@phdthesis{Tabares2011, author = {Tabares, Paula}, title = {Antimicrobial, anti-protease and immunomodulatory activities of secondary metabolites from Caribbean sponges and their associated bacteria}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67000}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Marine sponges and their associated bacteria have been proven to be a rich source of novel secondary metabolites with therapeutic usefulness in infection and autoimmunity. This Ph.D. project aimed to isolate bioactive secondary metabolites from the marine sponges Amphimedon compressa, Aiolochroia crassa and Theonella swinhoei as well as from bacteria associated with different Caribbean sponges, specifically actinomycetes and sphingomonads. In this study, amphitoxin was isolated from the crude methanol extract of the sponge A. compressa and it was found to have antibacterial and anti-parasitic activities. Amphitoxin showed protease inhibitory activity when tested against the mammalian protease cathepsin B and the parasitic proteases rhodesain and falcipain-2. Furthermore, miraziridine A was identified in the dichloromethane extract of the sponge T. swinhoei collected offshore Israel in the Red Sea. Miraziridine A, a natural peptide isolated previously from the marine sponge Theonella aff. mirabilis, is a potent cathepsin B inhibitor with an IC50 value of 1.4 g/mL (2.1 M). Secondary metabolites from sponge-derived bacteria were also isolated and identified. A total of 79 strains belonging to 20 genera of the order Actinomycetales and seven strains belonging to two genera of the order Sphingomonadales were cultivated from 18 different Caribbean sponges and identified by 16S rRNA gene sequencing. Seven of these strains are likely to represent novel species. Crude extracts from selected strains were found to exhibit protease inhibition against cathepsins B and L, rhodesain, and falcipain-2 as well as immunomodulatory activities such as induction of cytokine release by human peripheral blood mononuclear cells. The isolates Sphingobium sp. CO105 and Lapillicoccus sp. BA53 were selected for cultivation, extraction and purification of bioactive metabolites based on initial bioactive screening results. The isoalloxazine isolumichrome was isolated from the strain Sphingobium sp. CO105 which inhibited the protease rhodesain with an IC50 of 0.2 M. The strain Lapillicoccus sp. BA53 was found to produce p-aminosalicylic acid methyl ester, which showed activity against the proteases cathepsins B and L, falcipain-2 and rhodesain. These results highlight the significance of marine sponge-associated bacteria to produce bioactive secondary metabolites with therapeutic potential in the treatment of infectious diseases and disorders of the immune system.}, subject = {Schw{\"a}mme}, language = {en} } @phdthesis{Shityakov2011, author = {Shityakov, Sergey}, title = {Molecular modelling and simulation of retroviral proteins and nanobiocomposites}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-56960}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Molecular modelling and simulation are powerful methods in providing important in-formation on different biological systems to elucidate their structural and functional proper-ties, which cannot be determined in experiment. These methods are applied to analyse versa-tile biological systems: lipid membrane bilayers stabilized by an intercalated single wall carbon nanotube and retroviral proteins such as HIV protease and integrase. HIV-1 integrase has nuclear localization signals (NLS) which play a crucial role in nuclear import of viral preintegration complex (PIC). However, the detailed mechanisms of PIC formation and its nuclear transport are not known. Previously it was shown that NLSs bind to the cell transport machinery e.g. proteins of nuclear pore complex such as transportins. I investigated the interaction of this viral protein HIV-1 integrase with proteins of the nuclear pore complex such as transportin-SR2 (Shityakov et al., 2010). I showed that the transportin-SR2 in nuclear import is required due to its interaction with the HIV-1 integrase. I analyzed key domain interaction, and hydrogen bond formation in transportin-SR2. These results were discussed in comparison to other retroviral species such as foamy viruses to better understand this specific and efficient retroviral trafficking route. The retroviral nuclear import was next analyzed in experiments regarding the retroviral ability to infect nondividing cells. To accomplish the gene transfer task successfully, ret-roviruses must efficiently transduce different cell cultures at different phases of cell cycle. However, promising and safe foamy viral vectors used for gene transfer are unable to effi-ciently infect quiescent cells. This drawback was due to their inability to create a preintegra-tion complex (PIC) for nuclear import of retroviral DNA. On the contrary, the lentiviral vec-tors are not dependant on cell cycle. In the course of reverse transcription the polypurine tract (PPT) is believed to be crucial for PIC formation. In this thesis, I compared the transduction frequencies of PPT modified FV vectors with lentiviral vectors in nondividing and dividing alveolar basal epithelial cells from human adenocarcinoma (A549) by using molecular cloning, transfection and transduction techniques and several other methods. In contrast to lentiviral vectors, FV vectors were not able to effi-ciently transduce nondividing cell (Shityakov and Rethwilm, unpublished data). Despite the findings, which support the use of FV vectors as a safe and efficient alternative to lentiviral vectors, major limitation in terms of foamy-based retroviral vector gene transfer in quiescent cells still remains. Many attempts have been made recently to search for the potential molecules as pos-sible drug candidates to treat HIV infection for over decades now. These molecules can be retrieved from chemical libraries or can be designed on a computer screen and then synthe-sized in a laboratory. Most notably, one could use the computerized structure as a reference to determine the types of molecules that might block the enzyme. Such structure-based drug design strategies have the potential to save off years and millions of dollars compared to a more traditional trial-and-error drug development process. After the crystal structure of the HIV-encoded protease enzyme had been elucidated, computer-aided drug design played a pivotal role in the development of new compounds that inhibit this enzyme which is responsible for HIV maturation and infectivity. Promising repre-sentatives of these compounds have recently found their way to patients. Protease inhibitors show a powerful sustained suppression of HIV-1 replication, especially when used in combi-nation therapy regimens. However, these drugs are becoming less effective to more resistant HIV strains due to multiple mutations in the retroviral proteases. In computational drug design I used molecular modelling methods such as lead ex-pansion algorithm (Tripos®) to create a virtual library of compounds with different binding affinities to protease binding site. In addition, I heavily applied computer assisted combinato-rial chemistry approaches to design and optimize virtual libraries of protease inhibitors and performed in silico screening and pharmacophore-similarity scoring of these drug candidates. Further computational analyses revealed one unique compound with different protease bind-ing ability from the initial hit and its role for possible new class of protease inhibitors is dis-cussed (Shityakov and Dandekar, 2009). A number of atomistic models were developed to elucidate the nanotube behaviour in lipid bilayers. However, none of them provided useful information for CNT effect upon the lipid membrane bilayer for implementing all-atom models that will allow us to calculate the deviations of lipid molecules from CNT with atomistic precision. Unfortunately, the direct experimental investigation of nanotube behaviour in lipid bilayer remains quite a tricky prob-lem opening the door before the molecular simulation techniques. In this regard, more de-tailed multi-scale simulations are needed to clearly understand the stabilization characteristics of CNTs in hydrophobic environment. The phenomenon of an intercalated single-wall carbon nanotube in the center of lipid membrane was extensively studied and analyzed. The root mean square deviation and root mean square fluctuation functions were calculated in order to measure stability of lipid mem-branes. The results indicated that an intercalated carbon nanotube restrains the conformational freedom of adjacent lipids and hence has an impact on the membrane stabilization dynamics (Shityakov and Dandekar, 2011). On the other hand, different lipid membranes may have dissimilarities due to the differing abilities to create a bridge formation between the adherent lipid molecules. The results derived from this thesis will help to develop stable nanobiocom-posites for construction of novel biomaterials and delivery of various biomolecules for medi-cine and biology.}, subject = {Kohlenstoff}, language = {en} } @phdthesis{Pletinckx2011, author = {Pletinckx, Katrien}, title = {Dendritic cell maturation and instruction of CD4+ T cell tolerance in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67375}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Effective T cell immunity was believed to occur by mature DC, whereas tolerogenicity was attributed strictly to immature DC phenotypes. However, intermediate DC maturation stages were identified conditioned by inflammatory mediators like TNF. Furthermore, the T cell tolerance mechanisms are dependent on distinct modes and intensities of co-stimulation. Therefore, in this study it was addressed how distinct DC maturation signatures instruct CD4+ T cell tolerance mechanisms. DC acquire antigens from apoptotic cells for self-peptide-MHC presentation and functionally adapt presumed tolerogenic DC phenotypes. Here, immature murine bone-marrow derived DC representing both inflammatory and conventional DC subsets adapted a maturationresistant DC signature upon apoptotic cell recognition but no additional tolerogenic features. Immature DC instruct CD4+ FoxP3+ regulatory T cells in a TGF-β prone micro-environment or generate anergic CD4+ T cells hampered in the TCR-induced proliferation and IL-2 secretion. Secondary stimulation of such anergic CD4+ T cells by immature DC increased primarily IL-10 production and conferred regulatory function. These IL-10+ regulatory T cells expressed high levels of CTLA-4, which is potently induced by immature DC in particular. Data in this work showed that anergic T cells can be re-programmed to become IL-10+ regulatory T cells upon ligation of CTLA-4 and CD28 signalling cascades by B7 costimulatory ligands on immature DC. In contrast, semi-mature DC phenotypes conditioned by the inflammatory mediator TNF prevented autoimmune disorders by induction of IL-10+ Th2 responses as demonstrated previously. Here, it was shown that TNF as an endogenous maturation stimulus and pathogenic Trypanosoma brucei variant-specific surface glycoproteins (VSG) induced highly similar DC gene expression signatures which instructed default effector Th2 responses. Repetitive administration of the differentially conditioned semi-mature DC effectively skewed T cell immunity to IL-10+ Th2 cells, mediating immune deviation and suppression. Collectively, the data presented in this work provide novel insights how immature and partially mature DC phenotypes generate T cell tolerance mechanisms in vitro, which has important implications for the design of effective DC-targeted vaccines. Unravelling the DC maturation signatures is central to the long-standing quest to break tolerance mimicked by malignant tumours or re-establish immune homeostasis in allergic or autoimmune disorders.}, subject = {Dendritische Zelle}, language = {en} } @article{OberlaenderPletinckxDaehleretal.2011, author = {Oberl{\"a}nder, Uwe and Pletinckx, Katrien and D{\"a}hler, Anja and M{\"u}ller, Nora and Lutz, Manfred and Arzberger, Thomas and Riederer, Peter and Gerlach, Manfred and Koutsilieri, Eleni and Scheller, Carsten}, title = {Neuromelanin is an Immune Stimulator for Dendritic Cells in vitro}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69210}, year = {2011}, abstract = {Background: Parkinson's disease (PD) is characterized at the cellular level by a destruction of neuromelanin (NM)-containing dopaminergic cells and a profound reduction in striatal dopamine. It has been shown recently that antimelanin antibodies are increased in sera of Parkinson patients, suggesting that NM may act as an autoantigen. In this study we tested whether NM is being recognized by dendritic cells (DCs), the major cell type for inducing Tand B-cell responses in vivo. This recognition of NM by DCs is a prerequisite to trigger an adaptive autoimmune response directed against NM-associated structures. Results: Murine DCs were treated with NM of substantia nigra (SN) from human subjects or with synthetic dopamine melanin (DAM). DCs effectively phagocytized NM and subsequently developed a mature phenotype (CD86high/MHCIIhigh). NM-activated DCs secreted the proinflammatory cytokines IL-6 and TNF-a. In addition, they potently triggered T cell proliferation in a mixed lymphocyte reaction, showing that DC activation was functional to induce a primary T cell response. In contrast, DAM, which lacks the protein and lipid components of NM but mimics the dopamine-melanin backbone of NM, had only very little effect on DC phenotype and function. Conclusions: NM is recognized by DCs in vitro and triggers their maturation. If operative in vivo, this would allow the DC-mediated transport and presentation of SN antigens to the adaptive immune system, leading to autoimmmunity in susceptible individuals. Our data provide a rationale for an autoimmune-based pathomechanism of PD with NM as the initial trigger.}, subject = {Immunstimulation}, language = {en} } @article{LindemannRethwilm2011, author = {Lindemann, Dirk and Rethwilm, Axel}, title = {Foamy Virus Biology and Its Application for Vector Development}, series = {Viruses}, volume = {3}, journal = {Viruses}, number = {5}, doi = {10.3390/v3050561}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139811}, pages = {561-585}, year = {2011}, abstract = {Spuma- or foamy viruses (FV), endemic in most non-human primates, cats, cattle and horses, comprise a special type of retrovirus that has developed a replication strategy combining features of both retroviruses and hepadnaviruses. Unique features of FVs include an apparent apathogenicity in natural hosts as well as zoonotically infected humans, a reverse transcription of the packaged viral RNA genome late during viral replication resulting in an infectious DNA genome in released FV particles and a special particle release strategy depending capsid and glycoprotein coexpression and specific interaction between both components. In addition, particular features with respect to the integration profile into the host genomic DNA discriminate FV from orthoretroviruses. It appears that some inherent properties of FV vectors set them favorably apart from orthoretroviral vectors and ask for additional basic research on the viruses as well as on the application in Gene Therapy. This review will summarize the current knowledge of FV biology and the development as a gene transfer system.}, language = {en} } @article{KasangKalluvyaMajingeetal.2011, author = {Kasang, Christa and Kalluvya, Samuel and Majinge, Charles and Stich, August and Bodem, Jochen and Kongola, Gilbert and Jacobs, Graeme B. and Mllewa, Mathias and Mildner, Miriam and Hensel, Irina and Horn, Anne and Preiser, Wolfgang and van Zyl, Gert and Klinker, Hartwig and Koutsilieri, Eleni and Rethwilm, Axel and Scheller, Carsten and Weissbrich, Benedikt}, title = {HIV drug resistance (HIVDR) in antiretroviral therapy-naive patients in Tanzania not eligible for WHO threshold HIVDR survey is dramatically high}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69024}, year = {2011}, abstract = {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.}, subject = {Tansania}, language = {en} } @article{KasangKalluvyaMajingeetal.2011, author = {Kasang, Christa and Kalluvya, Samuel and Majinge, Charles and Stich, August and Bodem, Jochen and Kongola, Gilbert and Jacobs, Graeme B. and Mlewa, Mathias and Mildner, Miriam and Hensel, Irina and Horn, Anne and Preiser, Wolfgang and van Zyl, Gert and Klinker, Hartwig and Koutsilieri, Eleni and Rethwilm, Axel and Scheller, Carsten and Weissbrich, Benedikt}, title = {HIV Drug Resistance (HIVDR) in Antiretroviral Therapy-Na{\"i}ve Patients in Tanzania Not Eligible for WHO Threshold HIVDR Survey Is Dramatically High}, series = {PLoS One}, volume = {6}, journal = {PLoS One}, number = {8}, doi = {10.1371/journal.pone.0023091}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137988}, pages = {e23091}, year = {2011}, abstract = {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-na{\"i}ve population. Methods and Findings HIVDR was determined in 88 sequentially enrolled ART-na{\"i}ve 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-na{\"i}ve 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-na{\"i}ve 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-na{\"i}ve HIV-infected population.}, language = {en} } @phdthesis{Jacobs2011, author = {Jacobs, Graeme Brendon}, title = {HIV-1 resistance analyses from therapy-na{\"i}ve patients in South Africa, Tanzania and the characterization of a new HIV-1 subtype C proviral molecular clone}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-67319}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {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-na{\"i}ve 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-na{\"i}ve. In Mwanza, Tanzania the HIV drug resistance (HIVDR) prevalence in the therapy-na{\"i}ve 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-na{\"i}ve 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.}, subject = {HIV}, language = {en} } @article{HartlBodemJochheimetal.2011, author = {Hartl, Maximilian J. and Bodem, Jochen and Jochheim, Fabian and Rethwilm, Axel and R{\"o}sch, Paul and W{\"o}hrl, Birgitta M.}, title = {Regulation of foamy virus protease activity by viral RNA}, series = {Retrovirology}, volume = {8}, journal = {Retrovirology}, number = {Suppl. 1}, doi = {10.1186/1742-4690-8-S1-A228}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142248}, pages = {A228}, year = {2011}, abstract = {No abstract available.}, language = {en} } @article{BeyersdorfWernerWolfetal.2011, author = {Beyersdorf, Niklas and Werner, Sandra and Wolf, Nelli and Herrmann, Thomas and Kerkau, Thomas}, title = {Characterization of a New Mouse Model for Peripheral T Cell Lymphoma in Humans}, series = {PLoS One}, volume = {6}, journal = {PLoS One}, number = {12}, doi = {10.1371/journal.pone.0028546}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137946}, pages = {e28546}, year = {2011}, abstract = {Peripheral T cell lymphomas (PTCLs) are associated with a poor prognosis due to often advanced disease at the time of diagnosis and due to a lack of efficient therapeutic options. Therefore, appropriate animal models of PTCL are vital to improve clinical management of this disease. Here, we describe a monoclonal CD8\(^+\) CD4\(^-\) αβ T cell receptor Vβ2\(^+\) CD28\(^+\) T cell lymphoma line, termed T8-28. T8-28 cells were isolated from an un-manipulated adult BALB/c mouse housed under standard pathogen-free conditions. T8-28 cells induced terminal malignancy upon adoptive transfer into syngeneic BALB/c mice. Despite intracellular expression of the cytotoxic T cell differentiation marker granzyme B, T8-28 cells appeared to be defective with respect to cytotoxic activity as read-out in vitro. Among the protocols tested, only addition of interleukin 2 in vitro could partially compensate for the in vivo micro-milieu in promoting growth of the T8-28 lymphoma cells.}, language = {en} }