TY - JOUR A1 - Sporbert, Anje A1 - Cseresnyes, Zoltan A1 - Heidbreder, Meike A1 - Domaing, Petra A1 - Hauser, Stefan A1 - Kaltschmidt, Barbara A1 - Kaltschmidt, Christian A1 - Heilemann, Mike A1 - Widera, Darius T1 - Simple Method for Sub-Diffraction Resolution Imaging of Cellular Structures on Standard Confocal Microscopes by Three-Photon Absorption of Quantum Dots JF - PLoS ONE N2 - This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts. KW - HIV KW - stem-cell KW - infection Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-130963 VL - 8 IS - 5 ER - TY - JOUR A1 - Spinner, Christoph D A1 - Wille, Florian A1 - Schwerdtfeger, Christiane A1 - Thies, Philipp A1 - Tanase, Ursula A1 - Von Figura, Guido A1 - Schmid, Roland M A1 - Heinz, Werner J A1 - Klinker, Hartwig Hf T1 - Pharmacokinetics of chewed vs. swallowed raltegravir in a patient with AIDS and MAI infection: some new conflicting data JF - AIDS Research and Therapy N2 - Background: While HIV, AIDS and atypical Mycobacterium infections are closely linked, the use of Integrase-Inhibitor based cART, notably raltegravir-based regimens is more widespread. RAL should be double-dosed to 800 mg semi-daily in situation of rifampicin co-medication, because RAL is more rapidly metabolized due to rifampicin-induced Uridine-5'-diphosph-gluronosyl-transferase (UGT1A1). Recently, it was speculated that chewed RAL might lead to increased absorption, which might compensate the inductive effect of rifampicin-rapid metabolized RAL, as part of cost-saving effects in countries with high-tuberculosis prevalence and less economic power. Methods: We report measurement of raltegravir pharmacokinetics in a 34-year AIDS-patient suffering from disseminated Mycobacterium avium infection with necessity of parenteral rifampicin treatment. RAL levels were measured with HPLC (internal standard: carbamazepine, LLQ 11 ng/ml, validation with Valistat 2.0 program (Arvecon, Germany)). For statistical analysis, a two-sided Wilcoxon signed rank test for paired samples was used. Results: High intra-personal variability in raltegravir serum levels was seen. Comparable C\(_{max}\) concentrations were found for 800 mg chewed and swallowed RAL, as well as for 400 mg chewed and swallowed RAL. While C\(_{max}\) seems to be more dependent from overall RAL dosing than from swallowed or chewed tablets, increased AUC(12) is clearly linked to higher RAL dosages per administration. Anyway, chewed raltegravir showed a rapid decrease in serum levels. Conclusions: We found no evidence that chewed 400 mg semi-daily raltegravir in rifampicin co-medication leads to optimized pharmacokinetics. There is need for more data from randomized trials for further recommendations. KW - pharmacology KW - drug KW - HIV KW - chewed KW - Mycobacterium avium KW - raltegravir KW - pharmacokinetic Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-144058 VL - 12 IS - 1 ER - TY - THES A1 - Sippel, Martin T1 - Computational Structure-based Design Approaches: Targeting HIV-1 Integrase and the Macrophage Infectivity Potentiator of Legionella pneumophila T1 - Computergestütztes strukturbasiertes Design bei HIV-1 Integrase und dem Macrophage Infectivity Potentiator (MIP) von Legionella pneumophila N2 - Die vorliegende Arbeit thematisiert das computergestützte strukturbasierte Design auf dem Gebiet der HIV-1-Integrase und des Macrophage Infectivity Potentiator (MIP) von Legionella pneumophila. Die durchgeführten Studien geben wertvolle Aufschlüsse über den Wirk-mechanismus einer bekannten Integrase-Inhibitorenklasse and zeigt darüber hinaus einen neuartigen Ansatz zur Integrase-Inhibition auf. Im Falle des MIP-Enzyms konnten zwei niedermolekulare Inhibitoren ermittelt werden. Die Integrase-Studien ergaben wertvolle Informationen im Hinblick auf das Design neuer Inhibitoren. Docking-Experimente konnten die Hypothese weiter untermauern, nach der die Klasse der Diketosäure-Inhibitoren nicht als freie Liganden, sondern als Metallion-Komplexe an das aktive Zentrum der Integrase binden. Die Ergebnisse dieser Studie helfen dabei, das Verständnis über den Wirkmechanismus dieser wichtigen Klasse von Integrase-Inhibitoren weiter zu vertiefen. Um der Entwicklung von Integrase-Inhibitoren einen neuen Impuls zu geben, wurde eine neue Strategie zur Inhibition dargelegt: Anstatt an das aktive Zentrum soll eine neue Inhibitor-Klasse an das Dimerisierungs-Interface eines Integrase-Monomers binden, die katalytisch notwendige Dimerisierung verhindern und somit die enzymatische Aktivität stören. Das Hauptproblem hierbei bestand in den fehlenden Strukturdaten des freien Monomers. Hierzu wurden Molekulardynamik-Simulationen durchgeführt, um nähere strukturelle Informationen zu erhalten. Momentaufnahmen unterschiedlicher Konformationen dienten als Input-Strukturen für eine Docking-Studie mit dem peptidischen Inhibitor YFLLKL, um dessen Bindemodus aufzuklären. Hierbei zeigte sich, dass dieser Ligand an eine Interface-Konformation bindet, die durch eine Y-förmige Bindestelle charakterisiert ist. Im nächsten Schritt sollte diese Protein-Konformation mit kleinen, nicht-peptidischen Molekülen adressiert werden. Die erste Strategie bestand darin, ein Pharmakophor-Modell zu erstellen, das zur Suche nach Molekülen mit einer guten Komplementarität zur Y-förmigen Bindetasche geeignet ist. Das folgende virtuelle Screening ergab zehn Verbindungen, die eine gute Komplementarität und günstige hydrophobe Wechselwirkungen aufwiesen. Leider zeigte keine der Verbindungen eine reproduzierbare Aktivität im Integrase-Assay. Hierbei verbleiben jedoch gewisse Zweifel, da in dem Assay die Zugabe von BSA vorgeschrieben war, das möglicherweise die hydrophoben Inhibitor-Kandidaten gebunden hat. Die erwähnte erste Strategie wurde überdacht: In einem zweiten Ansatz galt die Hauptaufmerksamkeit der Absättigung von wasserstoffbrückenbildenden Resten. Diese waren zuvor von den eher hydrophoben Verbindungen nicht optimal abgesättigt worden. Zwei Pharmakophor-Modelle wurden erstellt und in einem virtuellen Screening eingesetzt: Docking-Studien der Hits zeigten jedoch, dass nach wie vor viele wasserstoffbrückenbildende Reste des Proteins nicht vom Liganden abgesättigt wurden. Nach abschließender eingehender Betrachtung der Bindemoden der verbliebenen Moleküle aus dem virtuellen Screening konnten nur acht für weitere Testungen ausgewählt werden (Ergebnisse der experimentellen Testung durch Kooperationspartner stehen noch aus). Diese geringe „Ausbeute“ an geeigneten Verbindungen für das Integrase-Dimerisierungsinterface zeigt, wie schwer dieses Target zu adressieren ist: Das Interface weist eine schnell wechselnde Abfolge von basischen, sauren und hydrophoben Resten auf. Im Gegensatz zu anderen Protein-Protein-Interfaces zeigt das Integrase-Interface keine „aufgeräumte“ Bindetasche mit klar voneinander getrennten hydrophoben und hydrophilen Bereichen. Für das zweite Enzym, MIP, konnten mit Hilfe des strukturbasierten Designs zwei niedermolekulare Inhibitoren gefunden werden. Beide Verbindungen führten zu einer deutlichen Abnahme der katalytischen Aktivität. Soweit bekannt, sind bisher keinerlei niedermolekulare MIP-Inhibitoren veröffentlicht. Der Vergleich von MIP mit der humanen PPIase FKBP12 zeigte eine größtenteils ähnliche Tasche, die jedoch einen entscheidenden Unterschied aufweist, nämlich in der Orientierung des Restes Tyr109. Die detaillierte Betrachtung der Strukturdaten beider Enzyme konnte schließlich eine Erklärung liefern, warum ein ketoacyl-substituiertes Pipecolinderivat nicht an MIP bindet, ein sulfonsubstituiertes Pipecolinderivat hingegen das Enzym inhibiert. Die Erkenntnisse über das Inhibitoren-Design für Legionella-MIP können auch auf andere Organismen (z.B. Trypanosomen) übertragen werden, bei denen ebenfalls (homologes) MIP ein Pathogenitätsfaktor ist. N2 - In this thesis, computational structure-based design approaches were employed to target the HIV-1 integrase and the macrophage infectivity potentiator (MIP) of Legionella pneumophila. The thesis yields valuable information about the mechanism of action of a known class of integrase inhibitors and a novel approach towards enzyme inhibition, which still is mainly unaddressed in current integrase research. For the MIP enzyme, two small-molecule MIP inhibitors were discovered. The computational studies of HIV-1 integrase have provided valuable information for IN inhibitor design. Docking experiments supported the hypothesis that the well-known diketo acid inhibitors enter the IN active site not as free ligands, but rather as metal complexes. These results help to reveal the mechanism of action of this important class of IN inhibitors.To give an impulse for the development of a novel class of inhibitors, a new strategy towards IN inhibition was introduced: An alternative binding site, the dimerization interface of an IN catalytic core domain monomer, was explored for inhibitor design. The lack of structural data of the free monomer was overcome by extensive MD studies. Snapshots derived from the MD simulation were used as protein input structures in a docking study with the inhibitory peptide YFLLKL to reveal its potential binding mode. The docking procedure showed that the peptidic ligand binds to a dimerization interface conformation which shows a Y-shaped binding site.. The next step was to address this protein conformation with small, non-peptidic molecules. The first strategy towards finding small-molecule interface binders was to create a pharmacophore model with hydrophobic features and shape constraints, aiming to find molecules with a good complementarity to the Y-shaped dimerization interface. Virtual screening yielded a total of 10 compounds, which all displayed good shape complementarity and favorable hydrophobic interactions. Unfortunately, none of the compounds showed a reproducible inhibitory activity in biological assays. Some doubts remain about the validity of the assay results: The use of BSA was critical, since it is not unlikely that BSA “intercepted” the hydrophobic candidate compounds. The first strategy towards finding small-molecule dimerization inhibitors was reconsidered: In the second approach, the satisfaction of hydrogen bonding residues at the dimerization interface, was of major interest. Two pharmacophore models were employed, which retrieved several hundred hit molecules. However, docking of these molecules showed that still many hydrogen bonding groups of the protein remained unaddressed by the ligands. Eventually, after visual inspection, only eight molecules were selected as candidate compounds for further testing (results pending). This small “yield” underlines the difficulties in finding interface binders: The IN dimerization interface is a peculiar target with frequently alternating basic, acidic, and hydrophobic residues. It is not a well-ordered binding site with continuous hydrophobic areas and distinct hydrogen bond donors / acceptors. Other protein-protein interfaces show such well-ordered binding sites. Accordingly, the peculiarity of the IN dimerization interface, in addition to the delicate task of disrupting protein-protein interactions at all, makes the development of IN dimerization inhibitors very challenging. For MIP, the studies revealed two experimentally validated MIP inhibitors, which significantly reduce MIP enzymatic activity. To our knowledge, no small-molecule MIP inhibitor has been reported in the literature so far. A detailed analysis of the available structural data of MIP and a comparison to the human PPIase counterpart, FKBP12, pointed out a conformational diversity among the MIP structures and a crucial difference between the two PPIases, which could be traced to mainly one residue (Tyr109). The detailed comparison of FKBP12 and MIP complex structures made it possible to give an explanation, why a ketoacyl-substituted pipecoline derivative most probably does not bind to MIP, but a sulfone-substituted pipecoline derivative does bind to MIP. Knowledge of Legionella MIP inhibitors could be transferred also to other organisms (e.g. trypanosoms), where homologous MIP proteins are also pathological factors. KW - Legionella pneumophila KW - Integrasen KW - HIV KW - Arzneimitteldesign KW - Molekulardesign KW - Legionärskrankheit KW - Arzneimitteldesign KW - Molecular modelling KW - HIV KW - Legionnaires' Disease KW - drug design Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-51247 ER - TY - JOUR A1 - Shityakov, Sergey A1 - Förster, Carola A1 - Rethwilm, Axel A1 - Dandekar, Thomas T1 - Evaluation and Prediction of the HIV-1 Central Polypurine Tract Influence on Foamy Viral Vectors to Transduce Dividing and Growth-Arrested Cells N2 - Retroviral vectors are potent tools for gene delivery and various biomedical applications. To accomplish a gene transfer task successfully, retroviral vectors must effectively transduce diverse cell cultures at different phases of a cell cycle. However, very promising retroviral vectors based on the foamy viral (FV) backbone lack the capacity to efficiently transduce quiescent cells. It is hypothesized that this phenomenon might be explained as the inability of foamy viruses to form a pre-integration complex (PIC) with nuclear import activity in growth-arrested cells, which is the characteristic for lentiviruses (HIV-1). In this process, the HIV-1 central polypurine tract (cPPT) serves as a primer for plus-strand synthesis to produce a “flap” element and is believed to be crucial for the subsequent double-stranded cDNA formation of all retroviral RNA genomes. In this study, the effects of the lentiviral cPPT element on the FV transduction potential in dividing and growth-arrested (G1/S phase) adenocarcinomic human alveolar basal epithelial (A549) cells are investigated by experimental and theoretical methods. The results indicated that the HIV-1 cPPT element in a foamy viral vector background will lead to a significant reduction of the FV transduction and viral titre in growth-arrested cells due to the absence of PICs with nuclear import activity. KW - Evaluation KW - Prognose KW - HIV KW - Spumaviren KW - Einfluss Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112763 ER - TY - JOUR A1 - Seif, Michelle A1 - Einsele, Hermann A1 - Löffler, Jürgen T1 - CAR T cells beyond cancer: hope for immunomodulatory therapy of infectious diseases JF - Frontiers in Immunology N2 - Infectious diseases are still a significant cause of morbidity and mortality worldwide. Despite the progress in drug development, the occurrence of microbial resistance is still a significant concern. Alternative therapeutic strategies are required for non-responding or relapsing patients. Chimeric antigen receptor (CAR) T cells has revolutionized cancer immunotherapy, providing a potential therapeutic option for patients who are unresponsive to standard treatments. Recently two CAR T cell therapies, Yescarta® (Kite Pharma/Gilead) and Kymriah® (Novartis) were approved by the FDA for the treatments of certain types of non-Hodgkin lymphoma and B-cell precursor acute lymphoblastic leukemia, respectively. The success of adoptive CAR T cell therapy for cancer has inspired researchers to develop CARs for the treatment of infectious diseases. Here, we review the main achievements in CAR T cell therapy targeting viral infections, including Human Immunodeficiency Virus, Hepatitis C Virus, Hepatitis B Virus, Human Cytomegalovirus, and opportunistic fungal infections such as invasive aspergillosis. KW - infectious diseases KW - mAb engineering KW - CAR T cells KW - HIV KW - HCV KW - CMV KW - invasive aspergillosis KW - HBV Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-195596 SN - 1664-3224 VL - 10 IS - 2711 ER - TY - CHAP A1 - Schwinn, Andreas A1 - Rethwilm, Axel A1 - Esers, Stefan A1 - Borisch, Bettina A1 - ter Meulen, Volker T1 - Interaction of HIV-1 and HHV-6 N2 - No abstract available. KW - HIV KW - Herpesviren Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86415 ER - TY - JOUR A1 - Schneider-Schaulies, Sibylle A1 - Schumacher, Fabian A1 - Wigger, Dominik A1 - Schöl, Marie A1 - Waghmare, Trushnal A1 - Schlegel, Jan A1 - Seibel, Jürgen A1 - Kleuser, Burkhard T1 - Sphingolipids: effectors and Achilles heals in viral infections? JF - Cells N2 - As viruses are obligatory intracellular parasites, any step during their life cycle strictly depends on successful interaction with their particular host cells. In particular, their interaction with cellular membranes is of crucial importance for most steps in the viral replication cycle. Such interactions are initiated by uptake of viral particles and subsequent trafficking to intracellular compartments to access their replication compartments which provide a spatially confined environment concentrating viral and cellular components, and subsequently, employ cellular membranes for assembly and exit of viral progeny. The ability of viruses to actively modulate lipid composition such as sphingolipids (SLs) is essential for successful completion of the viral life cycle. In addition to their structural and biophysical properties of cellular membranes, some sphingolipid (SL) species are bioactive and as such, take part in cellular signaling processes involved in regulating viral replication. It is especially due to the progress made in tools to study accumulation and dynamics of SLs, which visualize their compartmentalization and identify interaction partners at a cellular level, as well as the availability of genetic knockout systems, that the role of particular SL species in the viral replication process can be analyzed and, most importantly, be explored as targets for therapeutic intervention. KW - glycosphingolipids KW - ceramides KW - sphingosine 1-phosphate KW - sphingomyelinase KW - HIV KW - SARS-CoV-2 KW - measles Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-245151 SN - 2073-4409 VL - 10 IS - 9 ER - TY - JOUR A1 - Rudovick, Ladius A1 - Brauner, Jan M. A1 - Englert, Johanna A1 - Seemann, Carolina A1 - Plugaru, Karina A1 - Kidenya, Benson R. A1 - Kalluvya, Samuel E. A1 - Scheller, Carsten A1 - Kasang, Christa T1 - Prevalence of pretreatment HIV drug resistance in Mwanza, Tanzania JF - Journal of Antimicrobial Chemotherapy N2 - Background: In a 2008-10 study, we found a pretreatment HIV drug resistance (PDR) prevalence of 18.2% in patients at Bugando Medical Centre (BMC) in Mwanza, Tanzania. Objectives: To determine the prevalence of PDR and transmitted HIV drug resistance (TDR) in patients visiting the BMC from 2013 to 2015. Methods: Adult outpatients were sequentially enrolled into two groups, separated by whether they were initiating ART. Previous exposure to antiretroviral drugs, except for prevention of mother-to-child transmission, was an exclusion criterion. HIV pol sequences were analysed according to WHO guidelines for surveillance of PDR and TDR. Results: Two hundred and thirty-five sequences were analysed (138 ART initiators, 97 non-initiators). The prevalence of PDR was 4.7% (95% CI 2.6%-8.2%) overall, 3.1% (95% CI 1.1%-8.7%) for non-initiators and 5.8% (95% CI 3.0%-11.0%) for ART initiators. PDR to NNRTIs and nucleoside or nucelotide reverse transcriptase inhibitors was found in 3.0% (95% CI 1.5%-6.0%) and 1.7% (95% CI 0.7%-4.3%) of patients, respectively. Resistance to PIs was not observed. The prevalence of TDR was 6.0% (95% CI 3.6%-9.8%). Conclusions: Prevalence of PDR significantly decreased compared with 2008-10 and was below the WHO-defined threshold for triggering a public health response. National and systematic surveillance is needed to inform Tanzania's public health strategy. KW - HIV KW - adult KW - anti-hiv agents KW - child KW - mothers KW - nucleosides KW - outpatients KW - reverse transcriptase inhibitors KW - tanzania KW - world health organization KW - guidelines KW - public health medicine KW - anti-retroviral agents KW - non-nucleoside reverse transcriptase inhibitors KW - surveillance KW - medical KW - exclusion criteria KW - prevention Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-227124 VL - 73 IS - 12 ER - TY - CHAP A1 - Rethwilm, Axel A1 - Baunach, Gerald A1 - Mori, Kazuyasu A1 - ter Meulen, Volker T1 - Transactivation of HIV by human spumaretrovirus N2 - To study the activation of HIV by human spumaretrovirus (HSRV) the long terminal repeats (LTRs) of HSRV, HIVl and HIV2 were examined with respect to their ability to function as transcriptional promoters in virus infected and uninfected cells. Transient transfections using plasmids in which the L TRs of the three viruses were coupled to the bacterial chloramphenicol acetyltransferase (CA T) gene revealed (i) the level of cat gene expression directed by the HSRV LTR was markedly increased in HSRV infected cells compared to uninfected cells, (ii) cat gene expression driven by the HIV1 LTR, but not by the HIV2 LTR could be enhanced upon HSRV infection, whereas (iii) neither in HIV1 nor in HIV2 infected cells an effect on HSRV LTR driven cat geneexpression was detected. KW - HIV Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86436 ER - TY - JOUR A1 - Prottengeier, Johannes A1 - Koutsilieri, Eleni A1 - Scheller, Carsten T1 - The effects of opioids on HIV reactivation in latently-infected T-lymphoblasts JF - AIDS Research and Therapy N2 - Background: Opioids may have effects on susceptibility to HIV-infection, viral replication and disease progression. Injecting drug users (IDU), as well as anyone receiving opioids for anesthesia and analgesia may suffer the clinical consequences of such interactions. There is conflicting data between in vitro experiments showing an enhancing effect of opioids on HIV replication and clinical data, mostly showing no such effect. For clarification we studied the effects of the opioids heroin and morphine on HIV replication in cultured CD4-positive T cells at several concentrations and we related the observed effects with the relevant reached plasma concentrations found in IDUs. Methods: Latently-infected ACH-2 T lymphoblasts were incubated with different concentrations of morphine and heroine. Reactivation of HIV was assessed by intracellular staining of viral Gag p24 protein and subsequent flow cytometric quantification of p24-positive cells. The influence of the opioid antagonist naloxone and the antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH) on HIV reactivation was determined. Cell viability was investigated by 7-AAD staining and flow cytometric quantification. Results: Morphine and heroine triggered reactivation of HIV replication in ACH-2 cells in a dose-dependent manner at concentrations above 1 mM (EC50 morphine 2.82 mM; EC50 morphine 1.96 mM). Naloxone did not interfere with heroine-mediated HIV reactivation, even at high concentrations (1 mM). Opioids also triggered necrotic cell death at similar concentrations at which HIV reactivation was observed. Both opioid-mediated reactivation of HIV and opioid-triggered cell death could be inhibited by the antioxidants GSH and NAC. Conclusions: Opioids reactivate HIV in vitro but at concentrations that are far above the plasma levels of analgesic regimes or drug concentrations found in IDUs. HIV reactivation was mediated by effects unrelated to opioid-receptor activation and was tightly linked to the cytotoxic activity of the substances at millimolar concentrations, suggesting that opioid-mediated reactivation of HIV was due to accompanying effects of cellular necrosis such as activation of reactive oxygen species and NF-kB. KW - naloxone KW - ACH-2 KW - HIV KW - reactivation KW - opioids KW - heroine KW - morphine KW - human immunodeficiency virus KW - human peripheral blood KW - injecting drug users Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-115860 SN - 1742-6405 VL - 11 IS - 17 ER - TY - CHAP A1 - Mori, Kazuyasu A1 - Rethwilm, Axel A1 - Schwinn, Andreas A1 - Horak, Ivan T1 - Replication of human immunodeficiency virus type 1 in human t-cells expressing antisense RNA N2 - No abstract available. KW - HIV Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-86426 ER - TY - JOUR A1 - Lee, Marcel A1 - Eyer, Florian A1 - Felgenhauer, Norbert A1 - Klinker, Hartwig H. F. A1 - Spinner, Christoph D. T1 - Overdose of dolutegravir in combination with tenofovir disaproxil fumarate/emtricitabine in suicide attempt in a 21-year old patient JF - AIDS Research and Therapy N2 - A 21 year old MSM patient with newly diagnosed HIV infection was hospitalized in our department after ingestion of an overdose of his antiretroviral therapy (ART) comprising dolutegravir (DTG - Tivicay\(^{®}\)) and tenofovir disaproxil fumarate/emtricitabine (Truvada\(^{®}\)) in suicidal intention. On admission, the patient did not show any clinical signs of intoxication and laboratory findings were unremarkable. After 6 hours of intensive care monitoring, the patient was referred to a psychiatric clinic. 5 days after the day of intoxication, serum creatinine levels increased to high normal values (1.2 mg/dl). However, levels never exceeded the upper threshold. 8 and 12 weeks later, serum creatinine normalized to levels measured prior to the intoxication. No other adverse events occurred, and the patient does not suffer from permanent impairments. KW - integrase inhibitor KW - HIV KW - AIDS KW - suicide attempt KW - dolutegravir KW - tenofovir disaproxil fumarate KW - emtricitabine KW - overdose Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-151994 VL - 12 IS - 18 ER - TY - THES A1 - Kleen, Thomas Oliver T1 - Dissociated expression of granzyme B and IFN-gamma by T lymphocytes in HIV-1 infected individuals and its implications for Tc1 effector diversity T1 - Seperate Expression von Ganyme B und IFN-gamma durch T-Zellen in HIV Infizierten und die Implikationen für Tc1 Effektor Diversität N2 - A CD8+ cell-mediated host defense relies on cognate killing of infected target cells and on local inflammation induced by the secretion of IFN-g. Using assays of single cell resolution, it was studied to what extent these two effector function of CD8+ cells are linked. Granzyme B (GzB) is stored in cytolytic granules of CD8+ cells and its secretion is induced by antigen recognition of these cells. Following entry into the cytosol GzB induces apoptosis in the target cells. It was measured whether GzB release by individual CD8+ cells is accompanied by the secretion of IFN-gƒnƒnand of other cytokines. HIV peptide libraries were tested on bulk peripheral blood mononuclear cells and on purified CD4+ and CD8+ cells obtained from HIV infected individuals. The library included a panel of previously defined HLA class I restricted HIV peptides and an overlapping 20-mer peptide-series that covered the entire gp120 molecule. To characterize the in vivo differentiation state of the T-cells, freshly isolated lymphocytes were tested in assays of 24h duration. The data showed that only ~20% of the peptides triggered the release of both GzB and IFN-g from CD8+ cells. The majority of the HIV peptides induced either GzB or IFN-g, ~40% in each category. The GzB positive, IFN-g negative CD8+ cells did not produce IL-4 or IL-5, which suggests that they do not correspond to Tc2 cells but represent a novel Tc1 subclass, which was termed Tc1c. Also the IFN-g positive, GzB negative CD8+ cell subpopulation represents a yet undefined CD8+ effector cell lineage that was termed Tc1b. Tc1b and Tc1c cells are likely to make different, possibly antagonistic contributions to the control of HIV infection. Since IFN-g activates HIV replication in latently infected macrophages, the secretion of this cytokine by Tc1b cells in the absence of killing may have adverse effects on the host defense. In contrast, cytolysis by Tc1c cells in the absence of IFN-g production might represent the protective class of response. Further studies in the field of Tc1 effector cell diversity should lead to valuable insights for management of infections and developing rationales for vaccine design. N2 - Im Verlauf von Infektionskrankheiten basiert die Verteidigung durch CD8+-Zellen auf der direkten Tötung von infizierten Zellen über die Perforin/Granzyme-Kaskade und auf der Entzündungsbildung verursacht durch die Sekretion von Interferon-Gamma (IFN-g). In der vorliegenden Arbeit wurde die Granzyme B (GzB) Sezernierung als direkter Nachweis für das Abtöten von Zellen und parallel die Produktion von IFN-g durch HIV peptid-spezifische T-Zellen in chronisch HIV-infizierten Patienten gemessen. Eine Auswahl von in vorhergehenden Arbeiten definierten HLA-Klasse-I spezifischen HIV-Peptiden und eine HIV-Peptide-Bibiliotek, bestehend aus sich überlappenden 20-mer Peptiden, die das gesamte Gp120 Molekül von HIV-1 darstellten, wurden benutzt, um T-Lymphozyten aus frisch von HIV-infizierten Patienten gewonnenen mononukleären Zellen aufgereinigte CD8+-Zellen zu stimulieren. ELISPOT-Assays wurden benutzt, um die Sekretion von GzB und IFN-g mit einer Auflösung auf der Ebene der Einzelzelle zu messen. Nur ~20% der Peptide lösten die Freisetzung von sowohl GzB als auch IFN-g von CD8+ Zellen aus. Die Mehrheit der Peptide induzierte entweder GzB oder IFN-g, je ~40% in der jeweiligen Kategorie. Granzyme B-positive Zellen produzierten in parallel gemessen kein IL-4 oder IL-5. Da sie aus diesem Grunde keine Tc2 Zellen darstellen, wurden sie als neue Untergruppe Tc1c definiert. Diese GzB+/IFN-g- CD8+ Zellen vermutlich eine durch Apoptose induziertes Absterben von infizierten Zellen auslösen, ohne gleichzeitig eine Entzündungsreaktion zu verursachen. Die GzB-/IFN-g+ CD8+-Zellen stellen ebenfalls eine neue bisher unbeschriebene Zelluntergruppe dar und wurden als Tc1b Zellen bezeichnet. Diese könnten Entzündungsreaktion verursachen, ohne gleichzeitig direkt das Abstreben von Zellen zu induzieren und im Verlauf der HIV Infektion eine antagonistische Rolle zu den Tc1c Zellen einnehmen. Diese Tc1-CD8+ Effektorzell-diversität könnte die Implementierung und Feineinstellung von fundamental verschieden Verteidigungsstrategien gegen HIV und andere Infektionskrankheiten ermöglichen. Die hier vorgelegten Studien liefern eindeutige Indizien dafür, dass es in HIV-Infizierten eine signifikante Population von GzB sezernierenden CD8+-Zellen gibt, die weder IFN-g noch IL-4 oder IL-5 produzieren und daher in der Lage sind, mit Hilfe der Perforin/Granzyme-Kaskade zu töten, ohne dabei klassische Tc1 oder Tc2-Zellen darzustellen. Die höhere Sensitivität des GzB ELISPOT-Assays Zytotoxizität im Vergleich zum Chromium-Release-Assay zu messen, bietet eine hilfreiche Methode zum besseren Verständnis CD8+-Zell vermittelter Immunität. Die Ergebnisse führen zu der Schlussfolgerung, dass es im Menschen die von uns erstmals beschriebenen GzB+/IFN-g- und GzB-/IFN-g+ Untergruppen von CD8+-Zellen gibt. Aufgrund der verschiedenen Effektorfunktionen die diese vermutlich ausüben, erscheint es wichtig ihre jeweiligen Anteile im Kampf gegen HIV und andere Infektionskrankheiten zu ermitteln. Bei der Beurteilung von Immunantworten, ausgelöst durch experimentelle HIV-Impfstoffe, dürfte es sich als ausgesprochen wichtig erweisen, diese GzB produzierenden CD8+-Zellen neben den konventionell gemessenen IFN-g sezernierden CD8+-Zellen zu berücksichtigen. Diese Vorgehensweise sollte wertvolle Einblicke gewähren, in wie weit der jeweilige Phänotyp von Effektorzellen den höheren oder effektiveren Schutz gewährt und daher wertvolle Hilfe beim Management von Infektionen und im Bereich des Impfstoffdesign liefern. KW - Antigen CD8 KW - Flymphozyt KW - HIV-Infektion KW - granzyme B KW - Interferon gamma KW - HIV KW - AIDS KW - Cytotoxizität KW - Granzyme B KW - Interferon gamma KW - HIV KW - AIDS KW - Cytotoxicity Y1 - 2003 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-8460 ER - TY - JOUR A1 - Kerkau, T. A1 - Gernert, S. A1 - Kneitz, C. A1 - Schimpl, A. T1 - Mechanism of MHC class I downregulation in HIV infected cells N2 - HIV infection of CD4+ peripheral blood lymphocytes leads to a loss of MHC dass I molecules on the surface of the infected cells as detectable by monodonal antibody staining and flow cytometry. Incubation of the infected cells at 26 oe or treatment at 37 oe with peptides leads to upregulation of MHC dass I to levels equal to those found on uninfected cells cultured und er the same conditions. The data suggest that, after HIV infection, the mechanisms responsible for peptide generation, peptide transport and thus stable association between peptides and MHC dass I molecules are severely affected. KW - HIV Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-56849 ER - TY - JOUR A1 - Kasang, Christa A1 - Ulmer, Albrecht A1 - Donhauser, Norbert A1 - Schmidt, Barabara A1 - Stich, August A1 - Klinker, Hartwig A1 - Kalluvya, Samuel A1 - Koutsilieri, Eleni A1 - Rethwilm, Axel A1 - Scheller, Carsten T1 - HIV patients treated with low-dose prednisolone exhibit lower immune activation than untreated patients N2 - Background: HIV-associated general immune activation is a strong predictor for HIV disease progression, suggesting that chronic immune activation may drive HIV pathogenesis. Consequently, immunomodulating agents may decelerate HIV disease progression. Methods: In an observational study, we determined immune activation in HIV patients receiving low-dose (5 mg/day) prednisolone with or without highly-active antiretroviral therapy (HAART) compared to patients without prednisolone treatment. Lymphocyte activation was determined by flow cytometry detecting expression of CD38 on CD8(+) T cells. The monocyte activation markers sCD14 and LPS binding protein (LBP) as well as inflammation markers soluble urokinase plasminogen activated receptor (suPAR) and sCD40L were determined from plasma by ELISA. Results: CD38-expression on CD8+ T lymphocytes was significantly lower in prednisolone-treated patients compared to untreated patients (median 55.40% [percentile range 48.76-67.70] versus 73.34% [65.21-78.92], p = 0.0011, Mann-Whitney test). Similarly, we detected lower levels of sCD14 (3.6 μg/ml [2.78-5.12] vs. 6.11 μg/ml [4.58-7.70]; p = 0.0048), LBP (2.18 ng/ml [1.59-2.87] vs. 3.45 ng/ml [1.84-5.03]; p = 0.0386), suPAR antigen (2.17 μg/ml [1.65-2.81] vs. 2.56 μg/ml [2.24-4.26]; p = 0.0351) and a trend towards lower levels of sCD40L (2.70 pg/ml [1.90-4.00] vs. 3.60 pg/ml [2.95-5.30]; p = 0.0782). Viral load in both groups was similar (0.8 × 105 ng/ml [0.2-42.4 × 105] vs. 1.1 × 105 [0.5-12.2 × 105]; p = 0.3806). No effects attributable to prednisolone were observed when patients receiving HAART in combination with prednisolone were compared to patients who received HAART alone. Conclusions: Patients treated with low-dose prednisolone display significantly lower general immune activation than untreated patients. Further longitudinal studies are required to assess whether treatment with low-dose prednisolone translates into differences in HIV disease progression. KW - HIV KW - Prednisolon Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75100 ER - TY - JOUR A1 - Kasang, Christa A1 - Kalluvya, Samuel A1 - Majinge, Charles A1 - Stich, August A1 - Bodem, Jochen A1 - Kongola, Gilbert A1 - Jacobs, Graeme B. A1 - Mllewa, Mathias A1 - Mildner, Miriam A1 - Hensel, Irina A1 - Horn, Anne A1 - Preiser, Wolfgang A1 - van Zyl, Gert A1 - Klinker, Hartwig A1 - Koutsilieri, Eleni A1 - Rethwilm, Axel A1 - Scheller, Carsten A1 - Weissbrich, Benedikt T1 - HIV drug resistance (HIVDR) in antiretroviral therapy-naive patients in Tanzania not eligible for WHO threshold HIVDR survey is dramatically high N2 - Background: The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients ,25 years is representative for HIVDR in the rest of the therapy-naive population. Methods and Findings: HIVDR was determined in 88 sequentially enrolled ART-naive patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged, 25 years and 68 patients were aged 25–63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072–0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients .25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095–0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma. Conclusions: ART-naive patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naive population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naive HIV-infected population. KW - Tansania KW - HIV Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-69024 ER - TY - JOUR A1 - Kasang, Christa A1 - Kalluvya, Samuel A1 - Majinge, Charles A1 - Stich, August A1 - Bodem, Jochen A1 - Kongola, Gilbert A1 - Jacobs, Graeme B. A1 - Mlewa, Mathias A1 - Mildner, Miriam A1 - Hensel, Irina A1 - Horn, Anne A1 - Preiser, Wolfgang A1 - van Zyl, Gert A1 - Klinker, Hartwig A1 - Koutsilieri, Eleni A1 - Rethwilm, Axel A1 - Scheller, Carsten A1 - Weissbrich, Benedikt T1 - HIV Drug Resistance (HIVDR) in Antiretroviral Therapy-Naïve Patients in Tanzania Not Eligible for WHO Threshold HIVDR Survey Is Dramatically High JF - PLoS One N2 - Background The World Health Organization (WHO) has recommended guidelines for a HIV drug resistance (HIVDR) survey for resource-limited countries. Eligibility criteria for patients include age below 25 years in order to focus on the prevalence of transmitted HIVDR (tHIVDR) in newly-infected individuals. Most of the participating sites across Africa have so far reported tHIVDR prevalences of below 5%. In this study we investigated whether the rate of HIVDR in patients <25 years is representative for HIVDR in the rest of the therapy-naïve population. Methods and Findings HIVDR was determined in 88 sequentially enrolled ART-naïve patients from Mwanza, Tanzania (mean age 35.4 years). Twenty patients were aged <25 years and 68 patients were aged 25–63 years. The frequency of HIVDR in the study population was 14.8% (95%; CI 0.072–0.223) and independent of NVP-resistance induced by prevention of mother-to-child transmission programs. Patients >25 years had a significantly higher HIVDR frequency than younger patients (19.1%; 95% CI 0.095–0.28) versus 0%, P = 0.0344). In 2 out of the 16 patients with HIVDR we found traces of antiretrovirals (ARVs) in plasma. Conclusions ART-naïve patients aged over 25 years exhibited significantly higher HIVDR than younger patients. Detection of traces of ARVs in individuals with HIVDR suggests that besides transmission, undisclosed misuse of ARVs may constitute a significant factor in the generation of the observed high HIVDR rate. The current WHO tHIVDR survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may therefore result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Similar studies should be performed also in other areas to test whether the so far reported optimistic picture of low HIVDR prevalence in young individuals is really representative for the rest of the ART-naïve HIV-infected population. KW - Tanzania KW - antimicrobial resistance KW - antiretroviral therapy KW - HIV KW - sequence databases KW - mutation databases KW - antiretrovirals KW - HIV diagnosis and management Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-137988 VL - 6 IS - 8 ER - TY - JOUR A1 - Kasang, Christa A1 - Kalluvya, Samuel A1 - Majinge, Charles A1 - Kongola, Gilbert A1 - Mlewa, Mathias A1 - Massawe, Irene A1 - Kabyemera, Rogatus A1 - Magambo, Kinanga A1 - Ulmer, Albrecht A1 - Klinker, Hartwig A1 - Gschmack, Eva A1 - Horn, Anne A1 - Koutsilieri, Eleni A1 - Preiser, Wolfgang A1 - Hofmann, Daniela A1 - Hain, Johannes A1 - Müller, Andreas A1 - Dölken, Lars A1 - Weissbrich, Benedikt A1 - Rethwilm, Axel A1 - Stich, August A1 - Scheller, Carsten T1 - Effects of Prednisolone on Disease Progression in Antiretroviral-Untreated HIV Infection: A 2-Year Randomized, Double-Blind Placebo-Controlled Clinical Trial JF - PLoS One N2 - Background HIV-disease progression correlates with immune activation. Here we investigated whether corticosteroid treatment can attenuate HIV disease progression in antiretroviral-untreated patients. Methods Double-blind, placebo-controlled randomized clinical trial including 326 HIV-patients in a resource-limited setting in Tanzania (clinicaltrials.gov NCT01299948). Inclusion criteria were a CD4 count above 300 cells/μl, the absence of AIDS-defining symptoms and an ART-naïve therapy status. Study participants received 5 mg prednisolone per day or placebo for 2 years. Primary endpoint was time to progression to an AIDS-defining condition or to a CD4-count below 200 cells/μl. Results No significant change in progression towards the primary endpoint was observed in the intent-to-treat (ITT) analysis (19 cases with prednisolone versus 28 cases with placebo, p = 0.1407). In a per-protocol (PP)-analysis, 13 versus 24 study participants progressed to the primary study endpoint (p = 0.0741). Secondary endpoints: Prednisolone-treatment decreased immune activation (sCD14, suPAR, CD38/HLA-DR/CD8+) and increased CD4-counts (+77.42 ± 5.70 cells/μl compared to -37.42 ± 10.77 cells/μl under placebo, p < 0.0001). Treatment with prednisolone was associated with a 3.2-fold increase in HIV viral load (p < 0.0001). In a post-hoc analysis stratifying for sex, females treated with prednisolone progressed significantly slower to the primary study endpoint than females treated with placebo (ITT-analysis: 11 versus 21 cases, p = 0.0567; PP-analysis: 5 versus 18 cases, p = 0.0051): No changes in disease progression were observed in men. Conclusions This study could not detect any significant effects of prednisolone on disease progression in antiretroviral-untreated HIV infection within the intent-to-treat population. However, significant effects were observed on CD4 counts, immune activation and HIV viral load. This study contributes to a better understanding of the role of immune activation in the pathogenesis of HIV infection. KW - HIV KW - immune activation KW - viral load KW - drug adherence KW - viral replication KW - AIDS KW - HIV infections KW - highly-active antiretroviral therapy Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-146479 VL - 11 IS - 1 ER - TY - THES A1 - Kaiser, Fabian Marc Philipp T1 - Analysis of Cross-Clade Neutralizing Antibodies against HIV-1 Env Induced by Immunofocusing T1 - Analyse von breit neutralisierenden Antikörpern gegen HIV-1 Env, die durch Immunofocusing induziert wurden N2 - Despite intense research efforts, a safe and effective HIV-1/AIDS vaccine still remains far away. HIV-1 escapes the humoral immune response through various mechanisms and until now, only a few nAbs have been identified. A promising strategy to identify new epitopes that may elicit such nAbs is to dissect and analyze the humoral immune response of sera with broadly reactive nAbs. The identified epitopes recognized by these antibodies might then be incorporated into a vaccine to elicit similar nAbs and thus provide protection from HIV-1 infection. Using random peptide phage display libraries, the Ruprecht laboratory has identified the epitopes recognized by polyclonal antibodies of a rhesus monkey with high-titer, broadly reactive nAbs that had been induced after infection with a SHIV encoding env of a recently transmitted HIV-1 clade C. The laboratory analyzed phage peptide inserts for conformational and linear homology with computational assistance. Several of the identified peptides mimicked domains of the original HIV-1 clade Env, such as conformational V3 loop epitopes and the conserved linear region of the gp120 C-terminus. As part of this work, these mimotopes were analyzed for cross-reactivity with other sera obtained from rhesus monkeys with nAbs and antibody recognition was shown for several mimotopes, particularly those representing the V3 loop. In addition, these mimotopes were incorporated into a novel DNA prime/phage boost strategy to analyze the immunogenicity of such phage-displayed peptides. Mice were primed only once with HIV-1 clade C gp160 DNA and subsequently boosted with mixtures of recombinant phages. This strategy was designed to focus the humoral immune response on a few, selected Env epitopes (immunofocusing) and induced HIV-1 clade C gp160 binding antibodies and cross-clade nAbs. Furthermore, the C-terminus of gp120, a conserved HIV Env region, was linked to the induction of nAbs for the first time. The identification of such conserved antigens may lead to the development of a vaccine that is capable of inducing broadly reactive nAbs that might confer protection form HIV-1 infection. N2 - Trotz enormer Forschungsleistungen liegt ein sicherer und effektiver Impfstoff gegen HIV-1/AIDS immer noch in weiter Ferne. HIV-1 entkommt der humoralen Immunantwort aufgrund mehrerer Mechanismen und daher wurden bis zu diesem Zeitpunkt nur wenige neutralisierende Antikörper identifiziert. Eine vielversprechende Strategie zur Identifizierung neuer Epitope, die neutralisierende Antikörper induzieren könnten, ist die Analyse von Seren mit solchen Antikörper. Die dabei identifizierten Epitope könnten dann zur Herstellung eines Impfstoffes verwendet werden, der ähnliche neutralisierende Antikörper induziert und damit vor einer Infektion mit HIV-1 schützt. Mittels Phage-Display hat das Labor von Ruth Ruprecht mehrere solcher Epitope von polyklonalen Antikörpern aus Rhesus Affen mit hochtitrigen, breit-neutralisierenden Antikörpern identifiziert. Diese Antikörper wurden nach einer Infektion mit einem SHIV induziert, das das virale Hüllprotein eines kürzlich übertragenen HIV-1 clade C Virus enthielt. Die Phagenpeptide wurden auf konformelle und lineare Homologie mittels einer Computer Software untersucht. Mehrere dieser Peptide entsprachen Domänen des viralen Hüllproteins, wie z.B. konformelle V3 loop Epitope and Epitope des linearen C-Terminus von gp120. Im Rahmen dieser Arbeit wurden diese Mimotope auf Kreuzreaktivität mit anderen Seren von Rhesus Affen mit neutralisierenden Antikörpern untersucht. Dabei wurden insbesondere die Mimotope des V3 loops von anderen Seren erkannt. Des Weiteren wurden diese Phagen-Mimotope im Rahmen einer neuen DNA prime/phage boost Strategie zur Immunisierung verwendet. Mäuse wurden einmalig mit HIV-1 clade C gp160 DNA immunisiert und anschließend mehrfach mit rekombinanten Phagen geboostet. Mittels dieser Strategie sollte das Immunsystem auf einige, spezielle Epitope des viralen Hüllproteins fokusiert werden (Immunofocusing). Hierbei wurden HIV-1 clade gp160-bindende Antikörper und breit-neutralisierende Antikörper induziert. Des Weiteren konnten zum ersten Mal neutralisierende Antikörper gegen den C-terminus von gp120, einer konservierten Region des viralen Hüllproteins, induziert werden. Die Identifikation solcher konservierter Mimotope kann zur Entwicklung von einem HIV-1 Impfstoff beitragen, der breit neutralisierende Antikörper induziert, die vor einer Infektion schützen können. KW - Antikörper KW - HIV KW - Phage Display KW - Impfstoff KW - Antibody KW - HIV KW - Phage Display KW - Vaccine Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-75494 ER - TY - THES A1 - Jacobs, Graeme Brendon T1 - HIV-1 resistance analyses from therapy-naïve patients in South Africa, Tanzania and the characterization of a new HIV-1 subtype C proviral molecular clone T1 - HIV-1 Resistenz-Analysen von nicht-therapierten Patienten aus Südafrika und Tansania und Charakterisierung eines neuen HIV-1 Subtyp C proviralen molekularen Klons N2 - The acquired immunodeficiency syndrome (AIDS) is currently the most infectious disease worldwide. It is caused by the human immunodeficiency virus (HIV). At the moment there are ~33.3 million people infected with HIV. Sub-Saharan Africa, with ~22.5 million people infected accounts for 68% of the global burden. In most African countries antiretroviral therapy (ART) is administered in limited-resource settings with standardised first- and second-line ART regimens. During this study I analysed the therapy-naïve population of Cape Town, South Africa and Mwanza, Tanzania for any resistance associated mutations (RAMs) against protease inhibitors, nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors. My results indicate that HIV-1 subtype C accounts for ~95% of all circulating strains in Cape Town, South Africa. I could show that ~3.6% of the patient derived viruses had RAMs, despite patients being therapy-naïve. In Mwanza, Tanzania the HIV drug resistance (HIVDR) prevalence in the therapy-naïve population was 14.8% and significantly higher in the older population, >25 years. Therefore, the current WHO transmitted HIVDR (tHIVDR) survey that is solely focused on the transmission of HIVDR and that excludes patients over 25 years of age may result in substantial underestimation of the prevalence of HIVDR in the therapy-naïve population. Based on the prevalence rates of tHIVDR in the study populations it is recommended that all HIV-1 positive individuals undergo a genotyping resistance test before starting ART. I also characterized vif sequences from HIV-1 infected patients from Cape Town, South Africa as the Vif protein has been shown to counteract the antiretroviral activity of the cellular APOBEC3G/F cytidine deaminases. There is no selective pressure on the HIV-1 Vif protein from current ART regimens and vif sequences was used as an evolutionary control. As the majority of phenotypic resistance assays are still based on HIV-1 subtype B, I wanted to design an infectious HIV-1 subtype C proviral molecular clone that can be used for in vitro assays based on circulating strains in South Africa. Therefore, I characterized an early primary HIV-1 subtype C isolate from Cape Town, South Africa and created a new infectious subtype C proviral molecular clone (pZAC). The new pZAC virus has a significantly higher transient viral titer after transfection and replication rate than the previously published HIV-1 subtype C virus from Botswana. The optimized proviral molecular clone, pZAC could be used in future cell culture and phenotypic HIV resistance assays regarding HIV-1 subtype C. N2 - Das erworbene Immundefektsyndrom (“acquired immunodeficiency syndrome”, AIDS), verursacht durch das Humane Immundefizienzvirus (HIV), ist derzeit die häufigste Infektionskrankheit weltweit. Zirka 33,3 Millionen Menschen sind gegenwärtig mit HIV infiziert, wobei hiervon etwa 22,5 Millionen Infizierte (68%) in den Ländern südlich der Sahara leben. In den meisten dieser Länder ist die antiretrovirale Therapie (ART) in nur zwei standardisierten Medikamentenkombinationen verfügbar. In dieser Arbeit wurden nichttherapierte Patienten aus Kapstadt (Südafrika) und Mwanza (Tansania) auf resistenzassoziierte Mutationen (RAMs) gegen Protease Inhibitoren, nukleosidische- und nichtnukleosidische Reverse Transkriptase Inhibitoren analysiert. Meine Ergebnisse zeigten, dass in 3,6 % der Patienten RAMs gefunden wurden, obwohl diese nicht vortherapiert waren. In der Patientengruppe aus Tansania wurden sogar in 14,8 % der Patientenviren RAMs gefunden. Dieses Patientenkollektiv war signifikant älter als 25 Jahre und damit außerhalb der von der WHO beobachteten Altersgruppe. Meine Studie legt nahe, dass die WHO-Kriterien zur Überwachung der Übertragung von resistenten HIVs die Weitergabe von resistenten Viren unterschätzt, da Patienten über 25 Jahre ausgeschlossen werden. Weiterhin wurden vif Sequenzen von HIV-1 infizierten Patienten aus Kapstadt charakterisiert, da bereits gezeigt wurde, dass das HIV Vif Protein die antiretrovirale Aktivität der Cytidin Deaminase APOBEC3G/F antagonisieren kann. Da jedoch keine Medikamenten induzierte Selektion auf diesen Sequenzen liegt, wurden diese zur Analyse der viralen Evolution verwendet. Phenotypische Resistenzanalysen basieren gegenwärtig meist auf dem HIV Subtyp B, jedoch sind die meisten Infizierten in Südafrika und sogar weltweit mit Subtyp C infiziert. Deshalb war es ein Ziel dieser Arbeit einen proviralen HIV Subtyp C Plasmid zu entwickeln. Dazu wurde das Virus aus einem frühen HIV Subtyp C Isolat kloniert. Das hier neu klonierte Virus (HIV-ZAC) zeigt sowohl einen höheren viralen Titer nach der Transfektion und auch eine höhere Replikationsrate als das zuvor publizierte HIV-1 Suptyp C Virus aus Botswana. Deshalb könnte der von mir optimierte und neu charakterisierte provirale molekulare Klon, pZAC, zukünftig in der Zellkultur und bei phenotypischen HIV Resistenztests als wildtypisches HIV-1 Suptyp C Virus eingesetzt werden. KW - HIV KW - Immunität KW - Südafrika KW - Tansania KW - HIV-1 KW - Subtyp C KW - HIV-1 KW - resistance KW - diversity KW - South Africa KW - Tanzania Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-67319 ER - TY - JOUR A1 - Jacobs, Graeme A1 - Bock, Stefanie A1 - Schuch, Anita A1 - Moschall, Rebecca A1 - Schrom, Eva-Maria A1 - Zahn, Juliane A1 - Reuter, Christian A1 - Preiser, Wolfgang A1 - Rethwilm, Axel A1 - Engelbrecht, Susan A1 - Krekau, Thomas A1 - Bodem, Jochen T1 - Construction of a high titer Infectious HIV-1 subtype C proviral clone from South Africa N2 - The Human Immunodeficiency Virus type 1 (HIV-1) subtype C is currently the predominant subtype worldwide. Cell culture studies of Sub-Saharan African subtype C proviral plasmids are hampered by the low replication capacity of the resulting viruses, although viral loads in subtype C infected patients are as high as those from patients with subtype B. Here, we describe the sequencing and construction of a new HIV-1 subtype C proviral clone (pZAC), replicating more than one order of magnitude better than the previous subtype C plasmids. We identify the env-region for being the determinant for the higher viral titers and the pZAC Env to be M-tropic. This higher replication capacity does not lead to a higher cytotoxicity compared to previously described subtype C viruses. In addition, the pZAC Vpu is also shown to be able to down-regulate CD4, but fails to fully counteract CD317. KW - HIV KW - HIV-1; subtype C; proviral plasmid; viral replication; resistance assays; Vpu; CD317; CD4 Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-76340 ER - TY - JOUR A1 - Horn, Anne A1 - Scheller, Carsten A1 - du Plessis, Stefan A1 - Arendt, Gabriele A1 - Nolting, Thorsten A1 - Joska, John A1 - Sopper, Sieghart A1 - Maschke, Matthias A1 - Obermann, Mark A1 - Husstedt, Ingo W. A1 - Hain, Johannes A1 - Maponga, Tongai A1 - Riederer, Peter A1 - Koutsilieri, Eleni T1 - Increases in CSF dopamine in HIV patients are due to the dopamine transporter 10/10-repeat allele which is more frequent in HIV-infected individuals JF - Journal of Neural Transmission N2 - Dysfunction of dopaminergic neurotransmission has been implicated in HIV infection. We showed previously increased dopamine (DA) levels in CSF of therapy-naïve HIV patients and an inverse correlation between CSF DA and CD4 counts in the periphery, suggesting adverse effects of high levels of DA on HIV infection. In the current study including a total of 167 HIV-positive and negative donors from Germany and South Africa (SA), we investigated the mechanistic background for the increase of CSF DA in HIV individuals. Interestingly, we found that the DAT 10/10-repeat allele is present more frequently within HIV individuals than in uninfected subjects. Logistic regression analysis adjusted for gender and ethnicity showed an odds ratio for HIV infection in DAT 10/10 allele carriers of 3.93 (95 % CI 1.72–8.96; p = 0.001, Fishers exact test). 42.6 % HIV-infected patients harbored the DAT 10/10 allele compared to only 10.5 % uninfected DAT 10/10 carriers in SA (odds ratio 6.31), whereas 68.1 versus 40.9 %, respectively, in Germany (odds ratio 3.08). Subjects homozygous for the 10-repeat allele had higher amounts of CSF DA and reduced DAT mRNA expression but similar disease severity compared with those carrying other DAT genotypes. These intriguing and novel findings show the mutual interaction between DA and HIV, suggesting caution in the interpretation of CNS DA alterations in HIV infection solely as a secondary phenomenon to the virus and open the door for larger studies investigating consequences of the DAT functional polymorphism on HIV epidemiology and progression of disease. KW - HIV KW - HAND KW - dopamine KW - DAT KW - polymorphism KW - CSF Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132385 VL - 120 ER - TY - JOUR A1 - Decloedt, Eric H. A1 - Freeman, Carla A1 - Howells, Fleur A1 - Casson-Crook, Martine A1 - Lesosky, Maia A1 - Koutsilieri, Eleni A1 - Lovestone, Simon A1 - Maartens, Gary A1 - Joska, John A. T1 - Moderate to severe HIV-associated neurocognitive impairment : A randomized placebo-controlled trial of lithium JF - Medicine N2 - Background: HIV-associated neurocognitive disorder (HAND) remains highly prevalent despite effective anti-retroviral therapy (ART). A number of adjunctive pharmacotherapies for HAND have been studied with disappointing results, but preliminary data suggest that lithium may provide clinical benefit. In addition, the low cost of lithium would facilitate access in low- and middle-income countries which carry the greatest burden of HIV. Methods: Our objective was to evaluate the 24-week efficacy and safety of lithium in patients with moderate to severe HAND. Our primary efficacy endpoint was the change in Global Deficit Score (GDS) from baseline to 24 weeks, whereas our secondary endpoint was the change in proton magnetic resonance spectroscopy (1H-MRS) brain metabolite concentrations. We conducted a 24-week randomized placebo-controlled trial of lithium as adjunctive pharmacotherapy. We enrolled participants with moderate to severe HAND, on ART for at least 6 months, with suppressed viral loads and attending public sector primary care clinics in Cape Town, South Africa. We randomized 66 participants to lithium (n = 32) or placebo (n = 34). Lithium or placebo was dosed 12-hourly and titrated to achieve the maintenance target plasma concentration of 0.6 to 1.0 mmol/L. Sham lithium concentrations were generated for participants receiving placebo. Results: Totally 61 participants completed the study (lithium arm = 30; placebo arm = 31). Participants at enrolment had a mean age of 40 years and a median CD4+ T-cell count of 500 cells/μL. The median change in GDS between baseline and week 24 for the lithium and placebo arms were –0.57 (95% confidence interval [CI] –0.77, –0.32) and –0.56 (–0.69, –0.34) respectively, with a mean difference of –0.054 (95% CI –0.26, 0.15); P = 0.716. The improvement remained similar when analyzed according to age, severity of impairment, CD4+ count, time on ART, and ART regimen. Standard 1H-MRS metabolite concentrations were similar between the treatment arms. The study drug was well tolerated in both study arms. Six serious adverse events occurred, but none were considered related to the study drug. Conclusion: Adjunctive lithium pharmacotherapy in patients on ART with HAND was well tolerated but had no additional benefit on neurocognitive impairment. KW - antiretroviral therapy KW - HIV KW - HIV neurocognitive impairment KW - lithium KW - Placebo KW - randomized controlled clinical trial KW - South Africa Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-165838 VL - 95 IS - 46 ER - TY - THES A1 - Brado, Dominik Alexander T1 - Genetic diversity and baseline drug resistance of South African HIV-1 Integrase sequences prior to the availability of Integrase strand-transfer inhibitors T1 - Genetische Variabilität und medikamentöse Resistenz südafrikanischer HIV-1 Integrase Sequenzen vor der Verfügbarkeit von Integrase Strang-Transfer Inhibitoren N2 - Background: Integrase strand transfer inhibitors (INSTIs) are the latest addition to the array of antiretroviral compounds used to treat an infection with Human Immunodeficiency Virus (HIV). Due to their high efficacy and increased tolerability, INSTIs have become an integral part of first-line therapy in most high-income countries over the past years. However, little is known about HIV-1’s genetic inter- and intra-subtype diversity on the Integrase (IN)-gene and its impact on the emergence of INSTI-resistance. In the absence of a functional cure, long-term efficacy of first-line compounds remains paramount for reducing virological failure and curbing on-going HIV transmissions. South Africa, harbouring more than 20% of the global HIV burden (7.7 / 37.9 million people), requires international attention in order to globally pursue UNAIDS’ (Joint United Nations Programme on HIV/AIDS) 90-90-90 goals and the road to ending the HIV/AIDS (Acquired immunodeficiency syndrome) pandemic by 2030. Methods: In this study, the prevalence of INSTI-resistance associated mutations (RAM) was investigated in a cohort of 169 archived drug-naïve blood samples from multiple collection sites around Cape Town, South Africa. Viral RNA was isolated from plasma samples, the integrase fragment amplified by RT-PCR and subsequently sequenced by Sanger-sequencing. Additionally, all publicly available drug-naïve, South African IN sequences, isolated before the availability of the first INSTIs in 2007, were retrieved from the Los Alamos HIV sequence database (n=284). All sequences were analysed for RAMs using the Stanford HIV Drug resistance database. The identification of polymorphism in the South African subtype C IN consensus sequence allowed for comparative analyses with global subtype B, as well as subtype C sequences, from countries other than South Africa. Results: The IN gene could be amplified and sequenced in 95/169 samples (56%). Phylogenetic inference revealed close homology between three sequence-pairs, warranting the exclusion of 3/95 sequences from further analyses. Of the 92 samples used for mutational analyses, 86/92 (93.5%) belonged to subtype C, 5/92 (5.4%) to subtype B and 1/92 (1.1%) to subtype A. The prevalence of major and accessory INSTI RAMs was 0/92 (0%) and 1/91 (1.1%), respectively, similar to the observed rates of 8/284 (2.8%) and 8/284 (2.8%) in the database sequences (p = 0.2076 and p = 0.6944, Fisher’s exact test). Compared to subtype B IN sequences, 15 polymorphisms were significantly enriched in South African subtype C sequences (corrected p<0.0015. Fisher’s exact test, Bonferroni post-hoc procedure). Compared to subtype C IN sequences isolated outside South Africa, four polymorphisms were significantly enriched in this study cohort (corrected p<0.0014, Fisher’s exact test, Bonferroni post-hoc procedure). The highest prevalence margin was observed for the polymorphism Met50Ile being present in 60.1% of South African subtype C sequences, compared to 37% in non-South African subtype C sequences. Conclusions: The low prevalence of major and minor RAMs in all South African Integrase sequences predicts a high susceptibility to INSTIs, however, the presence of natural polymorphisms, in particular Met50Ile, in the majority of sequences warrants further monitoring under therapeutic pressure, as their role in mutational pathways leading to INSTI- resistance is yet to be determined. Additionally, this study revealed the presence of substantial inter- and intra-subtype diversity within the HIV-1 Subtype C IN-gene. These results implicate the need for more research on a regional, potentially patient-specific level, as mutational insights from other diverse backgrounds may not accurately represent the South African context. The implementation of a national pre-treatment INSTI-resistance screening program may provide necessary insights into the development of mutational pathways leading to INSTI-resistance under therapeutic pressure for the South African context and thereby bring South Africa one step closer to achieving UNAIDS 90-90-90 goals and ending the AIDS epidemic by 2030. N2 - Hintergrund: Integrase Strang-Transfer Inhibitoren (INSTIs) sind die neuste medikamentöse Ergänzung in der Therapie einer HIV-Infektion. Auf Grund ihrer starken Wirksamkeit und eines guten Nebenwirkungsprofils sind INSTIs in den letzten Jahren ein integraler Bestandteil von Erstlinien-Therapieregimen in den meisten wirtschaftlich starken Ländern geworden. Allerdings ist wenig bekannt über die genetische Variabilität des IN-gens und über ihren Einfluss auf die Entwicklung von INSTI-Resistenzen. Mit einem Anteil von über 20% der globalen HIV-Last (7,7 / 37,9 Millionen Menschen) benötigt Südafrika einen internationalen Fokus, um die von UNAIDS formulierten 90-90-90 Ziele und das mögliche Ende der HIV/AIDS Pandemie bis 2030 auf globaler Ebene zu verfolgen. Methoden: In dieser Arbeit wurde die Prävalenz von INSTI RAMs in einer Kohorte von 169 archivierten, therapie-naiven Blutproben von mehreren Sammelstellen um Kapstadt, Südafrika, untersucht. Virale RNA wurde aus Plasmaproben isoliert, das Integrase-Fragment mittels RT-PCR amplifiziert und anschließend Sanger-sequenziert. Zusätzlich wurden alle in der Los Alamos HIV Sequenz Datenbank verfügbaren, therapie-naive, südafrikanische IN Sequenzen, die vor der Verfügbarkeit von INSTIs im Jahr 2007 isoliert wurden, der Analyse dieser Arbeit hinzugefügt (n=284). Die Interpretation der gefundenen Mutationen erfolgte mittels der HIV Therapie-Resistenz Datenbank der Stanford Universität. Durch Generierung eines südafrikanischen IN Subtyp C Consensus-Stranges und nachfolgendem Vergleich mit öffentlich verfügbaren Subtyp B und Subtyp C Sequenzen, die außerhalb Südafrikas isoliert wurden, erfolgte die Analyse von natürlich vorkommenden Polymorphismen. Ergebnisse: Das IN-Fragment konnte in 95/169 Plasmaproben (56%) erfolgreich amplifiziert und sequenziert werden. Phylogenetische Analysen zeigten eine enge Homologie zwischen drei Sequenz-Paaren, woraufhin 3/95 Sequenzen von weiteren Analysen ausgeschlossen wurden. Von den übrigen 92 Sequenzen gehörten 86/92 (93,5%) zu dem Subtyp C, 5/92 (5,4%) zu dem Subtyp B und 1/91 (1,1%) zu dem Subtyp A. Die Prävalenz von Haupt- und Nebenresistenz-Mutationen lag bei jeweils 0/92 (0%) und 1/92 (1,1%). Ähnliche Raten hierfür von 8/284 (2,8%) und 8/284 (2,8%) konnten in den Datenbank-Sequenzen beobachtet werden (p = 0,2076 und p = 0,6944, Fisher’s exact test). Im Vergleich zu Subtyp B IN Sequenzen waren 15 Polymorphismen signifikant erhöht in südafrikanischen Subtype C IN Sequenzen (korrigiertes p<0,0015, Fisher’s exact test, Bonferroni post-hoc Korrektur). Im Vergleich zu nicht-südafrikanischen Subtyp C Sequenzen zeigten sich vier Polymorphismen signifikant erhöht (korrigiertes p<0,0014, Fisher’s exact test, Bonferroni post-hoc Korrektur). Der größte Prävalenzunterschied konnte für den Polymorphismus Met50Ile beobachtet werden. Dieser war vorhanden in 217/361 (60,1%) der südafrikanischen Subtyp C Sequenzen, verglichen zu 203/548 (37.0%) der nicht-südafrikanischen Subtyp C Sequenzen. Schlussfolgerung: Die niedrige Prävalenz von Haupt- und Neben-RAMs in südafrikanischen IN-Sequenzen verspricht ein gutes Ansprechen von INSTIs in diesem Kontext. Allerdings bedingt das Vorhandensein von natürlichen Polymorphismen, insbesondere der Polymorphismus Met50Ile das weitere Beobachten dieser Mutationen unter dem Einfluss von therapeutischem Druck, da deren Bedeutung in der Entwicklung von INSTI-Resistenzen noch nicht abschließend geklärt werden konnte. Zudem impliziert die in dieser Arbeit gezeigte inter- und intra-subtyp Diversität auf dem IN-Gen, die Notwendigkeit von weiterer Forschung auf regionaler Ebene, da Beobachtungen, die auf verschiedenen polymorphistischen Kontexten beruhen, nicht notwendigerweise auf den südafrikanischen Kontext übertragen werden können. Mit der Einführung eines nationalen, prä-therapeutischen Screening- Programms für das Vorhandensein von INSTI-Resistenzen könnte Südafrika wichtige Einblicke in die Entwicklung von INSTI-Resistenzen gewinnen und somit den 90-90-90 Zielen und der Möglichkeit die AIDS-Pandemie bis zum Jahr 2030 zu beenden, einen Schritt näher sein. KW - HIV KW - Sequenzanalyse KW - HIV Drug resistance KW - HIV South Africa KW - Integrase inhibitor KW - Dolutegravir Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-216562 ER - TY - THES A1 - Aminake, Makoah Nigel T1 - Towards malaria combination therapy: Characterization of hybrid molecules for HIV/malaria combination therapy and of thiostrepton as a proteasome-targeting antibiotic with a dual mode of action T1 - Die Entwicklung von Malaria-Kombinationstherapien: Die Charakterisierung von Hybridmolekülen für eine HIV/Malaria-Kombinationstherapie und von Thiostrepton als ein gegen das Proteasom-gerichtetes Antibiotikum mit dualem Wirkmodus N2 - Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite’s proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease. N2 - Malaria und HIV gehören zu den wichtigsten weltweiten Gesundheitsproblemen unserer Zeit und verursachen jährlich zusammen fast drei Millionen Todesfälle. Das Verbreitungsgebiet beider Krankheit überschneidet sich in vielen Weltregionen wie Afrika südlich der Sahara, Südostasien und Südamerika, was zu einem erhöhten Risiko für Koinfektionen führt. Während der vorliegenden Arbeit stellten wir Hybridmoleküle her und charakterisierten diese in Bezug auf ihre gleichzeitige Wirksamkeit gegen P. falciparum und HIV mit dem Ziel einer möglichen Kombinationstherapie gegen beide Krankheiten. Diese Hybridmoleküle wurden durch kovalente Verbindung von Azidothymidin (AZT) mit Dihydroartemisinin (DHA), Tetraoxan und Chloroquin (CQ) hergestellt. Die dabei hergestellte kleine Molekülsammlung wurde auf antivirale Wirkung und Wirkung gegen Malaria getestet. In vitro ist, gemäß unserer Daten, Dihyate das wirksamste Molekül, mit einer dem DHA vergleichbaren Wirksamkeit gegen Plasmodium (IC50 = 26 nM, SI > 3000), einer mittelmäßigen Wirksamkeit gegen HIV (IC50 = 2.9 µM; SI > 35) und keiner Wirkung auf HeLa-Zellen bei den im Versuch verwendeten Konzentrationen (CC50 > 100 µM). Weiterhin ergaben pharmakokinetische Studien, dass Dihyate metabolisch instabil ist und nach einer O-Dealkylierung gespalten wird, sobald es in Kontakt mit Cytochrom P450 Enzymen kommt. Dies erklärt auch die Unwirksamkeit von Dihyate gegen dem CQ-sensitiven P. berghei N Stamm im Mausversuch bei oraler Gabe von 20mg/kg. Wir berichten hier von einem ersten Ansatz Hybridmoleküle gegen Malaria/ HIV zu entwickeln. Zukünftige Verbesserungen werden darauf abzielen Hybridmoleküle der zweiten Generation herzustellen um sowohl die Wirksamkeit gegen HIV als auch die Bioverfügbarkeit zu verbessern. Auf Grund der Entwicklung von Resistenzen gegenüber sämtliche Substanzen, die zusammen mit Artemisinin in Kombinationstherapien genutzt werden, hat die Verwendung von Antibiotika bei der Behandlung der Malaria das Interesse daran neu geweckt, Antibiotika mit starker Wirksamkeit gegenüber Plasmodium aufzuspüren. Während der vorliegenden Studie untersuchten wir die Wirksamkeit von Thiostrepton und seinen Derivaten gegenüber Plasmodium. Diese Derivate wurden durch Kombinationen von Verkürzung der Seitenkette, Oxidation und der Anbringung von lipophilen Thiolen an die endständige Dehydroaminosäure hergestellt. Wir konnten zeigen, dass die Derivate SS231 und SS234 (IC50 = 1 µM SI > 59 und SI > 77) eine bessere Wirksamkeit gegen Plasmodium besitzen als Thiostrepton (IC50 = 8.95 µM, SI = 1.7). Diese Wirksamkeit konnte bei Konzentrationen beobachtet werden, die nicht hämolytisch sind und ungiftig gegenüber menschlichen Zelllinien. Thiostrepton und seine Derivate zeigten transmissionsblockierende Eigenschaften, wenn sie in Konzentrationen, die ihren IC50- oder IC90-Werten entsprachen, eingesetzt wurden. Unsere Daten zeigen auch, dass diese Substanzen die Aktivität des Proteasoms von Plasmodium abschwächen, was zu einer Anreicherung von ubiquitinierten Proteinen führte, wenn die Parasiten mit den Substanzen in IC80-Konzentrationen inkubiert wurden. Unsere Ergebnisse sprechen dafür, dass das Proteasom ein attraktives Ziel für therapeutische Maßnahmen sein kann. In diesem Zusammenhang sind die Derivate des Thiostreptons vielversprechende Kandidaten, da sie gleichzeitig an zwei unabhängigen Zielstrukturen angreifen, dem Proteasom und dem Apicoplasten und die Fähigkeit besitzen, sowohl die asexuellen Blutstadien als auch diejenigen Blutstadien, die für die Weitergabe des Parasiten verantwortlich sind, zu beseitigen. Um unsere Ergebnisse weiter zu untermauern, untersuchten wir die Wirkung von Peptidyl-Sulfonyl-Fluoriden, einer neuen Klasse von Substanzen mit Wirksamkeit gegen Malaria und hemmender Wirkung gegenüber dem Proteasom auf die Reifung von Gametozyten. Die Substanzen AJ34 und AJ38 unterdrückten die Bildung von Gametozyten vollständig, wenn sie in Konzentrationen, die ihren IC50-Werten (0.23 µM und 0.17 µM) entsprachen, eingesetzt wurden. Dies spricht für ein starkes transmissionsblockierendes Potential dieser Substanzen. Das Proteasom, ein bedeutender proteinabbauender Komplex, der für den Abbau und die Wiedergewinnung nicht funktioneller Proteine verantwortlich ist, wurde bisher nur indirekt in P. falciparum untersucht. Zusätzlich wurde die Existenz eines, dem Proteasom-ähnlichen, Proteins mit Ähnlichkeiten zu bakteriellen ClpQ/hslV Threonin-Peptidasen in Plasmodium vermutet. Gegen die Untereinheiten alpha 5 (α5-SU), beta 5 (β5-SU) und gegen pfhslV wurden in Mäusen Antikörper generiert. Mit diesen konnten wir zeigen, dass das Proteasom sowohl in den asexuellen als auch in den sexuellen Blutstadien von P. falciparum exprimiert wird und im Zellkern und im Zytoplasma lokalisiert sind. Die Expression von PfhslV konnte jedoch nur in Trophozoiten und Schizonten beobachtet werden. Der Transport der Proteasomuntereinheiten wurde weiterhin durch die Herstellung von transgenen Parasiten, die GFP-markierte Proteine bilden, untersucht. Die Expression von α5-SU-GFP in transgenen Parasiten schien im Zytoplasma aller Blutstadien lokalisiert zu sein, wobei durch diese Parasiten keine zusätzlichen Informationen gewonnen werden konnten. Zusammengefasst sprechen unsere Daten für zwei neue Werkzeuge für Kombinationstherapien. Hybridmoleküle sind vielversprechende Werkzeuge zur Heilung von gleichzeitig mit Malaria und HIV infizierten Patienten. Sehr wirksame Antibiotika mit einem breiten Wirkungsspektrum könnten in Artemisinin-Kombinationstherapien nützlich werden, wenn es darum geht, resistente Parasiten zu beseitigen und die Übertragung sowohl der Resistenz als auch der Krankheit zu verringern. KW - Malaria KW - HIV KW - Thiostrepton KW - Arzneimitteldesign KW - Malaria KW - HIV KW - co-infection KW - drug KW - screening KW - hybrid KW - proteasome KW - thiostrepton Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-71841 ER - TY - JOUR A1 - Abimannan, Nagarajan A1 - Sumathi, G. A1 - Krishnarajasekhar, O. R. A1 - Sinha, Bhanu A1 - Krishnan, Padma T1 - Clonal Clusters and Virulence Factors of Methicillin-Resistant \(Staphylococcus\) \(Aureus\): Evidence for Community-Acquired Methicillin-Resistant \(Staphylococcus\) \(Aureus\) Infiltration into Hospital Settings in Chennai, South India JF - Indian Journal of Medical Microbiology N2 - Background and Objective: Staphylococcus aureus is one of the major pathogens of nosocomial infections as wells as community-acquired (CA) infections worldwide. So far, large-scale comprehensive molecular and epidemiological characterisation of S. aureus from very diverse settings has not been carried out in India. The objective of this study is to evaluate the molecular, epidemiological and virulence characteristics of S. aureus in both community and hospital settings in Chennai, southern India. Methods: S. aureus isolates were obtained from four different groups (a) healthy individuals from closed community settings, (b) inpatients from hospitals, (c) outpatients from hospitals, representing isolates of hospital-community interface and (d) HIV-infected patients to define isolates associated with the immunocompromised. Antibiotic susceptibility testing, multiplex polymerase chain reactions for detection of virulence and resistance determinants, molecular typing including Staphylococcal cassette chromosome mec (SCCmec) and agr typing, were carried out. Sequencing-based typing was done using spa and multilocus sequence typing (MLST) methods. Clonal complexes (CC) of hospital and CA methicillin-resistant S. aureus (MRSA) were identified and compared for virulence and resistance. Results and Conclusion: A total of 769 isolates of S. aureus isolates were studied. The prevalence of MRSA was found to be 7.17%, 81.67%, 58.33% and 22.85% for groups a, b, c and d, respectively. Of the four SCCmec types (I, III, IV and V) detected, SCCmec V was found to be predominant. Panton-Valentine leucocidin toxin genes were detected among MRSA isolates harbouring SCCmec IV and V. A total of 78 spa types were detected, t657 being the most prevalent. 13 MLST types belonging to 9 CC were detected. CC1 (ST-772, ST-1) and CC8 (ST238, ST368 and ST1208) were found to be predominant among MRSA. CA-MRSA isolates with SCCmec IV and V were isolated from all study groups including hospitalised patients and were found to be similar by molecular tools. This shows that CA MRSA has probably infiltrated into the hospital settings. KW - Community-acquired methicillin-resistant Staphylococcus aureus KW - HIV KW - hospital-acquired methicillin-resistant Staphylococcus aureus KW - innate immune evasions KW - MLST KW - microbial surface component recognising adhesive matrix molecules KW - spa typing KW - ST 772 KW - Inducible Clindamycin Resistance KW - Valentine Leukocidin Genes KW - Multiplex PCR KW - Nasal Carriage KW - Colonization KW - Prevalence KW - Emergence KW - Skin Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-226963 VL - 37 IS - 3 ER -