parasympathetic nerves inhibits sympathetic neurotransmission via an action on prejunctional muscarinic receptors situated on the noradrenergic nerve terminals. These heteroreceptors appear to be similar to the airway smooth muscle M3 receptors and differ from the M2 autoreceptors on parasympathetic nerve terminals. The implication of these results for the rational design of antimuscarinic drugs is discussed

Diprosopus: A Rare Case of Craniofacial Duplication and a Systematic Review of the Literature

In 1990, Gorlin et al. described four types of craniofacial duplications: (1) single mouth with duplication of the maxillary arch; (2) supernumerary mouth laterally placed with rudimentary segments; (3) single mouth with replication of the mandibular segments; and (4) true facial duplication, namely diprosopus. We describe a newborn born with wide-spaced eyes, a very broad nose, and two separate mouths. Workup revealed the absence of the corpus callosum and the presence of a brain midline lipoma, wide sutures, and a Chiari I malformation with cerebellar herniation. We conducted a systematic review of the literature and compared all the cases described as diprosopus. In 96% of these, the central nervous system is affected, with anencephaly being the most commonly associated abnormality. Other associated anomalies include cardiac malformations (86%), cleft palate (63%), diaphragmatic hernia (13%), and disorder of sex development (DSD) (13%). Although the facial features are those that first strike the eye, the almost obligate presence of cerebral malformations suggests a disruptive event in the cephalic pole of the forming embryo. No major monogenic contribution has been recognized today for this type of malformation

An Ultrastructural and Histological Study of the Equine Respiratory Tract in Health and Disease, Volume I

There were two main aims in the work presented in this thesis. First, to carry out a detailed scanning electron microscopic (SEM), histological and, where appropriate, transmission electron microscopic (TEM) study of the surface features of the entire respiratory tract of normal horses. The second aim was to investigate the effects of respiratory disease (Streptococcus equi infection or Strangles and chronic obstructive pulmonary disease) on equine respiratory tract surfaces

Mutationsanalysen in Genen des Acetylcholin-Rezeptor-Pathways in Patienten mit Fetal Akinesia Deformation Sequence (FADS)

Die Fetale Akinesia Deformation Sequence (FADS) umfasst ein breites klinisches Spektrum. Dieses reicht von Tot- und Fehlgeburten, fetalen Ödemen bis hin zu Kontrakturen, Pterygien und Atemschwäche. Die Ätiologie der FADS ist sehr heterogen. Der Fokus dieser Forschungsarbeit lag in der Mutationsanalyse in Genen des Acetylcholin-Rezeptor-Pathways. Bekannt war das homozygote missense und nonsense Mutationen in den Genen der fetalen Untereinheit CHRNG des Rezeptors mit dem klinischen Bild des multiple Pterygien-Syndrom (MPS) und Letalen multiplen Pterygien-Syndrom (LMPS) einhergehen. Missense Mutationen in weiteren Genen des AChR-Komplexes präsentieren sich klinisch als Congenitales Myasthenes Syndrom (CMS). Vermutet, aber nicht bewiesen war das homozygote nonsense Mutationen in diesen weiteren Genen letal verlaufen und ursächlich für das letale Pterygiensyndrom sein können. Wir konnten diese Hypothese bestätigen.:1. Einführung in das Thema 1.1. Prävalenz und Relevanz fetaler Bewegungsstörungen und angeborener Kontrakturen 1.2. Das klinische Bild 1.3. Ursachen verminderter fetaler Bewegung 1.4. Der nicotinerge Acetylcholinrezeptor 1.5. Mutationssuche in den Genen der α1-, β1- und δ-Untereinheit (CHRNA1, CHRNB1 und CHRND) sowie in dem Rezeptor assoziierten RAPSN-Gen 2. Publikation 3. Zusammenfassung der Arbeit Literaturverzeichnis Anlagen Darstellung des eigenen wissenschaftlichen Beitrags Erklärung über die eigenständige Abfassung der Arbei

Hydrogels for nucleus replacement: facing a biomechanical challenge

Hydrogels are considered promising for disc regeneration strategies. However, it is currently unknown whether the destruction of the natural interface between nucleus and surrounding structures caused by nucleotomy and an inadequate annulus closure diminishes the mechanical competence of the disc. This in vitro study aimed to clarify these mechanisms and to evaluate whether hydrogels are able to restore the biomechanical behaviour of the disc. Nucleus pressure in an ovine intervertebral disc was measured in vivo during day and night and adapted to an in vitro axial compressive diurnal (15min) and night (30min) load. Effects of different defects on disc height and nucleus pressure were subsequently measured in vitro using 30 ovine motion segments. Following cases were considered: intact; annulus incision repaired by suture and glue; annulus incision with removal and re-implantation of nucleus tissue; and two different hydrogels repaired by suture and glue. The intradiscal pressure in vivo was 0.75 MPa during day and 0.5 MPa during night corresponding to an in vitro axial compressive force of 130 and 58 N, respectively. The compression test showed that neither the implantation of hydrogels nor the re-implantation of the natural nucleus, assumed as being the ideal implant, was able to restore the mechanical functionality of an intact disc. Results indicate the importance of the natural anchorage of the nucleus with its surrounding structures and the relevance of an appropriate annulus closure. Therefore, hydrogels that are able to mimic the mechanical behaviour of the native nucleus may fail in restoring the mechanical behaviour of the disc.This work was funded by the EU project DISC REGENERATION (NMP3-LA-2008-213904), and by the German Research Foundation (WI 1352 14-1)