N-linked Glycosylation Is Required for Optimal Function of Kaposi's Sarcoma Herpesvirus–encoded, but Not Cellular, Interleukin 6

Kaposi's sarcoma–associated herpesvirus interleukin-6 (vIL-6) is a structural and functional homologue of the human cytokine IL-6 (hIL-6). hIL-6 and vIL-6 exhibit similar biological functions and both act via the gp130 receptor subunit to activate the Janus tyrosine kinase (JAK)1 and signal transducer and activator of transcription (STAT)1/3 pathway. Here we show that vIL-6 is N-linked glycosylated at N78 and N89 and demonstrate that N-linked glycosylation at site N89 of vIL-6 markedly enhances binding to gp130, signaling through the JAK1-STAT1/3 pathway and functions in a cytokine-dependent cell proliferation bioassay. Although hIL-6 is also N-glycosylated at N73 and multiply O-glycosylated, neither N-linked nor O-linked glycosylation is necessary for IL-6 receptor α–dependent binding to gp130 or signaling through JAK1-STAT1/3. As distinct from vIL-6, unglycosylated hIL-6 is as potent as glycosylated hIL-6 in stimulating B cell proliferation. These findings highlight distinct functional roles of N-linked glycosylation in viral and cellular IL-6

Prostaglandin E2 promotes survival of naive UCB T cells via the Wnt/β-catenin pathway and alters immune reconstitution after UCBT

The outcome of umbilical cord blood transplantation (UCBT) is compromised by low hematopoietic stem cell (HSC) doses leading to prolonged time to engraftment, delayed immunological reconstitution and late memory T-cell skewing. Exposure of UCB to dimethyl-prostaglandin E2 (dmPGE2) increases HSC in vivo. We determined that exposure of UCB T lymphocytes to dmPGE2 modified Wnt signaling resulting in T cell factor (TCF)-mediated transcription. Wnt signaling upregulated interleukin (IL)-7R and IL-2Rβ, resulting in enhanced survival mediated by the homeostatic cytokines IL-7 and IL-15. dmPGE2 also induced components of the Wnt pathway and Wnt receptors, thereby priming UCB T cells to receive signals via Wnt ligands in vivo. We observed that the Wnt transcription factor TCF7 and its target EOMES were elevated in the T cells of patients who received PGE2-treated UCBs. Consistent with the role of Wnt/β-catenin signaling to induce and maintain naive, memory precursors and long-lived central memory CD8+ cells, these patients also had increased fractions of CD8+CD45RO-CD62L+ plus CD8+CD45RO+CD62L+ subsets encompassing these T-cell populations. These effects of the PGE2/Wnt/β-catenin axis may have significant implications for harnessing immunity in the context of UCBT, where impaired immune reconstitution is associated with late memory T-cell skewing

A Bortezomib-Based Regimen Offers Promising Survival and Graft-versus-Host Disease Prophylaxis in Myeloablative HLA-Mismatched and Unrelated Donor Transplantation: A Phase II Trial

AbstractHematopoietic stem cell transplantation (HSCT) recipients lacking HLA-matched related donors have increased graft-versus-host disease (GVHD) and nonrelapse mortality (NRM). Bortezomib added to reduced-intensity conditioning can offer benefit in T cell–replete HLA-mismatched HSCT and may also benefit myeloablative conditioning (MAC) transplants. We conducted a phase II trial of short-course bortezomib plus standard tacrolimus/methotrexate after busulfan/fludarabine MAC in 34 patients with predominantly myeloid malignancies. Fourteen (41%) received 8/8 HLA-matched unrelated donor (MUD) and 20 (59%) received 7/8 HLA-mismatched related/unrelated donor peripheral blood stem cell grafts. Median age was 49 years (range, 21 to 60), and median follow-up was 25 months (range, 11 to 36). The regimen was well tolerated. No dose modifications were required. Neutrophil and platelet engraftment occurred at a median of 14 (range, 10 to 33) and 17 (range, 10 to 54) days, respectively. Median 30-day donor chimerism was 99% (range, 90 to 100), and 100-day grades II to IV and III to IV acute GVHD incidence was 32% and 12% respectively. One-year chronic GVHD incidence was 50%. Two-year cumulative incidence of both NRM and relapse was 16%. Two-year progression-free and overall survival rates were 70% and 71%, respectively. Outcomes were comparable to an 8/8 MUD MAC cohort (n = 45). Immune reconstitution was robust. Bortezomib-based MAC HSCT is well tolerated, with HLA-mismatched outcomes comparable with 8/8 MUD MAC HSCT, and is suitable for randomized evaluation. (clinicaltrials.gov: NCT01323920.

Genetics of tension-type headache

The objective of this study was to investigate the importance of genetics in tension-type headache. A MEDLINE search from 1966 to December 2006 was performed for “tension-type headache and prevalence” and “tension-type headache and genetics” The prevalence of tensiontype headache varies from 11 to 93%, with a slight female preponderance. Co-occurrence of migraine increases the frequency of tension-type headache. A family study of chronic tension-type headache suggests that genetic factors are important. A twin study analysing tension-type headache in migraineurs found that genetic factors play a minor role in episodic tension-type headache. Another twin study analysing twin pairs without co-occurrence of migraine showed a significantly higher concordance rate among monozygotic than same-gender dizygotic twin pairs with no or frequent episodic tension-type headache, while the difference was minor in twin pairs with infrequent episodic tensiontype headache. Frequent episodic and chronic tension-type headache is caused by a combination of genetic and environmental factors, while infrequent episodic tensiontype headache is caused primarily by environmental factors

Prevalence of headache in Europe: a review for the Eurolight project

The main aim of the present study was to do an update on studies on headache epidemiology as a preparation for the multinational European study on the prevalence and burden of headache and investigate the impact of different methodological issues on the results. The study was based on a previous study, and a systematic literature search was performed to identify the newest studies. More than 50% of adults indicate that they suffer from headache in general during the last year or less, but when asked specifically about tension-type headache, the prevalence was 60%. Migraine occurs in 15%, chronic headache in about 4% and possible medication overuse headache in 1–2%. Cluster headache has a lifetime prevalence of 0.2–0.3%. Most headaches are more prevalent in women, and somewhat less prevalent in children and youth. Some studies indicate that the headache prevalence is increasing during the last decades in Europe. As to methodological issues, lifetime prevalences are in general higher than 1-year prevalences, but the exact time frame of headache (1 year, 6 or 3 months, or no time frame stated) seems to be of less importance. Studies using personal interviews seem to give somewhat higher prevalences than those using questionnaires

Bortezomib-based immunosuppression after reduced-intensity conditioning hematopoietic stem cell transplantation: randomized phase II results

Aprior phase I/II trial of bortezomib/tacrolimus/methotrexate prophylaxis after human leukocyte antigen (HLA)-mismatched reduced intensity conditioning allogeneic hematopoietic stem cell transplantation documented low acute graft-versus-host disease incidence, with promising overall and progression-free survival. We performed an open-label three-arm 1:1:1 phase II randomized controlled trial comparing grade II–IV acute graft-versus-host disease between conventional tacrolimus/methotrexate (A) versus bortezomib/tacrolimus/methotrexate (B), and versus bortezomib/sirolimus/tacrolimus (C), in reduced intensity conditioning allogeneic transplantation recipients lacking HLA-matched related donors. The primary endpoint was grade II–IV acute graft-versus-host disease incidence rate by day +180. One hundred and thirty-eight patients (A 46, B 45, C 47) with a median age of 64 years (range: 24–75), varying malignant diagnoses and disease risk (low 14, intermediate 96, high/very high 28) received 7–8/8 HLA-mismatched (40) or matched unrelated donor (98) grafts. Median follow up in survivors was 30 months (range: 14–46). Despite early immune reconstitution differences, day +180 grade II-IV acute graft-versus-host disease rates were similar (A 32.6%, B 31.1%, C 21%; P=0.53 for A vs. B, P=0.16 for A vs. C). The 2-year non-relapse mortality incidence was similar (A 14%, B 16%, C 6.4%; P=0.62), as were relapse (A 32%, B 32%, C 38%; P=0.74), chronic graft-versus-host disease (A 59%, B 60% C 55%; P=0.66), progression-free survival (A 54%, B 52%, C 55%; P=0.95), and overall survival (A 61%, B 62%, C 62%; P=0.98). Overall, the bortezomib-based regimens evaluated did not improve outcomes compared with tacrolimus/methotrexate therapy. clinicaltrials.gov Identifier: 0175438

Splice Variants of the Stimulatory Gα-Protein Alpha Subunit and GPCR-mediated Activation of Adenylyl Cyclase

Titelblatt, Inhaltsverzeichnis und Abkürzungen 1\. Einleitung 1 2\. Zielstellung 23 3\. Material und Methoden 25 4\. Ergebnisse 57 5\. Diskussion 109 6\. Zusammenfassung 125 7\. Summary 127 8\. Literatur 129 AnhangDas stimulatorische Guaninnukleotid-bindende Protein (Gs) überträgt Signale von Gs-gekoppelten Rezeptoren (GPCR) zu Adenylylcyclasen und erhöht dadurch die Produktion des second messengers cAMP. In Geweben von Säugerorganismen werden vier Spleißvarianten der α-Untereinheit von Gs (Gαs) exprimiert. Die einzelnen Spleißvarianten sind in zahlreichen Untersuchungen charakterisiert worden, jedoch sind die Ergebnisse wegen unterschiedlicher Messparameter und -systeme weder einheitlich noch vergleichbar. Für heterolog exprimierte, aufgereinigte Spleißvarianten wurden Unterschiede in den biochemischen Eigenschaften beschrieben, aber es ist nicht bekannt, ob diese Unterschiede über die gesamte Signalkaskade verstärkt oder abgeschwächt werden. Daher wurde in der vorliegenden Arbeit die Effektivität der Signaltransduktion über die vier Formen des Gαs anhand variantenspezifischer Repression (knock down) oder durch Rekonstitution unmodifizierter Spleißvarianten in Gαs-defizienten Zellinien verglichen. Mehrere Methoden zur transienten Repression wurden mit Hilfe eines Luziferase-Reportersystems verglichen. Katalytisch aktive DNA (DNAzym) war wegen der geringen Aktivität bei physiologischen Magnesiumkonzentrationen ungeeignet. Dagegen wurde eine bis zu 90 %ige Inhibition der Luziferase-Expression durch die Verwendung von Propin- modifizierten Oligodeoxynukleotiden oder durch intrazelluläre Expression von small interfering RNA erreicht. Aufgrund ihrer größeren Flexibilität bei der Wahl der Zielsequenz wurden Propin-modifizierte Oligodeoxynukleotide zur Repression von Gαs ausgewählt. Sie verminderten die Expression von transient transfiziertem, aber nicht von endogenem Gαs. In Sf9-Insektenzellen wurden Gαs-Spleißvarianten mit unterschiedlichen GPCR koexprimiert. Die Wechselwirkung zwischen GPCR und Gαs wurde mit Hilfe von ligandenabhängigen Bindungskinetiken des GTP-Analogs [35S]GTPγS an Plasmamembranen analysiert. Die Spleißvarianten wurden mit vergleichbaren Nukleotid-Bindungskonstanten durch die β2-adrenergen, Glucagon-, Histamin- und Secretin-Rezeptoren aktiviert (kapp ≈ 0,1 min-1). In der Gαs-defizienten Maus-Fibroblastenzellinie 2B2 wurde die Signaltransduktionskaskade GPCR Gαs Adenylylcyclase durch transiente Expression der Gαs Spleißvarianten rekonstituiert und auf der Ebene der Adenylylcyclase untersucht. Die Interaktion der Spleißvarianten mit der Adenylylcyclase wurde durch direkte Aktivierung des G-Proteins in Plasmamembranen verglichen. Die vier Spleißvarianten aktivierten die Adenylylcyclase mit halbmaximal wirksamen Konzentrationen von 0,24-0,31 nM und waren vergleichbar effizient. Weil diese Werte äquivalent waren, konnte die Adenylylcyclase-Aktivität als Maß für die Interaktion zwischen GPCR und Gαs verwendet werden. Die Spleißvarianten waren vergleichbar potent in der Adenylylcyclase-Aktivierung durch β2-adrenergen, Glucagon-, Histamin-, Secretin-, Vasopressin- und Luteinisierungshormon-Rezeptor. Zusammenfassend zeigte diese Arbeit, dass sich die Gαs-Spleißvarianten in der GPCR- vermittelten Adenylylcyclase-Aktivierung vergleichbar verhalten. Daher muss der Grund für die Expression der vier Spleißvarianten noch gefunden werden; die bisher wenig untersuchten Interaktionen mit weiteren Signalmolekülen, wie Tubulin oder den Src-Kinasen, werden diskutiert.The stimulatory guanine nucleotide-binding protein (Gs) transmits signals from stimulatory G protein-coupled receptors (GPCR) to adenylyl cyclases, thereby increasing the production of the second messenger cAMP. Four splice variants of the α-subunit of Gs (Gαs) are expressed in mammalian tissues. Numerous studies have analysed their function, but due to differing experimental parameters and systems, the results were neither consistent nor comparable. Differences in the biochemical properties of heterologously expressed, purified splice variants have been described, but it is not known whether they are enhanced or weakened by transmission through the full signalling cascade. Therefore, the effectiveness of signal transduction via the four splice variants was compared using specific repression (knock down) or reconstitution of unmodified proteins in Gαs-deficient cell lines. Several methods of transient repression were compared using a luciferase reporter system. Catalytically active DNA (DNAzyme) was unsuitable because of its low activity at physiological magnesium concentrations. In contrast, up to 90 % inhibition of luciferase expression was achieved by either propyne-modified oligodeoxynucleotides or intracellular expression of small interfering RNA. The propyne-modified oligodeoxynucleotides were chosen for inhibition of Gαs on account of their greater flexibility in the choice of target sequences. They inhibited expression of transiently transfected but not of endogenous Gαs. In Sf9 insect cells, Gαs splice variants were co-expressed with various GPCR. The interaction between GPCR and Gαs in plasma membranes was compared by measuring ligand-dependent binding kinetics of the GTP analogue [35S]GTPγS. The four splice variants were activated with comparable apparent rate constants by the β2-adrenergic, glucagon, histamine and secretin receptors (kapp ≈ 0,1 min-1). In the Gαs-deficient mouse fibroblast cell line 2B2, the signal transduction cascade GPCR Gαs adenylyl cyclase was reconstituted by transiently expressing Gαs splice variants. Adenylyl cyclase activity was measured in the plasma membranes. Direct activation of Gαs splice variants showed them to be comparably effective and potent in activating adenylyl cyclase with half-maximal effective concentrations of 0.24-0.31 nM. Due to the similarity of these values, adenylyl cyclase activity could be taken as a measure of the interaction between GPCR and Gαs. The splice variants were comparably potent in receptor-mediated adenylyl cyclase activation by the β2-adrenergic, glucagon, histamine, secretin, vasopressin, and luteinising hormone receptors. However, the reason for the expression of four different splice variants remains to be elucidated; interactions with other signalling molecules such as tubulin and Src family tyrosine kinases are discussed