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- Abate, Ebba
(författare)
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The impact of helminth infection in patients with active tuberculosis
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The geographic distribution of helminth infection and tuberculosis (TB) overlap substantially. Experimental animal models and limited data from humans have shown that intestinal helminths could subvert the host immune response towards a T-helper 2 (Th2)-type immune response and an increased regulatory T-cell activity (Tregs). This in turn affects the host's ability to mount an effective Th1 immune-mediated protection against Mycobacterium tuberculosis. However, evidence for this hypothesis in the human setting from helminth infected TB patients is limited. This thesis primarily focuses on the immunological and clinical impact of helminth infection on pulmonary TB. The kinetics of the Quantiferon-Gold (QFN) assay, which measures IFN-³ response to TB-specific antigens in whole blood was assessed and showed a modest decline during TB treatment to the level observed for healthy blood donors. We further assessed another clinical monitoring tool, the-TB-score, composed of clinical signs and symptoms of TB, and found an early decline two weeks after initiation of TB- treatment where a failure of decline correlated with increased mortality. Overall, the helminth co-infection rate was significantly higher in TB patients compared to healthy controls. Helminth co-infection was associated to a significantly higher rate of eosinophilia and IgE-levels in healthy controls and patients with tuberculosis. During the first weeks of anti-TB treatment, a marked decrease in the rate of helminth infection was observed in HIV co-infected compared to HIV-negative TB patients. However, helminth co-infection was more common in HIV negative than HIV positive TB patients. There was no detectable impact of helminth infection on the clinical presentation of pulmonary tuberculosis. At baseline, helminth co-infected TB patients showed an increased frequency of Tregs compared to helminth negative TB patients and healthy controls. This was accompanied by an increased rate of PPD stimulated IL-5 and spontaneous production of IL-10 by peripheral blood mononuclear cells among helminth co-infected TB patients. A placebo controlled randomized trial was conducted in order to test the hypothesis that albendazole treatment of helminth positive TB patients may improve the clinical response of TB by reducing the immunmodulatory effect of helminthes on TB immunity. A total of 140 helminth co-infected TB patients were randomized to albendazole (400 mg per os for three consecutive days) or placebo. No significant difference was observed between the albendazole and placebo group in terms of the primary outcome (TB score change between baseline and week 8). Among the secondary outcomes, a significant decline of peripheral eosinophil cells was observed in the albendazole treated group, but no effect on other outcome variables (changes in chest x-ray findings, IgE level and sputum smear conversion). Regarding the immunological assessment no significant difference was observed for changes in Tregs, and PPD-induced production of IFN- ³ or IL-5 although a non-significant trend of a decrease in IL-10 expressing PBMCs were observed in the albendazole group. Taken together, the burden of helminth infection was higher in TB patients than in a healthy control group. Helminth co-infection during pulmonary TB in the human setting induces an immune response characterized by increased IgE production, eosinophilia as well as increased levels of Tregs and spontaneous IL-10 production. Thus, the immunological impact of helminth infection on the outcome and risk for developing TB merits further investigation.
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| 2. |
- Eklund, Daniel, 1984-
(författare)
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Mycobacterium tuberculosis and the human macrophage : shifting the balance through inflammasome activation
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mycobacterium tuberculosis is a very successful pathogen and tuberculosis constitutes a major threat to global health worldwide. The World Health Organization (WHO) estimates that almost nine million new cases and 1.5 million deaths occur annually and the situation is worsened by increased antibiotic resistance and an extreme synergism with the HIV pandemic. M. tuberculosis primarily affects the lungs where the infection can lead to either eradication of the bacteria or the initiation of an immune response that culminates in the formation of a large cluster of immune cells termed granulomas. In these granulomas, the bacteria can either replicate and cause disease with the ultimate goal of spreading to new hosts or cause latent tuberculosis, which can persist for decades. The tools available to manage the disease are currently suboptimal and include lengthy antibiotic treatments and an inefficient vaccine resulting in poor protection. On a cellular level, M. tuberculosis primarily infects the cell designed to recognize, ingest and eradicate bacteria, namely the human macrophage. Following recognition, the macrophage phagocytoses the bacterium and tries to kill it using an array of different effector mechanisms including acidification of the bacterium-containing vacuole, different degradative enzymes and the generation of radicals. However, the bacterium is able to circumvent many of these harmful effects, leading to a tug-of-war between the bacterium and host macrophage. This thesis aims at studying the interaction between the human macrophage and M. tuberculosis to identify host factors critical for controlling growth of the bacteria. More specifically, it focuses on the role of an intracellular receptor protein called NLRP3 and its downstream effects. NLRP3 is activated in human macrophages infected by M. tuberculosis and upon activation it forms a multi-protein complex known as the inflammasome. This protein complex is known to induce the production of the proinflammatory cytokine IL-1β and specialized forms of macrophage cell death. We hypothesized that stimulating this pathway would have a beneficial effect for the host macrophage during infection with M. tuberculosis.To allow us to follow interaction between M. tuberculosis and the human macrophage, we first developed a luminometry-based method of measuring bacterial numbers and following bacterial growth over several days in infected cells. With this new assay we showed that low numbers of bacteria induced very low levels of IL-1β and failed to induce any type of cell death in the macrophage. However, when a critical number of bacteria were reached, the infected macrophages underwent necrosis, which was accompanied by high levels of IL-1β. We were also able to show that addition of vitamin D, which has been implicated as an important factor for increased killing capacity of infected macrophages, increased the production of IL-1β, which coincided with increased killing of M. tuberculosis. This effect was seen specifically in cells from patients with active tuberculosis, suggesting that these cells are primed to respond to vitamin D and increased levels of IL-1β. Furthermore, we also showed that increasing production of IL-1β by stimulating infected macrophages with apoptotic neutrophils in turn drives the production of other proinflammatory cytokines. Lastly, we showed that gain-of-function polymorphisms in inflammasome components linked to increased inflammasome activation and IL-1β production promotes bacterial killing in human macrophages. In conclusion, the work presented in this thesis shows that by enhancing the functions of the inflammasome, it is possible to tip the balance between the human macrophage and M. tuberculosis in favor of the host cell.
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| 3. |
- Idh, Jonna, 1979-
(författare)
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The Role of Nitric Oxide in Host Defence Against Mycobacterium tuberculosis : Clinical and Experimental Studies
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), responsible for significant morbidity and mortality worldwide, especially in low-income countries. Considering aggravating factors, such as HIV co-infection and emerging drug resistance, new therapeutic interventions are urgently needed. Following exposure to M. tuberculosis, surprisingly few individuals will actually develop active disease, indicating effective defence mechanisms. One such candidate is nitric oxide (NO). The role of NO in human TB is not fully elucidated, but has been shown to have a vital role in controlling TB in animal models.The general aim of this thesis was to investigate the role of NO in the immune defence against M. tuberculosis, by combining clinical and experimental studies. In pulmonary TB patients, we found low levels of NO in exhaled air, and low levels of NO metabolites in urine. HIV coinfection decreased levels of exhaled NO even further, reflecting a locally impaired NO production in the lung. Low levels of exhaled NO were associated with a decreased cure rate in HIV-positive TB patients. Household contacts to sputum smear positive TB patient presented the highest levels of both urinary NO metabolites and exhaled NO. Malnutrition, a common condition in TB, may lead to deficiencies of important nutrients such as the amino acid L-arginine, essential for NO production. We therefore assessed the effect of an argininerich food supplement (peanuts) in a clinical trial including pulmonary TB patients, and found that peanut supplementation increased cure rate in HIV-positive TB patients.We also investigated NO susceptibility of clinical strains of M. tuberculosis, and its association to clinical outcome and antibiotic resistance. Patients infected with strains of M. tuberculosis with reduced susceptibility to NO in vitro, showed a tendency towards lower rate of weight gain during treatment. Moreover, there was a clear variability between strains in the susceptibility to NO, and in intracellular survival within NO-producing macrophages. A novel finding, that can be of importance in understanding drug resistance and for drug development, was that reduced susceptibility to NO was associated with resistance to firstline TB drugs, in particular isoniazid and mutations in inhA.Taken together, the data presented here show that NO plays a vital role in human immune defence against TB, and although larger multicentre studies are warranted, arginine-rich food supplementation can be recommended to malnourished HIV co-infected patients on TB treatment.
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| 4. |
- Welin, Amanda, 1983-
(författare)
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Survival strategies of Mycobacterium tuberculosis inside the human macrophage
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Mycobacterium tuberculosis (Mtb) is the bacterium responsible for tuberculosis (TB). For decades, it was believed that TB was a disease of the past, but the onset of the HIV epidemic resulting in a greatly increased number of TB cases, the emergence of antibiotic resistant Mtb strains, and the relative ineffectiveness of the BCG vaccine have put TB back on the agenda. With almost two million people being killed by TB each year, the World Health Organization has declared it a global emergency. TB is an especially big issue in low-income countries, where crowded living conditions accelerates spread of the disease, and where access to health care and medication is problematic. Mtb spreads by aerosol and infects its host through the airways. The bacterium is phagocytosed by resident macrophages in the lung, and when successful is able to replicate inside these cells, which are actually designed to kill invading microbes. Mtb is able to evade macrophage responses in part by inhibiting the fusion between the phagosome in which it resides and bactericidal lysosomes, as well as by dampening the acidification of the vacuole. The initial macrophage infection results in a pro-inflammatory response and the recruitment of other cells of the innate and adaptive immune systems, giving rise to the hallmark of Mtb infection – the granuloma. It is believed that in up to 50 % of exposed individuals, however, the infection is cleared without the involvement of the adaptive immune system, indicating that the innate immune system may be able to control or clear the infection if activated appropriately. This thesis focuses on the interaction between the host macrophage and Mtb, aiming to understand some of the mechanisms employed by the bacterium to evade macrophage responses to enable replication and spread to new host cells. Furthermore, mechanisms used by the macrophage to keep the infection under control were studied, and a method that could be used to measure the replication of the bacilli inside macrophages in vitro in an efficient way was developed. We found that a mycobacterial glycoprotein, mannose-capped lipoarabinomannan (ManLAM), which is shed from the bacilli during phagocytosis by macrophages, integrates into membrane raft domains of the host cell membrane via its GPI anchor. This integration leads to an inhibition of phagosomal maturation. Subsequently, we developed a luciferase-based method by which intracellular replication of Mtb as well as viability of the host macrophage could be measured in a rapid, inexpensive and quantitative way in a 96-well plate. This method could be used for drug screening as well as for studying the different host and bacterial factors that influence the growth of Mtb inside the host cell. Using this method, we discovered that infection of macrophages with Mtb at a low multiplicity of infection (MOI) led to effective control of bacterial growth by the cell, and that this was dependent on functional lysosomal proteases as well as phagosomal acidification. However, we found no correlation between controlled bacterial growth and the translocation of late endosomal membrane proteins to the phagosome, showing that these markers are poor indicators of phagosomal functionality. Furthermore, we discovered that infection of macrophages with Mtb at a higher MOI led to replication of the bacilli accompanied by host cell death within a few days. We characterized this cell death, and concluded that when replication of Mtb inside macrophages reaches a certain threshold and the bacteria secrete a protein termed ESAT-6, necrotic cell death of the host cell occurs. However, although the bacilli activated inflammasome complexes in the host cell and IL-1β was secreted during infection of macrophages, Mtb infection did not induce either of the recently characterized inflammasome-related cell death types pyroptosis or pyronecrosis. Thus, we have elucidated some of the strategies that Mtb uses to be able to survive and replicate inside the macrophage and spread to new cells, as well as studied the conditions under which the host cell is able to control infection. This knowledge could be used in the future for developing drugs that boost the innate immune system or targets bacterial virulence factors in the macrophage.
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