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

Promising activity of Anthemis austriaca Jacq. on the endometriosis rat model and isolation of its active constituents

© 2019, The Author(s). Heparin and heparan sulfate (Hp/HS) are linear complex glycosaminoglycans which are involved in diverse biological processes. The structural complexity brings difficulties in separation, making the study of structure-function relationships challenging. Here we present a separation method for Hp/HS oligosaccharide fractionation with cross-compatible solvent and conditions, combining size exclusion chromatography (SEC), ion-pair reversed phase chromatography (IPRP), and hydrophilic interaction chromatography (HILIC) as three orthogonal separation methods that do not require desalting or extensive sample handling. With this method, the final eluent is suitable for structure-function relationship studies, including tandem mass spectrometry and microarray printing. Our data indicate that high resolution is achieved on both IPRP and HILIC for Hp/HS isomers. In addition, the fractions co-eluted in IPRP could be further separated by HILIC, with both separation dimensions capable of resolving some isomeric oligosaccharides. We demonstrate this method using both unpurified reaction products from isomeric synthetic hexasaccharides and an octasaccharide fraction from enoxaparin, identifying isomers resolved by this multi-dimensional separation method. We demonstrate both structural analysis by MS, as well as functional analysis by microarray printing and screening using a prototypical Hp/HS binding protein: basic-fibroblast growth factor (FGF2). Collectively, this method provides a strategy for efficient Hp/HS structure-function characterization

カニクイザルを利用した子宮内膜症モデルの確立

この博士論文は内容の要約のみの公開（または一部非公開）になっています筑波大学 (University of Tsukuba)201

Epidemiologic characteristics of amniotic band sequence with limb malformations without body wall defect: data from the Polish Registry of Congenital Malformations

Abstract Amniotic Band Sequence (ABS) is a rare disruptive condition, with a variable spectrum of congenital defects caused by fibrous bands emerging as a result of amniotic rupture in the first trimester of gestation. Several factors, such as young parental age, primigravidity, febrile maternal illness, and drug use in the first trimester, were postulated to have substantial influence on ABS prevalence rate. We aimed our study to determine the prevalence of ABS with limb defects, but no body wall affectation, in a Polish population. We also examined the influence of different parental, gestational and environmental factors on the ABS prevalence value, and assessed the rate of gestational complications associated with this disorder. Among 1 706 639 births surveilled between 1998 and 2005, 36 liveborn infants with ABS-L were reported to the Polish Registry of Congenital Malformations, giving a global prevalence for a Polish population of 1 per 47 619 livebirths. We found that young maternal age, young paternal age, and primigravidity significantly increase the risk of ABS-L, when their effect was analyzed independently. However, because of a close relationship of these variables, we analyzed their mutually adjusted effect using conditional logistic regression models, and found that young maternal age proved the strongest risk factor for ABS-L (p = 0.0508). The condition was also more prevalent in infants with low birthweight (OR = 5.71; p < 0.0001). Since gestational complications are often relevant to maternal age and birth order, we introduced an adjustment for these variables, and found that respiratory tract infections and vaginal bleeding/spotting convey approximately fourfold increased risk of ABS-L (OR = 3.72/p = 0.0058 and OR = 3.70/p = 0.0014 respectively)

Life-long Programming Implications of Exposure to Tobacco Smoking and Nicotine Before and Soon After Birth: Evidence for Altered Lung Development

Tobacco smoking during pregnancy remains common, especially in indigenous communities, and likely contributes to respiratory illness in exposed offspring. It is now well established that components of tobacco smoke, notably nicotine, can affect multiple organs in the fetus and newborn, potentially with life-long consequences. Recent studies have shown that nicotine can permanently affect the developing lung such that its final structure and function are adversely affected; these changes can increase the risk of respiratory illness and accelerate the decline in lung function with age. In this review we discuss the impact of maternal smoking on the lungs and consider the evidence that smoking can have life-long, programming consequences for exposed offspring. Exposure to maternal tobacco smoking and nicotine intake during pregnancy and lactation changes the genetic program that controls the development and aging of the lungs of the offspring. Changes in the conducting airways and alveoli reduce lung function in exposed offspring, rendering the lungs more susceptible to obstructive lung disease and accelerating lung aging. Although it is generally accepted that prevention of maternal smoking during pregnancy and lactation is essential, current knowledge of the effects of nicotine on lung development does not support the use of nicotine replacement therapy in this group

The Unmet Needs for Studying Chronic Pelvic/Visceral Pain Using Animal Models

The different definitions of chronic pelvic/visceral pain used by international societies have changed over the years. These differences have a great impact on the way researchers study chronic pelvic/visceral pain. Recently, the role of systemic changes, including the role of the central nervous system, in the perpetuation and chronification of pelvic/visceral pain has gained weight. Consequently, researchers are using animal models that resemble those systemic changes rather than using models that are organ- or tissue-specific. In this review, we discuss the advantages and disadvantages of using bladder-centric and systemic models, enumerating some of the central nervous system changes and pain-related behaviors occurring in each model. We also present some drawbacks when using animal models and pain-related behavior tests and raise questions about possible, yet to be demonstrated, investigator-related bias. We also suggest new approaches to study chronic pelvic/visceral pain by refining existing animal models or using new ones.</jats:p

СУЧАСНІ ПОГЛЯДИ НА ПАТОГЕНЕЗ ЕКСТРАГЕНІТАЛЬНОГО ЕНДОМЕТРІОЗУ (Огляд літератури)

SUMMARY. Endometriosis is a chronic benign hormone-dependent condition in which endometrium spreads and grows outside the uterine mucosa, with identical to eutopic endometrium morphological and functional properties. This review is dedicated to modern view on pathogenesis of extragenital endometriosis. The article presents both the theory of etiology and pathogenesis of this pathology and possible mechanisms that can potentially be considered as the key to pathogenetic therapy. Endometriosis is detected in 50 % of women with dysmenorrhea, in 50–80 % of patients with chronic pelvic pain and in 25–40 % of women with infertility, and in the structure of gynecological disease, endometriosis ranks third after inflammatory processes and uterine fibroids. Сonsidering the not-too-high effectiveness of treatment, a significant percentage of recurrence of the disease, the issue of updating views on pathogenetic mechanisms in order to optimize the therapeutic endometriosis tactic still remains relevant. The aim – to conduct the literature analysis in order to identify contemporary views on the pathogenesis of extragenital endometriosis and possible points of application in the development of targeted therapy. Conclusions. The most widely accepted theory of pathogenesis of endometriosis is the theory of retrospective menstruation of Sampson. This theory suggests that viable fragments of the endometrial tissue disintegrate into the peritoneal cavity or into the pelvic organs. The most probable evolution of this theory should be the theory of angiogenesis on the background of immune dysfunction, which contributes to the primary implantation of endometrioid ectopic and their subsequent development. Modern literature points to the role of angiogenesis in the formation and development of lesions, but at the same time, there is an immune dysfunction, which probably leads to the predominance of angiogenic factors locally.РЕЗЮМЕ. Эндометриоз – это хроническое доброкачественное гормонозависимое состояние, при котором за пределами слизистой оболочки матки происходит разрастание ткани, по морфологическим и функциональным свойствам идентичной эндометрию. Приведены данные обзора литературы, посвященные современным взглядам относительно патогенеза экстрагенитального эндометриоза. В статье приведены как теории этиологии и патогенеза данной патологии, так и возможные механизмы, которые могут рассматриваться как ключ к патогенетической терапии. Эндометриоз обнаруживают у 50 % женщин с дисменореей, у 50–80 % пациенток с хронической тазовой болью и у 25–40 % женщин с бесплодием, а в структуре гинекологической заболеваемости эндометриоз занимает третье место после воспалительных процессов и миомы матки. Учитывая не слишком высокую эффективность лечения, значительный процент рецидивов заболевания вопрос актуализации взглядов на патогенетические механизмы с целью оптимизации лечебной тактики эндометриоза до сих пор остается актуальным. Цель – провести анализ литературы с целью выявления современных взглядов относительно патогенеза екстрагениатльного эндометриоза и возможных точек приложения при разработке таргетной терапии. Выводы. Наиболее широко принятой теорией патогенеза эндометриоза является теория ретроградной менструации Сэмпсона. Данная теория предполагает, что жизнеспособные фрагменты эндометрия ткани дисеминируються в перитонеальную полость или в органы малого таза. Наиболее вероятным эволюционированием данной теории следует считать теорию ангиогенеза на фоне иммунной дисфункции, которая способствует первичной имплантации эндометриоидных эктопий и их дальнейшему развитию. Современные данные литературы ставят во главу угла роль ангиогенеза в становлении и развитии поражений, в то же время имеет место иммунная дисфункция, которая, вероятно, приводит к преобладанию ангиогенных факторов локально.РЕЗЮМЕ. Ендометріоз – це хронічний доброякісний гормонозалежний стан, при якому за межами слизової оболонки матки відбувається розростання тканини, за морфологічними та функціональними властивостями ідентичної ендометрію. Наведені дані огляду літератури присвячені сучасним поглядам на патогенез екстрагенітального ендометріозу. В статті висвітлені теорії щодо етіології та патогенезу цієї патології, а також описані можливі механізми, які потенційно можна розглядати як ключ до патогенетичної терапії. Ендометріоз виявляють у 50 % жінок з дисменореєю, у 50–80 % пацієнток із хронічним тазовим болем та у 25–40 % жінок із безпліддям, а в структурі гінекологічної захворюваності ендометріоз посідає третє місце після запальних процесів та міоми матки. Враховуючи не надто високу ефективність лікування та значний відсоток рецидивування захворювання питання актуалізації поглядів на патогенетичні механізми з метою оптимізації лікувальної тактики ендометріозу й досі залишається актуальним. Мета – провести аналіз літератури з метою виявлення сучасних поглядів щодо патогенезу екстрагеніатльного ендометріозу та можливі точки прикладання при розробці таргетної терапії. Висновки. Найширше прийнятою теорією патогенезу ендометріозу є теорія ретроградної менструації Семпсона. Ця теорія припускає, що життєздатні фрагменти ендометріальної тканини дисемінуються в перитонеальну порожнину або в органи малого таза. Найімовірнішим еволюціонуванням даної теорії слід вважати теорію ангіогенезу на тлі імунної дисфункції, яка сприяє первинній імплантації ендометріоїдних ектопій та їх подальшому розвитку. Сучасні дані літератури ставлять на чільне місце в становленні та розвитку уражень роль ангіогенезу, водночас має місце імунна дисфункція, яка, ймовірно, призводить до переважання ангіогенних факторів локально

The frequency of calcium oscillations induced by 5-HT, ACH, and KCl determine the contraction of smooth muscle cells of intrapulmonary bronchioles

Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca(2+)](i) of the SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH) induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of the airway SMCs but had little effect on airway size. 5-HT and ACH induced asynchronous oscillations in [Ca(2+)](i) that propagated as Ca(2+) waves within the airway SMCs. The frequency of the Ca(2+) oscillations was dependent on the agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular Ca(2+) or in the presence of Ni(2+), the frequency of the Ca(2+) oscillations declined and the airway relaxed. By contrast, KCl induced low frequency Ca(2+) oscillations that were associated with SMC twitching. Each KCl-induced Ca(2+) oscillation consisted of a large Ca(2+) wave that was preceded by multiple localized Ca(2+) transients. KCl-induced responses were resistant to neurotransmitter blockers but were abolished by Ni(2+) or nifedipine and the absence of extracellular Ca(2+). Caffeine abolished the contractile effects of 5-HT, ACH, and KCl. These results indicate that (a) 5-HT and ACH induce airway SMC contraction by initiating Ca(2+) oscillations, (b) KCl induces Ca(2+) transients and twitching by overloading and releasing Ca(2+) from intracellular stores, (c) a sustained, Ni(2+)-sensitive, influx of Ca(2+) mediates the refilling of stores to maintain Ca(2+) oscillations and, in turn, SMC contraction, and (d) the magnitude of sustained airway SMC contraction is regulated by the frequency of Ca(2+) oscillations

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

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