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| − | <h1>Autoimmune diseases</h1>
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| − | <p>Autoimmune disease are a range of disorders that result from attack of self-tissues by a dysregulated immune system. Examples are multiple sclerosis, Crohn's disease and type I diabetes. There are significant global inequalities in the treatment and prevalence of autoimmune disorders. 80% of diagnoses of autoimmune epilepsy are in the developing world. This is caused by the production of antibodies against proteins in the brain such as GABA receptors. However, it should be noted that prevalence of autoimmune diseases is greater in Europe and the USA. This is explained by the hygiene hypothesis which suggests that a lack of exposure to microorganisms, both pathogenic and symbiotic, prevents normal development of the immune system. <br> <br>
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| − | T helper (Th) cells produce immune responses and T regulatory (Treg) cells control the populations of Th cells. When Treg cell populations are low they are unable to stop excessive growth of helper cell populations. Th cells will produce superfluous inflammatory molecules that spread. This is the cellular basis of autoimmune disease. <br> <br>
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| − | We have focused on autoimmune diseases in developing countries where bacteria, such as segmented filamentous bacteria (SFB) are prevalent and typically found within water sources. SFB are gram positive bacteria that are tightly bound to the lining of the gut epithelia and have recently been implicated in modifying host immune responses by promoting the development of lymphocytes and the growth of Th17 populations. An additional effect of the signals required for Th17 differentiation is the inhibition of the production of Treg cells. Once fully differentiated, Th17 cells produce interleukin 17a (IL-17a) a proinflammatory cytokine. Elevated levels of IL-17a are associated with MS and the development of epilepsy and other autoimmune diseases. <br> <br> </p>
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| − | <h1>Current treatments</h1>
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| − | <p>Current treatments for autoimmune diseases are limited and act only to control the symptoms and do not attempt to cure the individual. Non- steroidal anti- inflammatory drugs (NSAIDs), such as ibuprofen, are the most commonly prescribed and reduce inflammation and accompanying symptoms. NSAIDs work to block the activity of cyclooxygenase enzymes which, in turn, halts the production of pro- inflammatory prostaglandins. However, there are a number of side effects associated with long- term use of NSAIDs, namely, dizziness, drowsiness and an increased risk of developing stomach ulcers. In addition, their use is restricted due to their tendency to react unpredictably with other common drugs.<br> <br>
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| − | Another common treatment is immunosuppressive drugs such as corticosteroids. These act to reduce expression of genes associated with an increase in inflammation, via the recruitment of histone deacetylases. These treatments are either inhaled, applied topically or ingested in tablet form. Prolonged use of this class of drugs and the consequent excessive exposure to cortisol can lead to the development of Cushing’ syndrome, which is characterised by high blood pressure, abdominal obesity and weak bones. In addition, immunosuppressive drugs can lead to excessive downregulation of the immune system.
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| − | Despite the widespread use of the aforementioned drugs in the treatment of autoimmune diseases it is clear that there are a number of undesirable side effects. Also, unlike our treatment, currently available drugs do not respond to the current state of the immune system and this can result in unrestricted dampening of immune response. <br> <br>
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| − | Current treatments for immunodeficiency are varied in their effects and success. One possible cure for immunodeficiency is a stem cell transplant. Stem cells are extracted from the bone marrow of the patient or of an appropriate donor with the same human leukocyte antigens as the patient. However, there are a number of risks associated with a stem cell transplant as any functional immune cells are removed prior to the transplant, via chemotherapy or radiotherapy, which leaves the patient highly susceptible to infection. Also, the transplant process is lengthy, with a total recovery time of around one to two years. This treatment is often unsuccessful, typically due to rejection of the donor cells. <br> <br>
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| − | In cases where a stem cell transplant is not possible, there are treatments directed at improving the function of the immune system. For example, immunoglobulin therapy is used to boost levels of antibodies. Gamma interferon therapy is used to boost interferon levels, which in turn act to stimulate the immune system. In addition, treatments using growth factors can be used to boost the size of the population of white blood cells. There are also a number of preventative measures such as the regular use of antibiotics, non- live vaccines and antivirals, which are used to manage exposure to infection. <br> </p>
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| − | <h1>Our solution</h1>
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| − | <p>Bacteria can’t detect the regulatory proteins due to their size and impermeability to the bacterial cell membrane, so we used two small molecules: adenosine and nitric oxide (NO). The concentrations of these are representative of the populations of T-reg cells and Th cells respectively. An imbalance in the levels correlates with the autoimmune response. We aim to restore these to healthy proportions by secreting Interleukin 10 (IL-10) - a signal protein which stimulates cell differentiation into T-reg cells. <br> <br>
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| − | Our bacteria produce personalised and easily administered doses of IL-10. This avoids impracticalities of current, injection based techniques. The gut has greater ease of introduction to the body over other sites of T-cell populations and no biological barriers need to be crossed for the E. coli to reach their target. Our focus is on autoimmune diseases in developing countries triggered by ingested pathogenic bacteria. In these cases the disease starts in the gut making it the obvious place for us to target. We propose the cultures could be ingested using a live yoghurt or tablet, this avoids the training needed for injections and the risk of infection. <br> <br>
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| − | NO is thought to be produced by Th cells as a signal to induce differentiation of cells into Th cells and control the populations of Th and Treg cells. Continuous production of NO makes it a suitable marker of the size of the population and severity of the autoimmune disease. <br> <br>
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| − | A range of purines are used by Treg cells as immune signals. ATP is released from cells during stress or injury and acts on almost all immune cells. To prevent it from causing excessive inflammation, ATP is rapidly metabolised into AMP then into Adenosine (Ado) by Treg cell surface enzymes. Ado is detected by GPCRs of Th cells, this triggers increased cAMP in the cells. Inflammatory signals are inhibited and IL10 production is enhanced in mature dendritic cells. Ado also induces semi-maturation of immature dendritic cells. Adenosine promotes Treg populations, adenosine generation and increases immunoregulatory activity. Production by Treg cells and its role in regulating T cell populations and IL10 production make it a suitable marker of Treg population size and degree of immunodeficiency. <br> <br>
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| − | In our circuit IL-10 is secreted in the presence of NO and absence of elevated adenosine levels. IL-10 expression is stimulated by the endogenous E. coli SoxR transcription factor, activated by free radicals and oxidative stress, while expression is inhibited in response to adenosine by means of a riboswitch controlled sRNA which will selectively prevent ribosome binding to IL-10 mRNA and thus protein synthesis. <br> <br>
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| − | We decided to use NO partly because of the Stanford 2009 iGEM team. They responded to NO by producing retinoic acid, an immune regulator. We used the same SoxR/SoxS promoter system to detect NO, this is used by E. coli to respond to oxidative stress. A single stimulus can result in false positives and excessive suppression, leading to immunodeficiency. An inhibitor signaling high Treg populations avoids oversuppression. We are using multiple signals to increase the specificity and accuracy. Instead of retinoic acid we are using it to stimulate IL-10 production. <br> <br> </p>
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| − | <h1>Parts</h1> <br>
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| − | <h2>Riboswitch</h2>
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| − | <p>No riboswitches have been characterised which selectively bind to adenosine so instead we have used the adenine-specific riboswitch pbuE. Thus, cleavage of the adenine moiety is necessary for detection of adenosine by E.coli. In order to reduce the impact of unregulated adenosine hydrolase on cellular metabolism, we decided to exclude it from the cytoplasm.</p>
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| − | <h2>Functional RNA</h2>
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| − | <p>The adenine riboswitch controls sRNA transcription, absence of adenine prevents RNA synthesis. The riboswitch, which is also transcribed, would hinder the sRNA binding to the target mRNA resulting in a loss of specificity and affinity. To prevent this the hepatitis delta virus ribozyme is between the features, this self-cleaves and liberates the sRNA. Modelling the effect of sRNA length on binding affinity allows us to create a tuneable inhibitory effect. This gives control over specificity and sensitivity to the adenosine stimulus. </p>
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| − | <h2>Hydrolase</h2>
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| − | <p>Adenosine is a key metabolite; to prevent unregulated adenosine hydrolase inhibiting cell growth, we decided to exclude it from the cytoplasm. This will minimise the impact on cellular metabolism and refine the signal by excluding cytoplasmic noise.
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| − | We have proposed two mechanisms for this exclusion. The first involves using the type-II TAT pathway to directly translocate the tagged hydrolase dimer into the periplasm while the second involves the targeting to the outer membrane, fused to an E.coli membrane anchor - YiaT. In the second approach, to promote dimer association, a flexible linker was used to join two hydrolase monomers together.</p>
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