TY - JOUR A1 - Vargas, Juan Gamboa A1 - Wagner, Jennifer A1 - Shaikh, Haroon A1 - Lang, Isabell A1 - Medler, Juliane A1 - Anany, Mohamed A1 - Steinfatt, Tim A1 - Mosca, Josefina Peña A1 - Haack, Stephanie A1 - Dahlhoff, Julia A1 - Büttner-Herold, Maike A1 - Graf, Carolin A1 - Viera, Estibaliz Arellano A1 - Einsele, Hermann A1 - Wajant, Harald A1 - Beilhack, Andreas T1 - A TNFR2-Specific TNF fusion protein with improved in vivo activity JF - Frontiers in Immunology N2 - Tumor necrosis factor (TNF) receptor-2 (TNFR2) has attracted considerable interest as a target for immunotherapy. Indeed, using oligomeric fusion proteins of single chain-encoded TNFR2-specific TNF mutants (scTNF80), expansion of regulatory T cells and therapeutic activity could be demonstrated in various autoinflammatory diseases, including graft-versus-host disease (GvHD), experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA). With the aim to improve the in vivo availability of TNFR2-specific TNF fusion proteins, we used here the neonatal Fc receptor (FcRn)-interacting IgG1 molecule as an oligomerizing building block and generated a new TNFR2 agonist with improved serum retention and superior in vivo activity. Methods Single-chain encoded murine TNF80 trimers (sc(mu)TNF80) were fused to the C-terminus of an in mice irrelevant IgG1 molecule carrying the N297A mutation which avoids/minimizes interaction with Fcγ-receptors (FcγRs). The fusion protein obtained (irrIgG1(N297A)-sc(mu)TNF80), termed NewSTAR2 (New selective TNF-based agonist of TNF receptor 2), was analyzed with respect to activity, productivity, serum retention and in vitro and in vivo activity. STAR2 (TNC-sc(mu)TNF80 or selective TNF-based agonist of TNF receptor 2), a well-established highly active nonameric TNFR2-specific variant, served as benchmark. NewSTAR2 was assessed in various in vitro and in vivo systems. Results STAR2 (TNC-sc(mu)TNF80) and NewSTAR2 (irrIgG1(N297A)-sc(mu)TNF80) revealed comparable in vitro activity. The novel domain architecture of NewSTAR2 significantly improved serum retention compared to STAR2, which correlated with efficient binding to FcRn. A single injection of NewSTAR2 enhanced regulatory T cell (Treg) suppressive activity and increased Treg numbers by > 300% in vivo 5 days after treatment. Treg numbers remained as high as 200% for about 10 days. Furthermore, a single in vivo treatment with NewSTAR2 upregulated the adenosine-regulating ectoenzyme CD39 and other activation markers on Tregs. TNFR2-stimulated Tregs proved to be more suppressive than unstimulated Tregs, reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, singular preemptive NewSTAR2 administration five days before allogeneic hematopoietic cell transplantation (allo-HCT) protected mice from acute GvHD. Conclusions NewSTAR2 represents a next generation ligand-based TNFR2 agonist, which is efficiently produced, exhibits improved pharmacokinetic properties and high serum retention with superior in vivo activity exerting powerful protective effects against acute GvHD. KW - agonist KW - GvHD KW - regulatory T cells KW - serum retention KW - TNF KW - TNFR2 Y1 - 2022 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-277436 SN - 1664-3224 VL - 13 ER - TY - THES A1 - Jazbutyte, Virginija T1 - Differential role of estrogen receptor isoforms in the cardiovascular system of young and senescent rats T1 - Differenzielle Rolle der Estrogen Rezeptor Isoformen in dem kardiovaskulären System von jungen and seneszenten Ratten N2 - Cardiovascular disease is the major cause of mortality morbidity in both men and women in industrialized countries. The incidence of cardiovascular diseases in pre-menopausal women is lower compared to age-matched men but the risk of heart diseases increases dramatically after the onset of menopause.Therefore, it has been postulated that female sex hormones play an important role in cardiovascular health in pre-menopausal women. In contrast to clinical data, which failed to show positive estrogen effects on cardiovascular system of post- menopausal women, extensive experimental studies indicated cardioprotective effects of estrogens in laboratory animals. The majority of experimental estrogen substitution studies were performed with young individuals, thus the effects of ageing remain neglected and are poorly understood. The present project is the first attempt to study the cardiac effects of each estrogen receptor isoform (estrogen receptor alpha (ERa) and estrogen receptor beta (ERb)) in adult (“menopausal”) and senescent (“post- menopausal”) hypertensive rats. The female senescent spontaneously hypertensive rats (SHR) served as a model system for age- associated hypertension in females whereas young individuals were used for control experiments. Young and senescent SHR rats were treated with 17b- estradiol as well as new estrogen receptor isoform selective ligands 16a-LE2 (ERa agonist) and 8b-VE2 (ERb agonist). The results showed different functions of both estrogen receptor isoforms in cardiovascular system: ERa attenuated cardiac hypertrophy but not hypertension whereas ERb could significantly reduce both, blood pressure and cardiac hypertrophy. Surprisingly, both agonists and 17b- estradiol were effective in young animals but not in senescent SHR rats. These findings match with the clinical data and could be related to altered estrogen metabolism in senescent rats, since estrogen plasma levels did not increase to measurable extent in senescent animals receiving estrogen. Estrogen is metabolized by several 17b- hydroxysteroid dehydrogenase isoforms. In the current study, 17b- hydroxysteroid dehydrogenase type 10 (17b- HSD10) was identified as a novel protein- protein interaction partner of estrogen receptor alpha ligand binding domain (ERaLBD) in human heart. Cellular localization experiments of ERa in the cardiac myocytes showed nuclear and cytosolic localization pattern which overlapped partially with that of cardiac mitochondria. 17b-HSD10 is localized only in mitochondria. Direct interaction of both proteins was confirmed by pull- down experiments where 17b-HSD10 could be co-precipitated with ERa. Interestingly, protein interaction could be detected only under estrogen- free conditions whereas the presence of estrogen in the system blocked this interaction. Enzymatic assay which was developed in our laboratory, helped to define functional relevance of this interaction. The data obtained from enzymatic assays and protein- protein interaction studies strongly suggest that estrogen receptor could play an important role in the control of intracellular (or mitochondrial) estogen metabolism. The second potential ERa interaction partner in the heart- bladder cancer associated protein 10 (BLCAP10) - was initially identified in non- invasive bladder cancer cell lines. BLCAP10 protein expression in the heart as well as its localization pattern in cardiac myocytes is shown in the last part of the theses. Due to perinuclear localization similarity with ERb, we conclude that BLCAP10 could interact with ERb rather than with ERa. Poor BLCAP10 protein overexpression and toxicity in both, bacteria and eukaryotic cells, suggested that BLCAP10 could be involved in cell- cycle and/ or protein expression control. In summary, the results showed that isoform selective activation of estrogen receptors exert divergent effects in the cardiovascular system both by upregulation of aMHC expression or by lowering blood pressure. Hormones were effective in young animals but had only minor effects in senescent rats. The new ERa protein- protein interaction partners identified during the project provide new information about estrogen receptor function in the heart and its possible role in the regulation of estrogen homeostasis. N2 - In den Industrieländern sind kardiovaskuläre Erkrankungen die Hauptursache von Mortalität und Morbidität bei Männer und Frauen. Das Auftreten kardiovaskulärer Erkrankungen ist bei pre-menopausalen Frauen niedriger als bei Männern, doch steigt das Risiko nach der Menopause dramatisch an. Aus diesem Grund wurde postuliert, dass weibliche Sexualhormone eine wichtige Rolle bei der Prävention kardiovaskulärer Erkrankungen spielen. Obwohl in klinischen Studien für das Estrogen kein Effekt nachgewiesen wurde, ist dessen kardioprotektiver Effekt experimentell belegt. Dies mag daran liegen, dass in aller Regel für diese Experimente Jungtiere verwendet wurden. Hierdurch wurden die noch unzureichend verstandenen Auswirkungen der Alterung vermieden. In der vorliegenden Arbeit wurden zum ersten Mal kardioprotektive Effekte der beiden Estrogenrezeptor (ER)-Isoformen ERa und ERb in erwachsenen („menopausalen“) und seneszenten („post-menopausalen) hypertensiven Ratten nachgewiesen. Weibliche seneszente spontan-hypertensive Ratten (SHR) wurden als Modell für den Alters-assoziierten Bluthochdruck bei Frauen verwendet. Junge Ratten dienten als Kontrolle. Junge und seneszente Ratten wurden mit 17β-Estradiol und den neuen ERa- und ERb-Agonisten 16a-LE2 bzw. 8b-VE2 behandelt. Beide Estrogenrezeptor-Isoformen wirkten unterschiedlich auf das kardiovaskuläre System. So minderte 16a-LE2 die kardiale Hypertrophie, nicht aber die Hypertension, während 8b-VE2 sowohl Hypertrophie als auch Hypertension verringerte. Beide Agonisten und 17b-Estradiol waren überraschenderweise nur in jungen, nicht aber in seneszenten Ratten effektiv. Diese Ergebnisse korrelieren mit klinischen Daten und lassen sich vermutlich mit dem geänderten Estrogen-Metabolismus älterer Ratten erklären. So waren ihre Estrogen-Plasmaspiegel nach Estrogen-Injektion nicht erhöht. Estrogen wird durch zahlreiche Isoformen der 17b-Hydroxysteroid-Dehydrogenase metabolisiert. In dieser Arbeit wurde die Isoform Typ 10 (17b- HSD10) als bisher unbekannter Interaktionspartner für die Ligandenbindungs-Domäne des Estrogenrezeptors in menschlichen Herzen identifiziert. Der Estrogenrezeptor ERα wurde im Kern und Zytosol humaner Kardiomyozyten nachgewiesen. Zytosolisches Lokalisationsmuster deckte sich teilweise mit dem von Herzmitochondrien. 17b- HSD10 ist ausschließlich in Mitochondrien vorhanden. Die direkte Interaktion zwischen ERa und 17b- HSD10 wurde mit pull down-Experimenten bestätigt, wo beide Proteine co-präzipitiert wurden. Die Interaktion von ERa und 17b- HSD10 wurde interessanterweise nur unter estrogenfreien Bedingungen nachgewiesen, während Estrogen diese Interaktion blockierte. Mit den in unserem Labor etablierten enzymatischen Untersuchungen wurde die funktionelle Bedeutung dieser Interaktion untersucht. Diese Ergebnisse sowie die Protein-Protein Interaktionsstudien lassen auf eine wichtige Rolle des Estrogen-Rezeptors ERa bei der Kontrolle des zellulären und mitochondrialen Estrogen-Metabolismus schließen. Der zweite Interaktionspartner von ERa ist BLCAP10, das „Bladder Cancer Associated Protein 10", das ursprünglich in Zelllinien nicht-invasiver Blasenkarzinome entdeckt wurde. Die Ergebnisse zur Expression von BLCAP10 im Herzen sowie das Lokalisationsmuster in Kardiomyozyten sind im letzten Teil der Arbeit vorgestellt. Wegen der Ähnlichkeit der perinukleären Lokalisation von BLCAP10 und ERb erscheint eine Interaktion beider Proteine möglich. Die geringe Überexpression von BLCAP10 in Bakterien und eukaryotischen Zellen deutet auf eine Funktion von BLCAP10 im Zellzyklus oder bei der Proteinexpression hin. Die Ergebnisse zeigen somit, dass eine Isoform-selektive Aktivierung von Estrogenrezeptoren divergente Effekte im kardiovaskulären System auslösen. So kommt es zu einer Hochregulierung der aMHC-Expression oder zur Senkung des Bluthochdrucks. Hormone, 17b-Estradiol bzw. die Agonisten 16a-LE2 und 8b-VE2, waren effektiv in jungen Tieren, zeigten aber nur geringe Effekte in alten Tieren. Der in dieser Arbeit identifizierte neue Interaktionspartner von ERa, das 17b-HSD10, liefert neue Informationen zur Funktion des Estrogenrezeptors im Herzen und zur möglichen Rolle bei der Regulierung der Estrogen-Homeostasis. KW - Östrogene KW - Kardiovaskuläres System KW - Östrogen- Rezeptor KW - Agonist KW - Estrogen receptor alpha KW - agonist KW - ligand binding domain KW - protein- protein interaction partner Y1 - 2007 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-24247 ER - TY - JOUR A1 - Schneider, Christof W. A1 - Tautz, Jürgen A1 - Grünewald, Bernd A1 - Fuchs, Stefan T1 - RFID Tracking of Sublethal Effects of Two Neonicotinoid Insecticides on the Foraging Behavior of Apis mellifera JF - PLoS One N2 - The development of insecticides requires valid risk assessment procedures to avoid causing harm to beneficial insects and especially to pollinators such as the honeybee Apis mellifera. In addition to testing according to current guidelines designed to detect bee mortality, tests are needed to determine possible sublethal effects interfering with the animal's vitality and behavioral performance. Several methods have been used to detect sublethal effects of different insecticides under laboratory conditions using olfactory conditioning. Furthermore, studies have been conducted on the influence insecticides have on foraging activity and homing ability which require time-consuming visual observation. We tested an experimental design using the radiofrequency identification (RFID) method to monitor the influence of sublethal doses of insecticides on individual honeybee foragers on an automated basis. With electronic readers positioned at the hive entrance and at an artificial food source, we obtained quantifiable data on honeybee foraging behavior. This enabled us to efficiently retrieve detailed information on flight parameters. We compared several groups of bees, fed simultaneously with different dosages of a tested substance. With this experimental approach we monitored the acute effects of sublethal doses of the neonicotinoids imidacloprid (0.15-6 ng/bee) and clothianidin (0.05-2 ng/bee) under field-like circumstances. At field-relevant doses for nectar and pollen no adverse effects were observed for either substance. Both substances led to a significant reduction of foraging activity and to longer foraging flights at doses of >= 0.5 ng/bee (clothianidin) and >= 1.5 ng/bee (imidacloprid) during the first three hours after treatment. This study demonstrates that the RFID-method is an effective way to record short-term alterations in foraging activity after insecticides have been administered once, orally, to individual bees. We contribute further information on the understanding of how honeybees are affected by sublethal doses of insecticides. KW - memory KW - nicotinic acetylcholine-receptors KW - unpaired median neurons KW - honey bees KW - learning performances KW - toxicity KW - hymenoptera KW - pesticides KW - relevance KW - agonist Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-131753 VL - 7 IS - 1 ER -