Browsing by discipline "Farmaceutisk biologi"

Chlamydia pneumoniae is an intracellular bacterium that causes a variety of respiratory infections to humans such as pneumonia and bronchitis. In addition C. pneumoniae -infection has been associated with multiple chronic diseases of which the most important are atherosclerosis and vascular diseases, asthma, chronic obstructive pulmonary disease and different kinds of neurological disorders. C. pneumoniae is a very common pathogen that has the ability to hide in the system in a persistent chronic form out of reach of the immune defences. C. pneumoniae has been shown to infect many other cell types besides bronchial epithelial cells. These cells include monocytes, macrophages and vascular endothelial cells. C. pneumoniae induces the secretion of different kinds of cytokines and cell signalling molecules and the expression of adhesion molecules in all of these cell types. Too strong cytokine and immune response is detrimental to cells and to whole system. Currently available antibiotics aren't effective enough against C. pneumoniae -infection, especially against its chronic form. Furthermore, the lack of effective anti-chlamydial drugs impairs the research of the association between C. pneumoniae and chronic diseases. The aim of this study was to investigate the effect of anti-chlamydial compounds on the release of cytokines and cell signaling molecule, nitric oxide, induced by C. pneumoniae -infection in different cell types. These anti-chlamydial compounds are currently under the investigation in the faculty of pharmacy. In addition the anti-inflammatory properties of the compounds were further investigated with the help of lipopolysaccharide of another gram-negative bacterium E. coli. The groups of compounds investigated in this study were β2,2-amino acid derivatives, Schisandra chinensis -lignans, TE-compounds synthesized in Vienna and benzimidazole compounds synthesized in the faculty of pharmacy. There were four cell types used in this study, HL- and BEAS-2B-epithelial cells, THP-1-monocytes/macrophages and RAW264.7-macrophages. The study focused on the determination of vascular endothelial growth factor VEGF and interleukins IL-8, IL-10 and IL-12. The concentrations of cytokines in the cell medium were measured after infection using ELISA-method. Nitric oxide measurements were also determined from the medium using Griess' reagent. Immunofluorescence labeling was used to confirm the infection and the infection was verified by fluorescence microscope. In addition some of the compounds were tested for the cell viability using resazurin assay. All the groups of compounds showed desired effects on the release of cytokines and nitric oxide. Especially β2,2-amino acid derivatives reduced clearly the release of both cytokines and nitric oxide. β2,2-amino acid derivatives could thus be potential drug candidates for the development of anti-chlamydial and anti-inflammatory drugs. Schisandra chinensis -lignans inhibited especially the release of nitric oxide in both C. pneumoniae -infected and LPS-stimulated cells which may tell about their broad anti-inflammatory properties. There were also found desired results with TE-compounds and benzimidazole compounds. Interleukins were not secreted by any of the studied cells so that part needs more research and further investigation. Based on the results found in this study it can be concluded that the studied compounds could be potential lead compounds in the discovery of anti-chlamydial drugs and drugs that specifically inhibit C. pneumoniae -infection. Further research is needed concerning the effects of these compounds on cytokines and especially on chronic infection.

Making the treatment of these infections even harder is the fact, that Chlamydia pneumoniae can produce persistent forms of itself, which are immune to antibiotic treatment. When the bacteria sense a stress factor, for example the presence of a β-lactam antibiotic or interferon γ, they start producing these persistent forms called aberrant bodies. When the stress factor is removed, the bacteria can switch back to their replicating form and start infecting the tissues again. It is also known, that C. pneumoniae bacteria will trigger persistence when the bacteria migrate from lung epithelia into monocytes. Interestingly the onset of this mode of persistence does not require any other triggers besides the invasion of the monocyte. These persistence mechanisms enable latent, quiet, and recurring infections. This master’s thesis aimed to study the coculture of lung epithelial (HL cells) and monocytes (THP-1 cells), and by utilising the magnetic separation method presented by Kortesoja et al, to find a positive control compound in the prevention of Chlamydia pneumoniae internalisation into the THP-1 cells for said protocol. In these cocultures the inhibitory effect of different compound groups such as lignans present in Schisandra chinensis plant, MAPK-inhibitors, and β2,2-amino acid derivatives in C. pneumoniae migration from HL cells to THP-1 cells was assessed. Statistic relevance was observed in JNK inhibitor SP600125, MAPKAP-kinase-2 inhibitor SB203580, and ERK1/2 inhibitor FR180204 compounds. These compounds inhibited the internalisation of Chlamydia pneumoniae into THP-1 cells in the cell coculture by 61,05 ± 16,63 % (p = 0,0001), 54,06 ± 16,02 % (p = 0,0002), and 36,76 ± 10,33 % (p = 0,009) respectively. SP600125 and SB 203580 compounds also had an inhibitory effect on the internalisation of C. pneumoniae into the THP-1 cells in a cell monoculture (39,98 ± 18,92 %, p = 0,026 and 37,89 ± 19,47 %, p = 0,035 respectively), whereas FR180204 had no statistical significance, even though it inhibited the internalisation of C. pneumoniae into the THP-1 cells in cell monoculture by 27,53 ± 21,17 %. From the compounds used in the experiments, only MAPK inhibitors had an effect in inhibiting the C. pneumoniae internalisation into the THP-1 cells. The most potent compound in said inhibition was the JNK inhibitor SP600125. JNK pathway has been thought to take part in chlamydial infections but only little research has been done. The results of this master’s thesis’ experiments support the thought of JNK enzyme taking part in chlamydial infections but determining how exactly it affects the infection cycle of C. pneumoniae bacteria still needs further investigation.

Susceptibility to antibiotics is constantly developing in bacteria due to selection pressure caused by use of antibiotics. For this reason, finding new antimicrobial substances is imperative. High-throughput screening (HTS) is an important tool to find new active substances. The need to analyse as many substances in as small time as possible is emphasised in modern drug development. Robust methods, suitable for fast throughput of substances, miniaturisation and automation, are particularly useful. In the context of antimicrobial screening, methods utilising bioluminescence can correspond this need, and genetic engineering can help in developing bacterial strains with beneficial features for screening. In this work, two screening methods were developed and optimised using genetically engineered Escherichia coli strains. The screening methods make use of the bioluminescent properties of the strains, and the methods can be used to screen compound libraries for antimicrobials rapidly enough to approach HTS. The strain E. coli WZM120/pCGLS 11 is constitutively luminescent, so weakening of luminescence means the cell viability weakens. The strain E. coli K12/pCSS305, where luminescence is produced by a heat-inducible runaway plasmid, can be used to especially detect compounds inhibiting DNA replication. In developing the method, workflow was optimised and conditions were validated so as to enable possible HTS campaigns. The target was to create as simple, fast and reproducible a method as possible. The Z' values calculated in assessing the performance are excellent for a cell-based method. The signal is readily distinguishable, the bacterial strains are in a stable manner, and the method is well reproducible. It is possible to continue assay development from 96-well format to 384-well format.

Multi-drug tolerance is a phenomenon, in which microorganisms normally susceptible to an antimicrobial agent are able to withstand a treatment via phenotypic alteration. The tolerance is conveyed by a microbial subpopulation that is in a non-replicative and metabolically inactive state also known as persistence. Through this kind of dormancy, the subpopulation may survive an otherwise appropriate course of antimicrobials, since the majority of the drugs target cellular division or metabolism. Upon the reduction of the surrounding antimicrobial concentration the multi-drug tolerant cells - persisters - become resuscitated thus allowing repopulation. As opposed to the more widely acknowledged challenge of antimicrobial resistance, the offspring of the specialist survivor cells are genetically identical to the susceptible majority. Persisters are especially abundant in biofilms, a microbial lifestyle characterized by aggregated microcolonies that are covered in a self-produced slimy matrix known as extracellular polymeric substance (EPS). Partly owning to this protective matrix, biofilms are inherently somewhat tolerant to antimicrobial chemotherapy. Moreover, microbes confined in a biofilm are additionally protected against the components of the host immune system. Conversely, it is assumed that persisters in planktonic, i.e. freely floating state, are easily cleared out by white blood cells. Combined, the immune evasive properties of biofilms and the remarkable multi-drug tolerance of persisters give rise to recalcitrant infections that are immensely difficult to eradicate. The described phenomenon constitutes crucially to the major healthcare challenge of chronic, treatment-resistant infections. Tuberculosis, cystic fibrosis lung disorder, bacterial endocarditis and infections related to indwelling medical devices are only a few examples of such problems. Despite the need for antimicrobials with anti-persister efficacy, no such therapeutics is available and very few are being investigated - one important factor being the lack of relevant drug discovery platforms. Therefore, the aim of this study was to develop an anti-persister assay and to carry out a pilot screening of natural product derived bioactive compounds. Based on the notion that persisters are enriched in bacterial cultures that have reached the stationary phase of growth, a persister model was designed using Staphylococcus aureus ATCC 25923 as the test strain. The bacteria were grown in liquid cultures until they reached the stationary phase and subsequent experimentation was carried out to confirm the tolerant state. After the stationary phase persister model was validated, a small pilot screening of natural products was undertaken in the hope of finding novel anti-persister activity. Mitomycin C, a cytotoxic drug with an existing anti-cancer indication was assigned as the positive control compound because of its previously established anti-persister activity. Since it is common for all of the persister-related diseases that the target microorganisms reside within a protective biofilm, an additional assay based on biofilm regrowth was designed to characterize the hit compounds on a more clinically relevant platform. The persister model culture was shown to be tolerant to conventional antibiotics. The re-induction of metabolic activity by diluting into fresh medium recovered the antimicrobial susceptibility expectedly. A total of 4 compounds were identified as anti-persister hits in the pilot screening campaign. Chromomycin A3, dehydroabietic acid, mithramycin A and oleanolic acid were all able to reduce the viable bacterial count in the stationary phase persister model more than 2 logarithmic units at 100 µM. Mithramycin A was the most potent, reducing the viability over 6 log units. The model compound mitomycin C reduced the viable counts 5.49 (± 0.96) logarithmic units. Out of the 4 hits, dehydroabietic acid was selected for the biofilm relapse assay because of its favourable biocompatibility properties. It reduced regrowth for the treated biofilms by 4 logarithmic.

Plant cells in plant cell cultures can be used for production of secondary metabolites and recombinant proteins. Producing the desired compounds can be problematic since cells grow slowly, yields can be low and sometimes plant cells do not produce the desired compounds. Yields can be increased by various methods, of which optimisation of growth conditions to favour growth and secondary metabolite biosynthesis is one of various strategies. Light quality is known to have an impact on growth of plants and on accumulation of secondary metabolites. Plants receive information of their environment with photoreceptors, which gives plants ability to alter their morphology and biochemistry to adapt to the prevailing conditions. One of the most important factors involved in controlling morphology and metabolism is activity of bZIP protein HY5, which levels are controlled by degradation by E3 ubiquitin ligase COP1. The photoreceptors are divided to three main groups. A group of Blue/UV-A photoreceptors consists of cryptochromes and phototropins. Phytochromes are photochrome photoreceptors of wavebands of red and far-red. UVR8 photoreceptors are specialized to sense UV-B wavebands. Activated photoreceptors reduce the activity of COP1 individually or inductively. Plant cells contain the same genetic information as intact plants. Object of this study is to investigate effects of different light spectra on plant cell mass pigment accumulation, lipid content and accumulation of secondary metabolites. Additionally, the obtained results can be utilized in designing new artificial light sources to enhance growth and nutritional value of horticultured plants grown under artificial light. VTT's callus cultures established from berries of Rubus (raspberry, cloudberry, arctic bramble) and Vaccinium (lingonberry, bilberry, cranberry) were used in this study. The cell cultures were grown in hormone balanced solid media. For this research Valoya provided four different LED light sources with different spectra, ranging between wavebands 400–800 nm. All berry callus cultures were grown for continuous period of 28–31 days under different light sources. Mass pigments, lipid composition, total phenolic concentration and anthocyanins were analysed from each cell cultures which received different light treatments. Samples were pooled and were by freeze dried and milled. Mass pigments were extracted with acetone and analysis was carried out with UPLC-DAD. Extraction of lipids was carried out with petroleum ether followed with transesterification of glycerolipids and silylation of free fatty acids. The lipid extracts were analysed with GC-MS. Phenolic compounds were extracted with methanol and the extracts were treated with Folin-Ciocalteu's reagent and then analysed with spectrophotometer. Anthocyanins were extracted with acidified methanol and a portion of the extracts were hydrolysed to qualify anthocyanidin moieties of anthocyanins. The extracts and the hydrolysed extract were analysed with UPLC-DAD. Analysis of volatile compounds from each light treated samples was carried out with SPME GC-MS. The obtained results were used to compare concentration differences of the analytes under different light treatments. Correlations between the concentrations of the analytes and different wavebands were possible to establish from the results. Activation of cryptochromes and phytochromes reduced certain lipids that are precursors in LOX-pathway which indicates to increased activity of the pathway. Same wavebands which activated the photoreceptors reduced accumulation of mass pigments, whereas, wavebands of far-red increased the concentrations of mass pigments. In some cases it was observed that small difference in light spectra reduced mass pigment accumulation significantly. The plant cell cultures produced mainly anthocyanins which anthocyanidin moieties were same as in intact plants. Cryptochrome and phytochrome activation increased accumulation of anthocyanins. Yields of anthocyanins can be increased significantly with certain spectra significantly. The effect of light spectra did not have as straightforward effect on total phenolic content. Specie- and linewise differences were observed in light conditions where the highest concentrations of total phenolics were obtained.