Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells

Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds

Investigation of the fos-expression in the paraventricular nucleus of the hypothalamus in male rats following a peripheral application of ghrelin

Einleitung Zahlreiche Studien haben gezeigt, dass der PVN an der Kontrolle der Nahrungsaufnahme beteiligt ist. Unterschiedliche Neurotransmitter, die die Nahrungsaufnahme regulieren, beeinflussen die neuronale Aktivität dieses Hirnkerns. Das Neuropeptid Ghrelin stimuliert nach zentraler Applikation die Kurzzeitnahrungsaufnahme bei Nagetieren und aktiviert Neurone des PVN und NPY- positive Neurone des NARC. Injektionen des Peptids NPY in den PVN wiederum beeinflussen die Plasmaspiegel von ACTH und Kortikosteroiden. Andere Studien zeigten die morphologische Assoziation zwischen NPY-positiven Fasern und CRH- positiven Neuronen im mpPVN. Vermutlich wird im PVN durch Interaktion von Neurotransmittern/-modulatoren die Freisetzung von NPY aktiviert bzw. gehemmt und dadurch die Nahrungsaufnahme reguliert. An dieser Regulation ist möglicherweise auch CRH beteiligt. Während die Aktivierung von Neuronen des PVN nach intrazerebroventrikulärer Injektion von Ghrelin demonstriert werden konnte, blieb sie in bisherigen Untersuchungen nach peripherer Applikation aus. In der vorliegenden Studie wurden die Effekte von intraperitoneal injiziertem Ghrelin auf die Nahrungsaufnahme und die neuronale Aktivierung im Hypothalamus und im Hirnstamm bei Ratten untersucht. Ferner wurden die entsprechenden Neurone und Fasern phänotypisiert. Methoden Zur Untersuchung des Einflusses von Ghrelin auf die Nahrungsaufnahme erhielten männliche Sprague-Dawley Ratten 1 oder 10nmol Ghrelin oder isotone Nariumchloridlösung intraperitoneal injiziert. Die kumulative Nahrungsaufnahme wurde über vier Stunden gemessen. Um eine neuronale Aktivierung im Hypothalamus und im Hirnstamm durch Ghrelin zu überprüfen, wurden die Gehirne männlicher Sprague- Dawley Ratten nach peripherer Ghrelinverabreichung immunhistologisch aufgearbeitet. Dabei wurde 1nmol Ghrelin intraperitoneal injiziert, die Injektion von 1ml Natriumchlorid-Lösung diente als Kontrolle. Nach einer transkardialen Perfusionsfixierung und der Unterteilung der Gehirne in koronare Gewebeschnitte, erfolgte die immunhistologische Markierung des Gewebes. Dabei diente Fos als Marker für neuronale Aktivität im PVN, NTS und NARC. Die Bestimmung des neuropeptidergen Phänotyps der aktivierten Neurone erfolgte mittels einer Doppelmarkierung gegen CRH. Mit einer weiteren Doppelmarkierung gegen NPY wurde der neuropeptiderge Phänotypus der Nervenfasern in den aktivierten Gehirnarealen bestimmt. Ergebnisse Nach ip. Injektion von Ghrelin wurde im Vergleich zur Kontrolle mit Natriumchlorid- Lösung eine signifikant erhöhte kumulative Nahrungsaufnahme beobachtet. Der Vergleich der beiden Versuchsgruppen, die mit 1 bzw. 10nmol Ghrelin/Ratte behandelt wurden, ergab keinen signifikanten Unterschied in der kumulativen Nahrungsaufnahme. Die Auswertung der neuronalen Aktivität im Hypothalamus und im Hirnstamm ergab eine erhöhte Fos-Dichte im PVN und NARC und geringfügig auch in der AP nach ip. Injektion von 1nmol Ghrelin im Vergleich zu isotoner Natriumchlorid-Lösung. Die Fos-Expression im NTS blieb unbeeinflusst durch Ghrelin. Die Doppelmarkierungen zur Bestimmung des neuropetidergen Phänotypus der Neurone und Axone im PVN demonstrierten im Grossteil der Fos-positiven Neurone eine CRH-Expression in deren Zytoplasma und eine NPY-Expression in den umgebenden Nervenfasern. Diskussion Im Gegensatz zu bisherigen Studien, wurde in dieser Arbeit die Aktivierung des PVN nach peripherer Injektion von Ghrelin demonstriert. Da in früheren Studien das Ghrelin intravenös, in der vorliegenden Arbeit jedoch intraperitoneal verabreicht wurde, kann eine Beeinflussung der Ergebnisse durch die unterschiedlichen Applikationswege nicht ausgeschlossen werden. Zum Nachweis der neuronalen Aktivität wurde der Marker Fos genutzt. Da Fos ein unspezifischer Marker ist, kommen andere Faktoren als Ghrelin, wie z.B. Nahrungsaufnahme oder Stress, für seine Induktion in Frage. Um eine sekundäre Fos-Bildung durch Nahrungsaufnahme zu verhindern, wurde den Versuchtieren während der Experimente die Nahrung entzogen. Ob die Versuchstiere starkem Stress ausgesetzt waren, kann nicht mit Sicherheit gesagt werden. Somit könnte Angst oder durch Hunger ausgelöster Stress bei den Versuchstieren Fos induziert haben. Was jedoch dagegen spricht ist die signifikant höhere Fos-Expression im PVN der ghrelinbehandelten Tiere im Vergleich zur Kontrolle mit Natriumchloridlösung. Der Nachweis von CRH und NPY in aktivierten Arealen des PVN lässt ein Zusammenspiel dieser Peptide mit Ghrelin vermuten. Dabei aktiviert Ghrelin möglicherweise Neurone des PVN indirekt über NPY-positive Projektionen zwischen dem NARC und dem PVN. Ferner wird angenommen, dass Ghrelin die Nahrungsaufnahme durch die Beeinflussung des CRH-Spiegels stimuliert. Um diese Hypothese zu bekräftigen, müssten weitere histochemische Untersuchungen und Tracingstudien durchgeführt werden. Die Bedeutung vagaler Afferenzen bezüglich der Wirkung von peripherem Ghrelin auf die Nahrungsaufnahme wurde in dieser Arbeit nicht untersucht.Introduction Several studies have shown the involvement of the PVN in the regulation of food intake. Different neurotransmitters regulating the food intake influence the activity of this nucleus. The neuropeptide ghrelin stimulates the food intake in rodents after central application and activates PVN neurons as well as NPY-positive neurons of the NARC. Injections of NPY into the PVN on the other side influence serum levels of ACTH and Corticosteroids. Morphological associations between NPY-positiv axons and CRH- positive neurons in the pmPVN have been shown by other studies. The food intake is most probably regulated by neurotransmitters/- modulators within the PVN, which by interacting with each other activate or inhibit the release of NPY. CRH might be involved in this regulatory circuit. Intracerebroventricular injections of ghrelin have been shown to activate neurons of the PVN, however peripheral applications failed to demonstrate such an activity in previous studies. In the present study the effect of intraperitoneally applied ghrelin on the food intake and the neuronal activity in the hypothalamus and brainstem in rats was investigated. Furthermore a phenotypical characterization of the relevant neurons was performed. Methods To investigate the effect of ghrelin on the food intake , male Sprague-Dawley rats were injected intraperitoneally 1 or 10 nmol ghrelin per animal or isotonic sodiumchlorid solution. The food intake was measured over a four hour period. For the investigation of the effect of ghrelin on the hypothalamus and the brainstem, male Sprague-Dawley rats were given 1nmol ghrelin intraperitoneally and immunohistological studies were performed on the brain tissue. The control group received isotonic sodiumchloride solution. Following a transcardiac perfusion fixation, the brain tissue was processed, cut into coronary tissue sections and immunohistologically stained. Fos was used as a marker for neuronal activity in the PVN, NTS and NARC. The phenotypical characterization of the activated neurons was performed via a double staining for CRH. A double staining for NPY was used to characterize the axons in the activated areas. Results Following an intraperitoneal injection of ghrelin the food intake rose significantly compared to the control group. The comparison between both ghrelin groups, which received either 1 nmol or 10 nmol ghrelin per animal did not reveal any significant difference regarding the food intake. The interpretation of the neuronal activity in the hypothalamus and in the brainstem demonstrated a significant increase in the fos-expression in the PVN and NARC and a slight increase in the AP after ghrelin administartion compared to the control group. No differnce in the neuronal activity was observed in the NTS. The doublestaining for the phenotypical characterization of the neurons and axons in the PVN demonstrated that the majority of the fos-positive neurons expressed CRH in their cytoplasm and that the surrounding axons were NPY- positive. Discussion In contrary to previous publications, the present study demonstrated the activation of the PVN by peripheral administration of ghrelin. Given the fact, that in previous studies ghrelin had been injected intravenously, and in the present study it was applied by intraperitoneal injection, a difference caused by the different application routes cannot be excluded. Fos is an unspecific marker for neuronal activity and many factors, such as food intake or stress, can stimulate its expression. To prevent a secondary fos induction by food intake, the animals were fasted during the experiments. It is difficult to say wether the animals were exposed to extreme stress. Fear or hunger could have induced fos. However, this does not explain the significantly higher fos-expression in the PVN of the ghrelin treated animals compared to the control group. The demonstration of CRH and NPY in the activated PVN areas is suggestive for an interaction between these peptides with ghrelin. Ghrelin most probably activates neurons of the PVN indirectly via NPY-positive projections between the NARC and the PVN. Furthermore, it is believed that ghrelin stimulates the food intake by influencing CRH levels. Further histochemical and tracing studies have to be done to prove this hypothesis. The relevance of afferent vagal fibers regarding the effects of ghrelin on the food intake was not investigated in this study

Posttraumatic Stress Symptoms and Related Variables in Families of Children with Disabilities

The purpose of this study was to examine the effects and relationships of variables related to the children with disabilities and their parents as well as the diagnosis process and informing parents after the diagnosis and variables which might be related to the traumatic stress levels of the parents of children with disabilities on parents' traumatic stress levels. This study which was conducted as relational research included a total of 347 parents of children with disabilities. Data collection forms included personal information form, Traumatic Stress Symptom Scale, and Multidimensional Scale of Perceived Social Support. The results showed that traumatic stress levels of parents of children with physical impairments were higher than other parents, there was a significant relationship between the time passed after the diagnosis and parents' traumatic stress levels; there was significant relationship between the parents' age and parents's traumatic stress levels; parents' traumatic stress levels didn't differ in terms of gender; traumatic stress levels of single parents were higher than married parents

Increased glutathione conjugate transport: a possible compensatory protection mechanism against oxidative stress in obesity?

Objective: To compare glutathione S-conjugate transport in obese and nonobese persons, and how glutathione S-conjugates are involved in the antioxidant status in obesity

Did the Red Sea - Mediterranean connection over the Dead Sea Fault Zone end in the Late Pliocene?

WOS: 000374478500010The delta plain of the Asi (Orontes) river in Hatay (Turkey) is located between two regional major tectonic zones, the Dead Sea Fault Zone from the Red Sea to Antakya and the East Anatolian Fault Zone from Karliova to Antakya. Sediment samples from five cores performed on the Asi delta plain to 15 m depth were studied, and three fossil groups were identified in 5 samples. They mainly consist of 51 foraminiferal species from 42 genera; ostracod genera representing marine, brackish and fresh water environments, and Gastropoda and Bivalvia taxa. The abundance of some benthic Foraminifera in the recent sediment samples, including Euuvigerina reineri (Bedford), Siphonina tubulosa Cushman and Siphonodosaria abyssorum (Brady), which are common in the Pacific Ocean, Indian Ocean, Arabian Sea, and Red Sea is remarkable. In addition, Euuvigerina reineri (Bedford) individuals about 70 ka have been collected from Magaracik village to the north of the drilling area. The existence of these species in the Quaternary sediments indicates that the water connection via the Gulf of Aqaba on the Dead Sea Fault Zone was still open in the Pleistocene. (C) 2015 Elsevier Ltd and INQUA. All rights reserved

Plasma lipids and lipoproteins during pregnancy and related pregnancy outcomes

Ustuner, Isik/0000-0002-3791-4071WOS: 000320383500008PubMed: 23400357To study the effect of maternal lipid profile changes in pregnancy in relation to fetal growth and development, prognosis, and complications of pregnancy. One thousand pregnant women between 17 and 48 years of age were included in this prospective longitudinal and uni-center study. Lipid profile tests [triglyceride (TG), total cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL)] were first requested as part of the routine pregnancy follow-up in first antenatal visit ( 28 weeks). the analysis included the medical, social-demographic, and nutritional status of the women as well. Primer outcome measures were defined as the association of the pregnancy-related lipid profile change to neonatal weight, the weight of the infant in third month and pregnancy complications (preeclampsia, gestational diabetes mellitus, IUGR, and preterm birth). the levels of TG, total cholesterol, HDL, LDL increased significantly as pregnancy progressed. the percentage of the change in the TG levels were higher in patients with well nutritional parameters (p = 0.033). As the percentage of change in the TG levels increased, the neonatal weight increased (p = 0.033) but no effect on the placental weight and the third month weight of the infant was seen. As the percent change in TG levels decreased, the risk of the preterm birth significantly increased. in women who were positive in 50 g screening test, but were uncomplicated with gestational diabetes mellitus, the percent change in cholesterol was lower (p = 0.010), the percent change in LDL was lower (p = 0.015), and the percent change in TG was higher (p = 0.032). in pregnancy, complex alterations occur in lipid metabolism. Percent change in TG is affected positively by the nutrition level. the neonatal weight also increases as well but postnatal weight is unaffected. Conversely TG levels significantly decrease in preterm birth. No association between preeclampsia and gestational diabetes mellitus with lipid profile changes were noted except in patients with glucose intolerance (> 140 mg/dl in 50 g screening test) in which change in cholesterol, LDL was low and TG was high

Single Liver Lobe Repopulation with Wildtype Hepatocytes Using Regional Hepatic Irradiation Cures Jaundice in Gunn Rats

Abstract Background and Aims: Preparative hepatic irradiation (HIR), together with mitotic stimulation of hepatocytes, permits extensive hepatic repopulation by transplanted hepatocytes in rats and mice. However, whole liver HIR is associated with radiation-induced liver disease (RILD), which limits its potential therapeutic application. In clinical experience, restricting HIR to a fraction of the liver reduces the susceptibility to RILD. Here we test the hypothesis that repopulation of selected liver lobes by regional HIR should be sufficient to correct some inherited metabolic disorders