Synthesis, characterization and in vitro biological evaluation of some new 1,3,5-triazine-chalcone hybrid molecules as Mycobacterium tuberculosis H37Rv inhibitors

A novel series of 1,3,5-triazine-chalcone hybrid molecules (4a-ii) have been synthesized and evaluated in vitro for Mycobacterium tuberculosis H37Rv inhibitory potency using Alamar blue assay and the activity expressed as the minimum inhibitory concentration (MIC) in µg/mL. The antitubercular activity screening data revealed that the compound 4z demonstrated comparatively the most potent inhibitory activity, with MIC value 3.125 µg/mL. It is noteworthy that the compounds 4e, 4p and 4bb also showed appreciable inhibitory activity with MIC value 6.25 µg/mL. Most of the compounds displayed significantly promising activity and their structure-activity relationships were also discussed. This could be the remarkable starting point to develop new lead molecules with potential antitubercular activity

Functional and phenotypic heterogeneity of Th17 cells in health and disease

Background Th17 cells have nonredundant roles in maintaining immunity, particularly at mucosal surfaces. These roles are achieved principally through the production of cytokines and the recruitment of other immune cells to maintain the integrity of mucosal barriers and prevent the dissemination of microorganisms. Th17 cells are heterogeneous and exhibit a considerable degree of plasticity. This allows these cells to respond to changing environmental challenges. However, Th17 cells also play pro‐inflammatory roles in chronic autoimmune diseases. The trigger(s) that initiate these Th17 responses in chronic autoimmune diseases remain unclear. Design In this report, we provide an overview of studies involving animal models, patient data, genome wide association studies and clinical trials targeting IL‐17 for treatment of patients to gain a better understanding of the pathogenic roles of Th17 cells play in a range of autoimmune diseases. Results The report sheds light on likely triggers that initiate or perpetuate Th17 responses that promote chronic inflammation and autoimmunity. The divergent effects of tumour necrosis factor alpha blockade on Th17 cells in patients, is explored. Furthermore, we highlight the role of Th17 cells in inducing autoreactive B cells, leading to autoantibody production. Pathogenic bacterial species can change Th17 cell phenotype and responses. These findings provide insights into how Th17 cells could be induced to promoting autoimmune disease pathogenesis. Conclusion This article provides an overview of the distinct roles Th17 cells play in maintaining immunity at mucosal surfaces and in skin mucosa and how their functional flexibility could be linked with chronic inflammation in autoimmune rheumatic diseases

The Role of Human Ephrin Receptor Tyrosine Kinase A2 (EphA2) in Chlamydia trachomatis Infection

Chlamydia trachomatis (Ctr), an obligate intracellular gram negative human pathogen, causes sexually transmitted diseases and acquired blindness in developing countries. The infectious elementary bodies (EB) of Ctr involved in adherence and invasion processes are critical for chlamydial infectivity and subsequent pathogenesis which requires cooperative interaction of several host cell factors. Few receptors have been known for this early event, yet the molecular mechanism of these receptors involvement throughout Ctr infection is not known. Chlamydial inclusion membrane serves as a signaling platform that coordinates Chlamydia-host cell interaction which encouraged me to look for host cell factors that associates with the inclusion membrane, using proteome analysis. The role of these factors in chlamydial replication was analyzed by RNA interference (RNAi) (in collaboration with AG Thomas Meyer). Interestingly, EphrinA2 receptor (EphA2), a cell surface tyrosine kinase receptor, implicated in many cancers, was identified as one of the potential candidates. Due to the presence of EphA2 in the Ctr inclusion proteome data, I investigated the role of EphA2 in Ctr infection. EphA2 was identified as a direct interacting receptor for adherence and entry of C. trachomatis. Pre-incubation of Ctr-EB with recombinant human EphA2, knockdown of EphA2 by siRNA, pretreatment of cells with anti-EphA2 antibodies or the tyrosine kinase inhibitor dasatinib significantly reduced Ctr infection. This marked reduction of Ctr infection was seen with both epithelial and endothelial cells used in this study. Ctr activates EphA2 upon infection and invades the cell together with the activated EphA2 receptor that interacts and activates PI3K survival signal, promoting chlamydial replication. EphA2 upregulation during infection is associated with Ctr inclusion membrane inside the cell and are prevented being translocated to the cell surface. Ephrins are natural ligands for Ephrin receptors that repress the activation of the PI3K/Akt pathway in a process called reverse signaling. Purified Ephrin-A1, a ligand of EphA2, strongly interferes with chlamydial infection and normal development, supporting the central role of these receptors in Chlamydia infection. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Ctr infection induces EphA2 upregulation and is mediated by activation of ERK signaling pathway. Interfering with EphA2 upregulation sensitizes Ctr-infected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-α) suggesting the importance of intracellular EphA2 signaling. Collectively, these results revealed the first Ephrin receptor “EphA2” that functions in promoting chlamydial infection. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism how Chlamydia subverts the host cell and induces apoptosis resistance. By applying the natural ligand Ephrin-A1 and targeting EphA2 offers a promising new approach to interfere with Chlamydia infection. Thus, the work provides the evidence for a host cell surface tyrosine kinase receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate the chlamydial replication

Die Rolle der humanen Rezeptor-Tyrosinkinase EphrinA2 (EphA2) in der Chlamydia trachomatis Infektion

Chlamydia trachomatis (Ctr), an obligate intracellular gram negative human pathogen, causes sexually transmitted diseases and acquired blindness in developing countries. The infectious elementary bodies (EB) of Ctr involved in adherence and invasion processes are critical for chlamydial infectivity and subsequent pathogenesis which requires cooperative interaction of several host cell factors. Few receptors have been known for this early event, yet the molecular mechanism of these receptors involvement throughout Ctr infection is not known. Chlamydial inclusion membrane serves as a signaling platform that coordinates Chlamydia-host cell interaction which encouraged me to look for host cell factors that associates with the inclusion membrane, using proteome analysis. The role of these factors in chlamydial replication was analyzed by RNA interference (RNAi) (in collaboration with AG Thomas Meyer). Interestingly, EphrinA2 receptor (EphA2), a cell surface tyrosine kinase receptor, implicated in many cancers, was identified as one of the potential candidates. Due to the presence of EphA2 in the Ctr inclusion proteome data, I investigated the role of EphA2 in Ctr infection. EphA2 was identified as a direct interacting receptor for adherence and entry of C. trachomatis. Pre-incubation of Ctr-EB with recombinant human EphA2, knockdown of EphA2 by siRNA, pretreatment of cells with anti-EphA2 antibodies or the tyrosine kinase inhibitor dasatinib significantly reduced Ctr infection. This marked reduction of Ctr infection was seen with both epithelial and endothelial cells used in this study. Ctr activates EphA2 upon infection and invades the cell together with the activated EphA2 receptor that interacts and activates PI3K survival signal, promoting chlamydial replication. EphA2 upregulation during infection is associated with Ctr inclusion membrane inside the cell and are prevented being translocated to the cell surface. Ephrins are natural ligands for Ephrin receptors that repress the activation of the PI3K/Akt pathway in a process called reverse signaling. Purified Ephrin-A1, a ligand of EphA2, strongly interferes with chlamydial infection and normal development, supporting the central role of these receptors in Chlamydia infection. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Ctr infection induces EphA2 upregulation and is mediated by activation of ERK signaling pathway. Interfering with EphA2 upregulation sensitizes Ctr-infected cells to apoptosis induced by tumor necrosis factor-alpha (TNF-α) suggesting the importance of intracellular EphA2 signaling. Collectively, these results revealed the first Ephrin receptor “EphA2” that functions in promoting chlamydial infection. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism how Chlamydia subverts the host cell and induces apoptosis resistance. By applying the natural ligand Ephrin-A1 and targeting EphA2 offers a promising new approach to interfere with Chlamydia infection. Thus, the work provides the evidence for a host cell surface tyrosine kinase receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate the chlamydial replication.Chlamydia trachomatis (Ctr) ist ein obligat intrazellulär lebendes Gram negatives Bakterium, das Geschlechtskrankheiten verursachen kann. In Entwicklungsländern führt es zudem häufig zu erworbener Blindheit. Die infektiösen Elementarkörper (EB) sind für die Anheftung an die Wirtszelle sowie die Aufnahme von Ctr in die Wirtzelle verantwortlich. Dies ist ein wichtiger Schritt, da nur so die sich anschließende Krankheitsentwicklung stattfinden kann. Diese ist auch abhängig vom engen Zusammenspiel der Ctr Proteine mit den Wirtszellfaktoren. Obgleich dieser Schritt so wichtig ist, wurden erst wenige Wirtszellrezeptoren gefunden und welche Rolle diese Rezeptoren im weiteren Verlauf der Infektion spielen, ist noch nicht richtig verstanden. Die chlamydiale Inklusionsmembran fungiert als Signalplattform, die das Zusammenspiel von Chlamydien und Wirtszelle koordiniert. In dieser Arbeit wurden die Wirtszellproteine, die an der Inklusionsmembran lokalisiert sind, mit Hilfe einer Proteomanalyse identifiziert. Anschließend wurde die Rolle dieser Proteine bei der Chlamydienvermehrung in einem RNAi screen untersucht (in Zusammenarbeit mit der AG Thomas Meyer). Hier wurde überraschenderweise der EphrinA2 Rezeptor, eine sich auf der Oberfläche der Zellen befindliche Rezeptor Tyrosin Kinase, die vor allem mit Krebs in Verbindung gebracht wird, als ein potentieller Kandidat identifiziert. Da die Proteomdaten gezeigt haben, dass EphrinA2 an der Inklusionsmembran lokalisiert ist, wurde die Rolle von EphrinA2 während der Ctr Infektion hier näher untersucht. Es konnte gezeigt werden, dass EphrinA2 ein direkter Rezeptor für Ctr ist, der sowohl die Adhärenz als auch die Aufnahme von Ctr in die Wirtszelle bewerkstelligt. Vorinkubation von Ctr- EB mit rekombinantem menschlichen EphrinA2, das herunterregulieren von EphrinA2 mit Hilfe einer siRNA oder das Vorinkubieren der menschlichen Zelle mit Antikörpern gegen EphrinA2 oder dem Tyrosinkinase Inhibitor Dasatinib, reduzierten die Ctr Infektion signifikant. Diese drastische Reduktion der Ctr Infektion wurde sowohl in Epithelzellen als auch in Endothelzellen beobachtet. Ctr aktiviert EphrinA2 während der Infektion und invadiert die Wirtszelle zusammen mit dem aktivierten Rezeptor, dieser interagiert mit dem aktivierten PI3K Überlebenssignal, was die Replikation der Chlamydien ermöglicht. An der Inklusionsmembran akkumuliert EphrinA2, da der Transport von neuem Rezeptor zur Zellmembran unterbunden ist. Ephrine sind die natürlichen Liganden der Ephrinrezeptoren, sie unterdrücken die Aktivierung des PI3K/Akt Signalweges in einem Prozess, der reverse Signalübertragung genannt wird. Aufgereinigtes Ephrin-A1, ein Ligand des EphrinA2 Rezeptors, verhindert eine normale Chlamydieninfektion, was eine zentrale Rolle dieses Rezeptors weiterhin bestätigt. Die Überexpression von EphrinA2, erhöhte die PI3K Aktivierung und Ctr Infektion. Dies war nicht der Fall, wenn eine Mutante, der die intrazelluläre Domäne fehlt, überexprimiert wurde. Eine Ctr Infektion induziert die Hochregulierung von EphrinA2, welche durch die Aktivierung des ERK Signalwegs bewerkstelligt wird. Wenn die Hochregulierung von EphrinA2 verhindert wird, werden Ctr infizierte Zellen sensitiver für Apoptose induziert durch tumor necrosis factor-alpha (TNF-α), was ein weiter Hinweis für die Bedeutung der intrazellulären EphrinA2 Signalübermittlung ist. Insgesamt haben diese Ergebnisse den ersten Ephrin Rezeptor "EphA2" offenbart, der in der Förderung chlamydialer Infektionen fungiert. Hinzu kommt, dass die Bindung eines Oberflächenrezeptors an die Inklusionsmembran ein neuer Mechanismus ist, die Wirtszelle zu verändern und eine Apoptoseresistenz in der Zelle zu induzieren. Die Zugabe des natürlichen Liganden Ephrin-A1 eröffnet eine neue vielversprechende Möglichkeit Chlamydieninfektionen zu bekämpfen. Daher liefert diese Arbeit erste Hinweise, das eine Wirtszelltyrosinkinase, die sich an der Zelloberfläche befindet, notwendig ist für die Invasion und die intrazelluläre Signalübermittlung, welche für die chlamydiale Replikation notwendig ist, essentiell ist

Characterization of allergen-specific antibodies induced by specific immunotherapy

Birkenpollen sind eine häufige Ursache für IgE-mediierte Allergien in Zentral- und Nordeuropa sowie Nordamerika. Birkenpollen-AllergikerInnen entwickeln häufig Birkenpollen-assoziierte Nahrungsmittelallergien. Insbesondere Apfel und Haselnuss sind häufige Auslöser dieser Kreuzallergie, da diese Nahrungsmittel Proteine enthalten, die strukturelle Ähnlichkeiten mit dem Hauptallergen in Birkenpollen, Bet v 1, aufweisen. In Folge können Bet v 1-spezifische IgE-Antikörper mit Mal d 1 in Apfel und Cor a 1 in Haselnuss kreuzreagieren und allergische Symptome, wie z.B. das Orale Allergiesyndrom, auslösen. Spezifische Immuntherapie (SIT) ist derzeit die einzige kausale Behandlungsform der IgE-mediierten Allergie. Im Immunsystem der Behandelten löst diese Therapie Veränderungen auf der T-Zellebene aus, wie z.B. die Induktion von allergen-spezifischen Th1-Zellen und regulatorischen T-Zellen. Weiters kommt es zu einem Anstieg der allergen-spezifischen IgG Antikörper, insbesondere der Subklasse IgG4. Diese Antikörper können das Allergen binden und dadurch dessen Bindung an IgE hemmen (blockierende Antikörper). Derzeit ist wenig bekannt darüber, ob eine SIT mit Birkenpollen (BP-SIT) Bet v 1-spezifische IgG4 Antikörper induziert, die mit Bet v 1-verwandten Nahrungsmittelallergenen kreuzreagieren. In der vorliegenden Dissertation wurden die durch BP-SIT induzierten Bet v 1-spezifischen IgG4 Antikörper bezüglich ihrer Kinetik, Kreuzreaktivität und IgE-blockierenden Aktivität charakterisiert. Um zu untersuchen, ob IgG4 Antikörper dieselben Epitope erkennen wie IgE-Antikörper wurde eine spezielle Variante des Phagedisplays entwickelt. Dieses System wurde ebenfalls eingesetzt, um die Diversität des IgG4-Repertoires über eine Behandlungsdauer von 3 Jahren zu untersuchen. In der Mehrzahl der PatientInnen konnte ein signifikanter Anstieg von Bet v 1-, Mal d 1- und Cor a 1-spezifischen IgG4 Antikörpern während der BP-SIT nachgewiesen werden. Diese Antikörper blockierten die IgE-Bindung an diese Allergene sowie die IgE-mediierte Aktivierung von Basophilen und T-Zellen. Unsere Ergebnisse weisen darauf hin, dass die von IgG4 erkannten Epitope mit den von IgE erkannten Bindungsstellen überlappen. Weiters zeigen unsere Daten, dass die Vielfalt der allergen-spezifischen IgG4-Antikörper bereits nach 6 Monaten etabliert zu sein scheint und auch nach längerer Therapiedauer nicht mehr ansteigt. Zusammenfassend konnten wir zeigen, dass BP-SIT Bet v 1-spezifische IgG4 Antikörper induziert, welche die IgE-Bindung an Bet v 1-homologe Nahrungsmittelallergene blockieren können. Weiters weisen unsere Ergebnisse darauf hin, dass das Bet v 1-spezifische IgG4-Repertoire durch Langzeit-SIT mit Birkenpollen nicht vergrößert wird. In unserer Studie fiel auf, dass über 15% der mit BP-SIT Behandelten keine kreuzreaktiven Bet v 1-spezifischen Antikörper entwickelt haben, obwohl sie kreuzreaktive IgE-Antikörper aufwiesen. Diese Beobachtung könnte erklären, warum BP-SIT häufig zu keiner Verbesserung der Birkenpollen-assoziierten Nahrungsmittelallergie führt.Birch pollen is an important cause for IgE-mediated allergy in Central and Northern Europe and North America. Birch pollen-allergic patients often develop hypersensitivity reactions to certain foods. These aliments contain proteins highly homologous with the major birch pollen allergen, Bet v 1. Apple and hazelnut were identified as the most common triggers for birch pollen-related food allergy. Apple contains Mal d 1 and hazelnut contains Cor a 1, two food allergens that are structurally highly similar to Bet v 1. Thus, Bet v 1-specific IgE antibodies cross-react with these food allergens resulting in IgE-mediated immediate symptoms, e.g. the oral allergy syndrome. Specific immunotherapy (SIT) is the only causative treatment for IgE-mediated allergy. SIT induces immunological alterations that include the induction of allergen-specific Th1 cells, regulatory T cells and allergen-specific IgG antibodies. In particular, allergen-specific IgG4 antibodies rise during SIT and may intercept allergens before they cross-link FcϵRI-associated IgE. The pronounced induction of IgG4 gave rise to the hypothesis that a major part of the protective blocking activity resides within this Ig isotype. In birch pollen-SIT (BP-SIT) little is known about the cross reactivity between Bet v 1-specific IgG4 antibodies and Bet v 1-related food proteins. In this thesis, we investigated the kinetics, cross reactivity and IgE-blocking activity of Bet v 1-specific IgG4 antibodies induced by BP-SIT. Moreover, we developed a competitive immunoscreening of phage displayed peptides to study whether SIT-induced IgG4 antibodies overlap with IgE antibodies in epitope recognition and whether their epitope diversity changes during the course of treatment. BP-SIT significantly increased Bet v 1- and food allergen-reactive IgG4 antibodies in the majority of patients. These antibodies inhibited IgE-binding, basophil activation, and IgE-mediated allergen-induced T-cell proliferation to Bet v 1, Mal d 1 and Cor a 1. Results of the competitive immunoscreening of phage displayed peptides suggested that epitopes recognized by IgG4 on all three allergens strongly overlapped with epitopes recognized by IgE. Also, regarding the diversity of Bet v 1-specific IgG4 antibodies after 6 and 36 months of BP-SIT, our data suggested that the Bet v 1-specific IgG4 repertoire in the majority of patients was established early and did not broaden with prolonged treatment. In conclusion, BP-SIT induced Bet v 1-specific IgG4 antibodies that cross-reacted with related food allergens and inhibited IgE-binding by epitope competition. We also provide first evidence that the Bet v 1-specific IgG4 repertoire does not broaden with prolonged BP-SIT or develop specificities that recognize epitopes different from IgE Ab. Of note, more than 15% of patients failed to develop food allergen-reactive IgG4 after BP-SIT although they showed high cross reactivity of Bet v 1-specific IgE with Cor a 1 and Mal d 1. This lack of blocking antibodies might be one reason for the failure of BP-SIT to improve birch pollen-associated food allergy.submitted by Brinda SubbarayalAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. Sprachehttp://www.ncbi.nlm.nih.gov/pubmed/24053565Wien, Med. Univ., Diss., 2014OeBB(VLID)171490

EphrinA2 Receptor (EphA2) Is an Invasion and Intracellular Signaling Receptor for Chlamydia trachomatis

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance

Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

Chlamydia trachomatis is an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle of C. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress of C. trachomatis from human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens. IMPORTANCE Chlamydia trachomatis is a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections. C. trachomatis can develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal "coats" or "cages," whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell