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1.
  • Abate, Ebba, et al. (författare)
  • Asymptomatic Helminth Infection in Active Tuberculosis Is Associated with Increased Regulatory and Th-2 Responses and a Lower Sputum Smear Positivity
  • 2015
  • Ingår i: PLoS Neglected Tropical Diseases. - : PUBLIC LIBRARY SCIENCE. - 1935-2727 .- 1935-2735. ; 9:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Background The impact of intestinal helminth infection on the clinical presentation and immune response during active tuberculosis (TB) infection is not well characterized. Our aim was to investigate whether asymptomatic intestinal helminth infection alters the clinical signs and symptoms as well as the cell mediated immune responses in patients with active TB.Methodology Consecutive, newly diagnosed TB patients and healthy community controls (CCs) were recruited in North-west Ethiopia. TB-score, body mass index and stool samples were analyzed. Cells from HIV-negative TB patients (HIV-/TB) and from CCs were analyzed for regulatory T-cells (Tregs) and cytokine responses using flow cytometry and ELISPOT, respectively.Results A significantly higher ratio of helminth co-infection was observed in TB patients without HIV (Helm+/HIV-/TB) compared to HIV negative CCs, (40% (121/306) versus 28% (85/306), p = 0.003). Helm+/HIV-/TB patients showed significantly increased IL-5 secreting cells compared to Helm-/HIV-/TB (37 SFU (IQR:13-103) versus 2 SFU (1-50); p = 0.02, n = 30). Likewise, levels of absolute Tregs (9.4 (3.2-16.7) cells/mu l versus 2.4 (1.1-4.0) cells/mu l; p = 0.041) and IL-10 secreting cells (65 SFU (7-196) versus 1 SFU (0-31); p = 0.014) were significantly higher in Helm+/HIV-/TB patients compared to Helm-/HIV-/TB patients. In a multivariate analysis, a lower rate of sputum smear positivity for acid fast bacilli, lower body temperature, and eosinophilia were independently associated with helminth infection in TB patients.Conclusions Asymptomatic helminth infection is associated with increased regulatory T-cell and Th2-type responses and a lower rate of sputum smear positivity. Further studies are warranted to investigate the clinical and immunological impact of helminth infection in TB patients.
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2.
  • Abate, E., et al. (författare)
  • Effects of albendazole on the clinical outcome and immunological responses in helminth co-infected tuberculosis patients: a double blind randomised clinical trial
  • 2015
  • Ingår i: International Journal of Parasitology. - : Elsevier. - 0020-7519 .- 1879-0135. ; 45:2-3, s. 133-140
  • Tidskriftsartikel (refereegranskat)abstract
    • Despite several review papers and experimental studies concerning the impact of chronic helminth infection on tuberculosis in recent years, there is a scarcity of data from clinical field studies in highly endemic areas for these diseases. We believe this is the first randomised clinical trial investigating the impact of albendazole treatment on the clinical and immunological outcomes of helminth co-infected tuberculosis patients. A randomised, double-blind, placebo-controlled trial of albendazole (400 mg per day for 3 days) in helminth-positive tuberculosis patients was conducted in Gondar, Ethiopia. The primary outcome was clinical improvement (Delta TB score) after 2 months. Among secondary outcomes were changes in the levels of eosinophils, CD4+ T cells, regulatory T cells, IFN-gamma, IL-5 and IL-10 after 3 months. A total of 140 helminth co-infected tuberculosis patients were included with an HIV co-infection rate of 22.8%. There was no significant effect on the primary outcome (Delta TB score: 5.6 +/- 2.9 for albendazole versus 5.9 +/- 2.5 for placebo, P = 0.59). The albendazole-treated group showed a decline in eosinophil cells (P = 0.001) and IL-10 (P = 0.017) after 3 months. In an exploratory analysis after 12 weeks, the albendazole treated group showed a trend towards weight gain compared with the placebo group (11.2 +/- 8.5 kg versus 8.2 +/- 8.7 kg, P = 0.08)). The reductions in eosinophil counts and IL-10 show that asymptomatic helminth infection significantly affects host immunity during tuberculosis and can be effectively reversed by albendazole treatment. The clinical effects of helminth infection on chronic infectious diseases such as tuberculosis merit further characterisation. (C) 2014 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
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3.
  • Abate, Ebba, et al. (författare)
  • Effects of albendazole treatment on the clinical outcome and immunological responses in patients with helminth infection and pulmonary tuberculosis : a randomized clinical trial
  • 2013
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Background: The impact of helminth infection on the host immune response to tuberculosis (TB) has been characterized in experimental models but less so in the clinical setting. The objective of this study was to investigate the impact of deworming on the clinical outcome and cell mediated immune response in active TB.Methods: Newly diagnosed pulmonary TB patients in Gondar, Ethiopia were examined for helminth infection. Helminth-positive TB patients (W+/TB) were randomized to albendazole (400mg X III per os) or placebo. The primary outcome was change in TB-score after 2 months, and secondary outcomes were sputum smear conversion at the 2nd month, and changes in chest x-ray pattern, CD4+ T-cell count, eosinophil count, IgE-levels and immunological responses after 3 months. In a subset of W+/TB, W-/TB patients and healthy controls, flow cytometry and ELISPOT assays were used to characterize the regulatory T-cell population (Tregs) and the frequency of PPD- stimulated IFN-γ, IL-5 and IL-10 producing peripheral blood mononuclear cells (PBMCs).Results: A total of 140 helminth co-infected TB patients were included with an HIV coinfection rate of 22.8 %. Following albendazole treatment of the W+/TB patients, there was a significant decrease in helminth infection compared to placebo (8% (4/49) vs. 48 % (22/46), p<0.001). No significant effect was observed for albendazole compared to placebo on the primary outcome as evaluated by the TB-score (5.6 ±2.87 vs. 5.87 ±2.54, p=0.59). Eosinophil counts decreased significantly in the albendazole group. In a subgroup analysis of helminthnegative patients following albendazole treatment versus placebo, the albendazole group showed a trend for lower levels of IL-10 producing cells at month three (p=0.08). At baseline, W+/TB patients had a significantly higher mean level of Tregs (% Tregs/CD4+) compared to W-/TB patients and helminth-positive community controls. Additionally, the frequency of IFN-γ, IL-5 and spontaneous IL-10 levels was increased in helminth-positive compared to helminth-negative TB patients.Conclusions: No significant effects on the clinical outcome as measured with the TB-score was detected after albendazole treatment of helminth-positive TB patients compared to placebo. However, significant changes were observed in specific immunological responses such as reduced eosinophil counts and a trend towards lower levels of IL-10 producing cells. At baseline, helminth co-infected TB patients exhibited an increased Treg response as well as an increased IL-5 and spontaneous IL-10 production.
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4.
  • Abate, Ebba, et al. (författare)
  • Impact of helminth infection on the clinical presentation 1 of pulmonary tuberculosis
  • 2013
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Background: The effects of helminth infection on chronic infectious diseases such as HIV and tuberculosis (TB) merit further characterization. Thus, we assessed the baseline clinical characteristics of helminth infection in patients with active TB in a high endemic area.Methodology: Consecutive, newly diagnosed TB patients were recruited from three health institutions in the north Gondar administrative zone, Ethiopia. Structured questionnaires were used to collect socio-demographic and clinical characteristics. Additionally, the TB score, mid upper arm circumference, body mass index (BMI), BCG vaccination status, stool and sputum microscopy as well as HIV serology and CD4+T cells counts were evaluated.Results: A total of 377 pulmonary TB patients were included in the study. The helminth co infection rate was 33% (123/377) and the most prevalent parasite was Ascaris lumbricoides (53%, 65/123). The HIV co-infection rate was 29% (110/377). Seventy percent (77/110) of the HIV co-infected patients were on anti- retroviral therapy at the time of TB diagnosis. Helminth infection was more prevalent in HIV-negative TB patients compared to HIV-positive TB patients (p=0.025). Smoking and walking bare foot were independently associated to helminth infection in TB patients after adjusting for the influence of HIV. Other than increased eosinophilia, no other significant differences were observed between helminth positive and helminth negative TB patients in the clinical presentation including the TB score, CD4+T-cells, BMI or bacterial load.Conclusion: The clinical presentation of active pulmonary tuberculosis was not affected by helminth infection. Helminth infection was less frequent among HIV-positive TB patients and this finding merits further investigation.
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5.
  • Abate, Ebba, et al. (författare)
  • The Impact of Asymptomatic Helminth Co-Infection in Patients with Newly Diagnosed Tuberculosis in North-West Ethiopia
  • 2012
  • Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:8
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Areas endemic of helminth infection, tuberculosis (TB) and HIV are to a large extent overlapping. The aim of this study was to assess the impact of asymptomatic helminth infection on the immunological response among TB patients with and without HIV, their house hold contacts and community controls. less thanbrgreater than less thanbrgreater thanMethodology: Consecutive smear positive TB patients (n = 112), their household contacts (n = 71) and community controls (n = 112) were recruited in Gondar town, Ethiopia. Stool microscopy, HIV serology, serum IgE level, eosinophil and CD4 counts were performed and tuberculosis patients were followed up for 3 months after initiation of anti-TB treatment. less thanbrgreater than less thanbrgreater thanResults: Helminth co-infection rate was 29% in TB patients and 21% in both community control and household contacts (p = 0.3) where Ascaris lumbricoides was the most prevalent parasite. In TB patients the seroprevalence of HIV was 47% (53/112). Eosinophilia and elevated IgE level were significantly associated with asymptomatic helminth infection. During TB treatment, the worm infection rate of HIV+/TB patients declined from 31% (10/32) at week 0 to 9% (3/32) at week 2 of TB treatment, whereas HIV2/TB patients showed no change from baseline to week 2, 29% (13/45) vs. 22.2% (10/45). This trend was stable at week 8 and 12 as well. less thanbrgreater than less thanbrgreater thanConclusion: One third of smear positive TB patients were infected with helminths. Eosinophilia and elevated IgE level correlated with asymptomatic worm infection, indicating an effect on host immunity. The rate of worm infection declined during TB treatment in HIV+/TB co-infected patients whereas no decline was seen in HIV2/TB group.
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6.
  • Abate, Ebba (författare)
  • The impact of helminth infection in patients with active tuberculosis
  • 2013
  • 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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7.
  • Abate Waktola, Ebba Abate, et al. (författare)
  • Polymorphisms in CARD8 and NLRP3 are associated with extrapulmonary TB and poor clinical outcome in active TB in Ethiopia
  • 2019
  • Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 9
  • Tidskriftsartikel (refereegranskat)abstract
    • Innate immunity is a first line defense against Mycobacterium tuberculosis infection where inflammasome activation and secretion of the pro-inflammatory cytokine IL-1beta, plays a major role. Thus, genetic polymorphisms in innate immunity-related genes such as CARD8 and NLRP3 may contribute to the understanding of why most exposed individuals do not develop infection. Our aim was to investigate the association between polymorphisms in CARD8 and NLRP3 and active tuberculosis (TB) as well as their relationship to treatment outcome in a high-endemic setting for TB. Polymorphisms in CARD8 (C10X) and NLRP3 (Q705K) were analysed in 1190 TB patients and 1990 healthy donors (HD). There was a significant association between homozygotes in the CARD8 polymorphism and extrapulmonary TB (EPTB), which was not the case for pulmonary TB or HDs. Among TB-patients, there was an association between poor treatment outcome and the NLRP3 (Q705K) polymorphism. Our study shows that inflammasome polymorphisms are associated with EPTB and poor clinical outcome in active TB in Ethiopia. The practical implications and determining causal relationships on a mechanistic level needs further study.
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8.
  • Abdalla, Hana, et al. (författare)
  • Effects of CNI-1493 on human granulocyte functions
  • 2006
  • Ingår i: Immunobiology. - : Elsevier BV. - 0171-2985 .- 1878-3279. ; 211:3, s. 191-197
  • Tidskriftsartikel (refereegranskat)abstract
    • During acute bacterial infections such as sepsis and meningitis, activation of inflammatory mediators such as nitric oxide (NO) plays a crucial role in both pathogenesis and host defense. We have previously reported that CNI-1493, a macrophage deactivator, reduced mortality in infant rats infected with Haemophilus influenzae type b (Hib) with associated decrease in the number of granulocytes in the infected tissue. The aim of the present study was to investigate how CNI-1493 affects granulocytes and macrophages in vitro. Murine macrophages (RAW 264.7) pre-incubated with CNI-1493 prior to activation with lipopolysaccharide (LPS)/interferon gamma (IFNγ) had decreased NO production measured as NO2−/NO3− levels and reduction in inducible NO-synthase (iNOS) expression. Reactive oxygen species (ROS) production was increased in formylmethionyl-leucyl-phenylalanine (FMLP)-stimulated granulocytes following CNI-1493 treatment, whereas F-actin content, motility and chemotaxis were decreased under the same conditions. The effects of CNI-1493 on both NO production in LPS/IFNγ-activated macrophages and ROS production, F-actin content, motility and chemotaxis in granulocytes, may contribute to the reduced inflammatory response and increased survival in Hib-infected animals treated with CNI-1493.
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9.
  • Abdalla, Hana, et al. (författare)
  • Expression of inducible nitric oxide synthases and nitrotyrosine during the course of Haemophilus influenzae type b meningitis in rat
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Bacterial meningitis continues to be a major health problem and despite great advances in antimicrobial therapy the fatality rate remains high. There is increasing evidence that leukocyte-endothelial interactions are involved in CNS damage during bacterial meningitis. Once leukocytes have entered the CSF they cause injury by releasing toxic molecules such as nitric oxide (NO) and reactive oxygen species (ROS). The induction of iNOS was examined by assessing intracerebral mRNA expression and protein production during the course of Haemophilus influenzae type b (Hib) meningitis in the rat. Induction of iNOS mRNA was detected 12h postinoculation (pi), followed by a gradual reduction. The increased number of intracerebral iN OS expressing cells was detected at 12h pi. followed by further elevation to peak expression at 72h pi. The iNOS positive tissue also bound antibodies specific for nitrotyrosine. The expression of iNOS and NO production, as shown by nitrotyrosine expression, correlated with disease severity, suggesting that activation of iNOS may play an important role in Haemophilus irifluenzae type b meningitis.
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10.
  • Andersson, Anna-Maria, et al. (författare)
  • Efferocytosis of Apoptotic Neutrophils Enhances Control of Mycobacterium tuberculosis in HIV-Coinfected Macrophages in a Myeloperoxidase-Dependent Manner
  • 2020
  • Ingår i: Journal of Innate Immunity. - : KARGER. - 1662-811X .- 1662-8128. ; 12:3, s. 235-247
  • Tidskriftsartikel (refereegranskat)abstract
    • Tuberculosis remains a big threat, with 1.6 million deaths in 2017, including 0.3 million deaths among patients with HIV. The risk of developing active disease increases considerably during an HIV coinfection. Alveolar macrophages are the first immune cells to encounter the causative agent Mycobacterium tuberculosis, but during the granuloma formation other cells are recruited in order to combat the bacteria. Here, we have investigated the effect of efferocytosis of apoptotic neutrophils by M. tuberculosis and HIV-coinfected macrophages in a human in vitro system. We found that the apo-ptotic neutrophils enhanced the control of M. tuberculosis in single and HIV-coinfected macrophages, and that this was dependent on myeloperoxidase (MPO) and reactive oxygen species in an autophagy-independent manner. We show that MPO remains active in the apoptotic neutrophils and can be harnessed by infected macrophages. In addition, MPO inhibition removed the suppression in M. tuberculosis growth caused by the apoptotic neutrophils. Antimycobacterial components from apoptotic neutrophils could thus increase the microbicidal activity of macrophages during an M. tuberculosis/HIV coinfection. This cooperation between innate immune cells could thereby be a way to compensate for the impaired adaptive immunity against M. tuberculosis seen during a concurrent HIV infection.
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11.
  • Andersson, Anna-Maria, 1990- (författare)
  • Mycobacterium tuberculosis and HIV coinfection : Effects on innate immunity and strategies to boost the immune response
  • 2019
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Tuberculosis (TB) still remains a big threat today, being the leading cause of death by a single infectious agent. The TB epidemic is fueled by HIV along with the increasing drug-resistance which prolongs the already long treatment duration and decreases the success rate for curing TB. In most cases an infection results in latency but HIV patients have a 20-30 times higher risk of developing active TB. There are around 36.9 million people living with HIV globally, with the highest burden in Africa. Although there are effective treatments against the disease, there is no cure for AIDS and the availability of the lifelong treatment is limited in low-income countries were the burden is highest. HIV infection causes an immunodeficiency characterized by the progressive loss of CD4 T cells which increases the risk of opportunistic infections, and infection by Mycobacterium tuberculosis (Mtb), the causative agent of TB. Mtb spreads through aerosols from one person with active tuberculosis to a healthy person. Upon inhalation the bacteria are phagocytosed by alveolar macrophages that secrete cytokines and chemokines to recruit more cells, such as dendritic cells, macrophages and lymphocytes, leading to the formation of a granuloma. During a single TB infection the bacteria are usually contained within the granuloma, but HIV can disrupt the stable granuloma, causing a rupture and dissemination of Mtb. This inflammatory site is also beneficial to HIV since it promotes replication of the virus within infected cells. HIV and Mtb are two successful intracellular pathogens able to avoid immune defense mechanisms both of the innate and adaptive immunity in order to persist and replicate. Their virulence factors can manipulate or inhibit cell signaling, phagosome maturation, autophagy, ROS production, apoptosis and antigen presentation, to promote survival. Boosting of immune defenses with host-directed therapies (HDT) has been proposed as a treatment strategy against TB, either alone or adjunctive to the current regimen.In this thesis, ways to boost the innate immune responses in Mtb and HIV coinfected macrophages were investigated, along with studies of the effect of HIV on Mtb antigen presentation in coinfected dendritic cells. The initial hypothesis was that autophagy induction through inhibition of mammalian target of rapamycin (mTOR) could suppress Mtb growth in HIV coinfected macrophages. However, during a low grade infection, autophagy induction increased Mtb replication due to a decreased autophagic flux and acidification of Mtb phagosomes. A general autophagic flux was induced, although not localized to the Mtb phagosomes, thus not inducing a xenophagy (autophagy of intracellular pathogens). Other ways of inducing autophagy or boosting the response in coinfected macrophages might be more beneficial and therefore the effect of efferocytosis was investigated. Uptake of apoptotic neutrophils by coinfected macrophages did not induce autophagy but enhanced the control of Mtb by other means. Upon efferocytosis, the macrophages acquired active myeloperoxidase (MPO) from the neutrophils that suppressed Mtb growth. The coinfected macrophages also produced more ROS after efferocytosis. The inhibition of Mtb growth could thus be mediated by MPO and the increased ROS production either directly or indirectly.The possibility to boost the innate immunity could prove to be important during an HIV coinfection, when the adaptive immunity is deficient. In addition to the well-known decline in CD4 T cells during the course of HIV progression, we found that HIV infection of dendritic cells inhibited antigen presentation by suppressing the expression of HLA-DR and co-stimulatory molecules on coinfected dendritic cells. Furthermore, HIV reduced secretion of pro-inflammatory cytokines and suppressed antigen processing through inhibition of autophagy. This impaired antigen presentation in coinfected dendritic cells resulted in a decreased activation and response of Mtb-specific CD4 T cells.In conclusion, this thesis shows how HIV can manipulate antigen presentation in Mtb coinfected dendritic cells and subsequently inhibit the adaptive immune response. It also contributes to insights on how efferocytosis of apoptotic neutrophils can boost the innate immune responses during coinfection. Lastly, autophagy induction through mTOR inhibition does not enhance protection against TB. Induction of autophagy should therefore be handled with care, particularly during HIV coinfection. 
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12.
  • Andersson, Henrik, et al. (författare)
  • Apoptotic neutrophils augment the inflammatory response to Mycobacterium tuberculosis infection in human macrophages
  • 2014
  • Ingår i: PLOS ONE. - : PLOS. - 1932-6203. ; 9:7
  • Tidskriftsartikel (refereegranskat)abstract
    • Macrophages in the lung are the primary cells being infected by Mycobacterium tuberculosis (Mtb) during the initial manifestation of tuberculosis. Since the adaptive immune response to Mtb is delayed, innate immune cells such as macrophages and neutrophils mount the early immune protection against this intracellular pathogen. Neutrophils are short-lived cells and removal of apoptotic cells by resident macrophages is a key event in the resolution of inflammation and tissue repair. Since anti-inflammatory activity is not compatible with effective immunity to intracellular pathogens, we therefore investigated how uptake of apoptotic neutrophils modulates the function of Mtb-activated human macrophages. We show that Mtb infection exerts a potent proinflammatory activation of human macrophages with enhanced gene activation and release of proinflammatory cytokines and that this response was augmented by apoptotic neutrophils. The enhanced macrophage response is linked to apoptotic neutrophil-driven activation of the NLRP3 inflammasome and subsequent IL-1β signalling. We also demonstrate that apoptotic neutrophils not only modulate the inflammatory response, but also enhance the capacity of infected macrophages to control intracellular growth of virulent Mtb. Taken together, these results suggest a novel role for apoptotic neutrophils in the modulation of the macrophage-dependent inflammatory response contributing to the early control of Mtb infection.
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13.
  • Andersson, Kerstin, et al. (författare)
  • Yersinia pseudotuberculosis-induced calcium signaling in neutrophils is blocked by the virulence effector YopH
  • 1999
  • Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 67:5, s. 2567-2574
  • Tidskriftsartikel (refereegranskat)abstract
    • Pathogenic species of the genus Yersinia evade the bactericidal functions of phagocytes. This evasion is mediated through their virulence effectors, Yops, which act within target cells. In this study we investigated the effect of Yersinia pseudotuberculosis on Ca 2+ signaling in polymorphonuclear neutrophils. The intracellular free calcium concentration in single adherent human neutrophils was monitored during bacterial infection and, in parallel, the encounter between the bacteria and cells was observed. When a plasmid-cured strain was used for infection, adherence of a single bacterium to the cellular surface induced a β 1 integrin-dependent transient increase in the intracellular concentration of free calcium. This was, however, not seen with Yop-expressing wild-type bacteria, which adhered to the cell surface without generating any Ca 2+ signal. Importantly, the overall Ca 2+ homeostasis was not affected by the wild-type strain; the Ca 2+ signal mediated by the G-protein-coupled formyl-methionyl-leucyl- phenylalanine receptor was still functioning. Hence, the blocking effect was restricted to certain receptors and their signaling pathways. The use of different Yop mutant strains revealed that the protein tyrosine phosphatase YopH was responsible for the inhibition. This virulence determinant has previously been implicated in very rapid Yersinia-mediated effects on target cells as the key effector in the blockage of phagocytic uptake. The present finding, that Y. pseudotuberculosis, via YopH, specifically inhibits a self- induced immediate-early Ca 2+ signal in neutrophils, offers more-detailed information concerning the effectiveness of this virulence effector and implies an effect on Ca 2+-dependent, downstream signals.
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14.
  • Augustinsson (Nilsdotter-Augustinsson), Åsa, 1962-, et al. (författare)
  • Interaction of staphylococcus epidermidis from infected hip prostheses with neutrophil granulocytes
  • 2001
  • Ingår i: Scandinavian Journal of Infectious Diseases. - : Informa UK Limited. - 0036-5548 .- 1651-1980. ; 33:6, s. 408-412
  • Tidskriftsartikel (refereegranskat)abstract
    • This study focuses on the interaction of Staphylococcus epidermis isolated from granulation tissue covering infected hip prostheses and neutrophil granulocytes. Bacterial strains isolated from normal flora were used as controls. The bacteria were well characterized with routine methods and further characterized with random amplified polymorphic DNA analyses and slime tests. Phagocytosis and chemiluminescence (CL) assays were used in the neutrophil interaction studies. The prostheses strains were ingested to a lesser extent than strains from normal flora (p ≤ 0.001). There was no significant difference between the prostheses strains and the normal flora strains in terms of total CL response. However, the extracellular CL response from the neutrophils was lower in comparison with the normal flora when interacting with the prostheses strains. The results of this study support the notion that S. epidermidis strains isolated from infected hip prostheses have an enhanced capacity to resist phagocytosis and that most of these strains elicit a reduced inflammatory response, measured as the production of extracellular oxidative metabolites from the neutrophils, compared to normal flora.
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15.
  • Bengtsson, T., et al. (författare)
  • Actin dynamics in human neutrophils during adhesion and phagocytosis is controlled by changes in intracellular free calcium
  • 1993
  • Ingår i: European Journal of Cell Biology. - Jena, Germany : Urban und Fischer Verlag. - 0171-9335 .- 1618-1298. ; 62:1, s. 19-58
  • Tidskriftsartikel (refereegranskat)abstract
    • The role of changes in cytosolic free calcium concentration ([Ca2+]i) in the assembly and disassembly of actin during adhesion and phagocytosis was evaluated. Rhodamine-phalloidin staining combined with quantitative fluorescence and confocal laser scanning microscopy was used to measure local F-actin changes in single adherent human neutrophils phagocytosing yeast particles on different surfaces and under different calcium conditions. Cells were suspended in a) calcium-containing medium (CCM) or b) calcium-free medium (CFM) or c) were first depleted of calcium (i.e., MAPT/AM-loaded in CFM) and then suspended in CFM (MAPT). In parallel, local [Ca2+]i changes were monitored using a fura-2 ratio imaging system. In CCM or CFM, attachment to the substrate and formation of pseudopods around a yeast particle generated, within a few seconds, rises in [Ca2+]i, both around the phagosome and in the cell body. During continued phagocytosis, [Ca2+]i was more elevated around the phagosome compared to the rest of the cell. No [Ca2+]i fluctuations were observed in MAPT cells. Adhesion and phagocytosis led to a several-fold increase in F-actin. The increase was transient in cells in CCM and CFM, but remained high in Ca-depleted neutrophils. A distinct ring of F-actin was formed around a phagosome with a yeast particle. Twenty min after ingestion the amount of this actin decreased more than 50% in CCM and CFM cells but increased by 40 to 100% in MAPT cells. The accumulation of F-actin in MAPT cells was reduced to resting levels by adding Ca2+ and ionomycin after ingestion. This treatment reestablished the periphagosomal [Ca2+]i rises, as observed in CCM cells. In conclusion, the present study shows that the actin polymerization, occurring in human neutrophils during adhesion and phagocytosis, is not influenced by changes in [Ca2+]i, whereas the subsequent depolymerization is. The accumulation of actin filaments around the phagosome in calcium-depleted cells could be involved in the inhibition of phagolysosome fusion seen in the absence of [Ca2+]i changes (Jaconi et al., J. Cell Biol. 110, 1555-1564 (1990)). This suggests that the actin network, controlled by [Ca2+]i, regulates the movement of granules during phagocytosis.
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16.
  • Bewket, Gezahegn, et al. (författare)
  • Helminth species specific expansion and increased TNF-alpha production of non-classical monocytes during active tuberculosis
  • 2021
  • Ingår i: PLoS Neglected Tropical Diseases. - : Public Library of Science. - 1935-2727 .- 1935-2735. ; 15:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Author summary Monocytes are important cells for the early innate immune response and play an integral part during inflammation and infection. Classical monocytes, the dominant monocyte subset during homeostasis and health, have been linked to efficient TB protection. Intermediate or non-classical monocytes have instead been associated with uncontrolled inflammation (TNF-alpha), cell death, and poor protection against Mycobacterium tuberculosis. In areas endemic for intestinal helminths, the immunoregulatory effects of monocytes may affect development or progression of TB disease. The role of monocyte subsets during helminth/TB coinfection have not been studied. In Gondar, Ethiopia, we show that in patients with helminth infection, a helminth species dependent expansion of non-classical monocytes is triggered, where Ascaris and hookworm had the strongest effect in coinfected pulmonary TB-patients. The increase in non-classical monocytes was mainly detected in coinfected patients with a low-to-intermediate disease severity. Only coinfection with helminths and TB induced an increased TNF-alpha response in monocytes. Thus, we found a helminth species-specific dysregulation of monocyte subset distribution and functionality in coinfected TB-patients which could affect TB pathogenesis. Both Mycobacterium tuberculosis infection and helminths may affect innate immune mechanisms such as differential effects on monocytes towards the non-classical and intermediate subsets that favor bacterial persistence. Our aim, was to investigate helminth species specific effects on the frequency and functional activity of monocyte subsets in patients with active tuberculosis and healthy subjects. HIV-negative patients with active pulmonary tuberculosis (PTB) and community controls (CCs) in Gondar, Ethiopia were screened for helminth infection by stool microscopy. Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs) and ex vivo stimulation with purified protein derivative (PPD) and helminth antigens were used to characterize the distribution of monocyte subsets and their function. A total of 74 PTB patients and 57 CCs with and without helminth infection were included. Non-classical monocytes were increased in PTB patients with Ascaris and hookworm infection but not in Schistosoma-infected patients. Ascaris had the strongest effect in increasing the frequency of non-classical monocytes in both PTB patients and CCs, whereas PTB without helminth infection did not affect the frequency of monocyte subsets. There was a helminth specific increase in the frequency of TNF-alpha producing non-classical monocytes in hookworm infected PTB patients, both with and without PPD-stimulation. Low-to-intermediate TB disease severity associated with increased frequency of non-classical monocytes only for helminth-positive PTB patients, and the frequency of TNF-alpha producing monocytes were significantly higher in intermediate and non-classical monocytes of helminth positive PTB patients with an intermediate disease score. Helminth infection affected the frequency of monocyte subsets and function both in TB patients and controls which was helminth species dependent in TB patients. The clinical role of this potential immunomodulatory effect needs further study and may affect the response and protection to tuberculosis in areas where helminth infections are endemic.
  •  
17.
  • Blomgran, Robert, et al. (författare)
  • Cathepsin-cleaved Bid promotes apoptosis in human neutrophils via oxidative stress-induced lysosomal membrane permeabilization
  • 2007
  • Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 0741-5400 .- 1938-3673. ; 81, s. 1213-1223
  • Tidskriftsartikel (refereegranskat)abstract
    • Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting increased production of reactive oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS-dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1-fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1-fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl-2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl-1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine-cathepsins but not caspases, and the differential effects of inhibitors of cysteine-cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe-induced apoptosis in neutrophils, ROS-dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.
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18.
  • Blomgran, Robert, et al. (författare)
  • Common Genetic Variations in the NALP3 Inflammasome Are Associated with Delayed Apoptosis of Human Neutrophils
  • 2012
  • Ingår i: PLOS ONE. - San Francisco, USA : Public Library of Science. - 1932-6203. ; 7:3
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Neutrophils are key-players in the innate host defense and their programmed cell death and removal are essential for efficient resolution of inflammation. These cells recognize a variety of pathogens, and the NOD-like receptors (NLRs) have been suggested as intracellular sensors of microbial components and cell injury/stress. Some NLR will upon activation form multi-protein complexes termed inflammasomes that result in IL-1 beta production. NLR mutations are associated with auto-inflammatory syndromes, and our previous data propose NLRP3 (Q705K)/CARD-8 (C10X) polymorphisms to contribute to increased risk and severity of inflammatory disease by acting as genetic susceptibility factors. These gene products are components of the NALP3 inflammasome, and approximately 6.5% of the Swedish population are heterozygote carriers of these combined gene variants. Since patients carrying the Q705K/C10X polymorphisms display leukocytosis, the aim of the present study was to find out whether the inflammatory phenotype was related to dysfunctional apoptosis and impaired clearance of neutrophils by macrophages. less thanbrgreater than less thanbrgreater thanMethods and Findings: Patients carrying the Q705K/C10X polymorphisms displayed significantly delayed spontaneous as well as microbe-induced apoptosis compared to matched controls. Western blotting revealed increased levels and phosphorylation of Akt and Mcl-1 in the patients neutrophils. In contrast to macrophages from healthy controls, macrophages from the patients produced lower amounts of TNF; suggesting impaired macrophage clearance response. less thanbrgreater than less thanbrgreater thanConclusions: The Q705K/C10X polymorphisms are associated with delayed apoptosis of neutrophils. These findings are explained by altered involvement of different regulators of apoptosis, resulting in an anti-apoptotic profile. Moreover, the macrophage response to ingestion of microbe-induced apoptotic neutrophils is altered in the patients. Taken together, the patients display impaired turnover and clearance of apoptotic neutrophils, pointing towards a dysregulated innate immune response that influences the resolution of inflammation. The future challenge is to understand how microbes affect the activation of inflammasomes, and why this interaction will develop into severe inflammatory disease in certain individuals.
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19.
  • Blomgran, Robert, 1975- (författare)
  • Microbe-induced apoptosis in phagocytic cells and its role in innate immunity
  • 2006
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Apoptosis, or programmed cell death, is a controlled process by which aged or damages cells are eliminated in multicellular organisms. Neutrophils, short-lived phagocytes of the innate immune system, are highly equipped effectors that can sense, locate, ingest and kill bacterial pathogens. Inflammatory mediators and the presence of bacterial products at the foci of infection regulate the function and life span of these cells. Modulation of neutrophil apoptosis and the subsequent clearance by scavenger cells, such as macrophages, is part of a balanced inflammatory process leading to resolution of inflammation. Many pathogens are capable of modulating host cell apoptosis, and thereby influence the progression of disease. Hence, this thesis was aiming at elucidating mechanisms involved in pathogen- and host-modulated apoptosis and its contribution to the inflammatory process.We found that different routes of bacterial entry, i.e. through invasion or by receptor-mediated phagocytosis, triggered different signaling pathways within phagocytes. Invasion of virulent Salmonella caused apoptosis, a process requiring activation of the Rho GTPases Rac1 and Cdc42. On the other hand, phagocytosis of the non-invasive Salmonella inhibited apoptosis despite similar intracellular survival as the invasive bacteria. Protection against phagocytosis-induced apoptosis was regulated by tyrosine- and PI3-kinase-dependent activation of AKT (also called PKB for protein kinase B). Furthermore, inhibiting the intraphagosomal production of reactive oxygen species (ROS) in neutrophils during phagocytosis of E. coli decreased apoptosis below spontaneous apoptosis, further indicating that both pro- and anti-apoptotic pathways are triggered by receptor-mediated phagocytosis.Type 1 fimbria-expressing E. coli adhering to neutrophils resisted ingestion, and induced a ROS-dependent apoptosis by a cooperative effect of the FimH adhesin and LPS. To explore how compartmentalization of ROS during neutrophil activation was involved in modulating apoptosis, we evaluated the stability of lysosomes. In contrast to phagocytosis of E. coli, the adhesive strain induced intracellular non-phagosomal ROS production which triggered early permeabilization and release of lysosomal enzymes to the cytosol. Cathepsin B and/or L were responsible for targeting of the pro-apoptotic Bcl-2 protein Bid, thereby inducing mitochondrial damage, and apoptosis. These data propose a novel pathway for ROS-induced apoptosis in human neutrophils, where the location of the ROS rather than production per se is important.Moreover, we found that pathogen-induced apoptotic neutrophils, in contrast to uninfected apoptotic neutrophils, activated blood-monocyte derived macrophages to increase their FcγRI surface expression and to produce large quantities of the pro-inflammatory cytokine TNF-α. This demonstrates that during the early phase of infection, pathogen-induced neutrophil apoptosis will help local macrophages to gain control over the microbes. Furthermore, we suggest that heat shock protein 60 and 70 represent a stress signal that enables macrophages to distinguish between, and react differently to, uninfected and inflammatory apoptotic neutrophils.
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20.
  • Blomgran, Robert, et al. (författare)
  • Uropathogenic Escherichia coli triggers oxygen-dependent apoptosis in human neutrophils through the cooperative effect of type 1 fimbriae and lipopolysaccharide
  • 2004
  • Ingår i: Infection and Immunity. - 0019-9567 .- 1098-5522. ; 72:8, s. 4570-4578
  • Tidskriftsartikel (refereegranskat)abstract
    • Type 1 fimbriae are the most commonly expressed virulence factor on uropathogenic Escherichia coli. In addition to promoting avid bacterial adherence to the uroepithelium and enabling colonization, type 1 fimbriae recruit neutrophils to the urinary tract as an early inflammatory response. Using clinical isolates of type 1 fimbriated E. coli and an isogenic type 1 fimbria-negative mutant (CN1016) lacking the FimH adhesin, we investigated if these strains could modulate apoptosis in human neutrophils. We found that E. coli expressing type 1 fimbriae interacted with neutrophils in a mannose- and lipopolysaccharide (LPS)-dependent manner, leading to apoptosis which was triggered by the intracellular generation of reactive oxygen species. This induced neutrophil apoptosis was abolished by blocking FimH-mediated attachment, by inhibiting NADPH oxidase activation, or by neutralizing LPS. In contrast, CN1016, which did not adhere to or activate the respiratory burst of neutrophils, delayed the spontaneous apoptosis in an LPS-dependent manner. This delayed apoptosis could be mimicked by adding purified LPS and was also observed by using fimbriated bacteria in the presence of D-mannose. These results suggest that LPS is required for E. coli to exert both pro- and antiapoptotic effects on neutrophils and that the difference in LPS presentation (i.e., with or without fimbriae) determines the outcome. The present study showed that there is a fine-tuned balance between type 1 fimbria-induced and LPS-mediated delay of apoptosis in human neutrophils, in which altered fimbrial expression on uropathogenic E. coli determines the neutrophil survival and the subsequent inflammation during urinary tract infections.
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21.
  • Braian, Clara, 1981- (författare)
  • Innate immune responses to Mycobacterium tuberculosis infection : How extracellular traps and trained immunity can restrict bacterial growth.
  • 2020
  • Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
    • Tuberculosis (TB) is an infectious disease caused by the bacterium Mycobacterium tuberculosis, and the cause of 1.5 million deaths in 2018. During a pulmonary TB infection, the bacterium reaches the lungs and is phagocytosed by cells of the innate immune system, primarily macrophages. The macrophages are either able to eradicate the bacteria or the bacteria start to replicate, and the following immune response leads to the formation of a large cluster of different cell types called a granuloma. In the granuloma the mycobacteria are contained in a latent infection, or they can start to replicate causing rupture of the granuloma and spread of the disease. Neutrophils are also innate immune cells that are rapidly recruited to the site of infection. They are phagocytes, but they also exert extracellular effector mechanisms by their release of microbicidal granule proteins, reactive oxygen species and neutrophil extracellular traps. M. tuberculosis has co-evolved and adapted to the human host making it ingenious at exploiting the human immune response, promoting its survival and replication in human host cells. The human immune system has also evolved mechanisms to limit M. tuberculosisreplication and spread. This thesis covers work on the innate immune response to TB and how neutrophils and macrophages respond to a mycobacterial infection and can control M. tuberculosis-replication.Neutrophils and macrophages can respond to M. tuberculosis by releasing extracellular traps. We demonstrated that neutrophil extracellular traps contain the danger signal heat-shock protein 72 when induced by mycobacteria, which subsequently mediate a proinflammatory activation of adjacent macrophages. Macrophages can also release extracellular traps, and we observed the release of macrophage extracellular traps in response to M. tuberculosis that grow in cord-structures. We further demonstrated that the induction of extracellular traps also required the mycobacterial virulence factor ESAT-6.Trained immunity is an epigenetically regulated memory of the innate immune system that results in a heightened response to a later encounter of the same or different pathogen. β-glucans are structural components of microbial cell walls and known inducers of trained immunity. We studied the effects of β-glucan from a bacterial source (curdlan from Alcaligenes faecalis), from yeast (WGP dispersible from Saccharomyces cerevisiae) and from the supernatant of a multicellular fungi (Alternaria) in search of functional changes in human macrophages which enhanced their anti-mycobacterial capacity. M. tuberculosis growth reduction was observed in WGP dispersible-trained macrophages when co-cultured with neutrophils. We also discovered that the interferon-gamma (IFNγ) signaling pathway, which is important for mycobacterial control, is hypomethylated in the WGP dispersible-trained macrophages. Since hypomethylation of genes typically is associated with gene activation, this suggests a more active IFNγ signaling in response to β-glucan innate immune training.Most of our studies were performed using in vitro culturing of primary human macrophages and neutrophils. However, an in vitro 3D tissue model is a valuable tool when studying complex events that occur during a TB infection that involves both multiple cell types and requires knowledge of the spatial movement of cells. In this thesis we also describe an in vitro lung tissue model, which we could use to observe the clustering of monocytes around mycobacteria and quantify the size and number of macrophage clusters.In conclusion, this thesis comprises work on innate immune functions during tuberculosis infection. We describe extracellular trap formation in macrophages and neutrophils in response to M. tuberculosis. We also explore trained immunity and how β-glucan training can enhance mycobacterial growth restriction.
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22.
  • Braian, Clara, 1981-, et al. (författare)
  • Mycobacterium tuberculosis-Induced Neutrophil Extracellular Traps Activate Human Macrophages
  • 2013
  • Ingår i: Journal of Innate Immunity. - : Karger. - 1662-811X .- 1662-8128. ; 5:6, s. 591-602
  • Tidskriftsartikel (refereegranskat)abstract
    • Neutrophils activated by Mycobacterium tuberculosis (Mtb) form neutrophil extracellular traps (NETs), containing DNA and several biologically active cytosolic and granular proteins. These NETs may assist in the innate immune defense against different pathogens. We investigated whether the NET-forming neutrophils mediate an activating signal to macrophages during the early multicellular inflammatory reaction and granuloma formation. Mtb-induced NETs were found to be reactive oxygen species dependent and phagocytosis dependent. A neutrophil elastase inhibitor also delayed NET formation. However, NET formation occurred independently of Mtb-induced apoptosis. We observed close interactions between macrophages and Mtb-activated neutrophils, where macrophages bound and phagocytosed NETs. Significant secretion of the cytokines interleukin (IL)-6, tumor necrosis factor-alpha, IL-1 beta and IL-10 were detected from macrophages cocultured with NETs from Mtb-activated but not phorbol myristate acetate-activated neutrophils. NETs binding heat shock protein 72 (Hsp72) or recombinant Hsp72 were able to trigger cytokine release from macrophages. Only Mtb-induced NETs contained Hsp72, suggesting that these NETs can transfer this danger signal to adjacent macrophages. We propose that Hsp72 sequestered in NETs plays an important role in the interaction between neutrophils and macrophages during the early innate immune phase of an Mtb infection. The immunomodulatory role of NETs and proteins derived from them may influence not only chronic inflammation during tuberculosis but also immune regulation and autoimmunity.
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23.
  • Eklund, Daniel, 1984-, et al. (författare)
  • Human Gene Variants Linked to Enhanced NLRP3 Activity Limit Intramacrophage Growth of Mycobacterium tuberculosis
  • 2014
  • Ingår i: Journal of Infectious Diseases. - Cary, USA : Oxford University Press. - 0022-1899 .- 1537-6613. ; 209:5, s. 749-753
  • Tidskriftsartikel (refereegranskat)abstract
    • Activation of the NLRP3 inflammasome and subsequent generation of interleukin 1 beta is initiated in macrophages upon recognition of several stimuli. In the present work, we show that gain-of-function gene variants of inflammasome components known to predispose individuals to inflammatory disorders have a host-protective role during infection with Mycobacterium tuberculosis. By isolation of macrophages from patients and healthy blood donors with genetic variants in NLRP3 and CARD8 and subsequent infection of the cells with virulent M. tuberculosis, we show that these gene variants, combined, are associated with increased control of bacterial growth in human macrophages.
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24.
  • Eklund, Daniel, 1984- (författare)
  • Mycobacterium tuberculosis and the human macrophage : shifting the balance through inflammasome activation
  • 2013
  • 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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25.
  • Eklund, Daniel, et al. (författare)
  • Validation of a Medium-Throughput Method for Evaluation of Intracellular Growth of Mycobacterium tuberculosis
  • 2010
  • Ingår i: Clinical and Vaccine Immunology. - : American Society for Microbiology. - 1556-6811 .- 1556-679X. ; 17:4, s. 513-517
  • Tidskriftsartikel (refereegranskat)abstract
    • Intracellular pathogens such as Mycobacterium tuberculosis have adapted to a life inside host cells, in which they utilize host nutrients to replicate and spread. Ineffective methods for the evaluation of growth of intracellular pathogens in their true environment pose an obstacle for basic research and drug screening. Here we present the validation of a luminometry-based method for the analysis of intramacrophage growth of M. tuberculosis. The method, which is performed in a medium-throughput format, can easily be adapted for studies of other intracellular pathogens and cell types. The use of host cells in drug-screening assays dedicated to find antimicrobials effective against intracellular pathogens permits the discovery of not only novel antibiotics but also compounds with immunomodulatory and virulence-impairing activities, which may be future alternatives or complements to antibiotics.
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26.
  • Eklund, Daniel, 1984-, et al. (författare)
  • Vitamin D enhances IL-1β secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients
  • 2013
  • Ingår i: International journal of mycobacteriology. - Netherlands : Wolters Kluwer. - 2212-5531. ; 2:1, s. 18-25
  • Tidskriftsartikel (refereegranskat)abstract
    • The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1β and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.
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27.
  • Favre, Cécile J., et al. (författare)
  • Organization of Ca2+ stores in myeloid cells: association of SERCA2b and the type-1 inositol-1,4,5-trisphosphate receptor
  • 1996
  • Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 316:1, s. 137-142
  • Tidskriftsartikel (refereegranskat)abstract
    • In this study, we have analysed the relationship between Ca2+ pumps and Ins(1,4,5)P3-sensitive Ca2+ channels in myeloid cells. To study whether sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA)-type Ca2+-ATPases are responsible for Ca2+ uptake into Ins(1,4,5)P3-sensitive Ca2+ stores, we used the three structurally unrelated inhibitors thapsigargin, 2,5-di-t-butylhydroquinone and cyclopiazonic acid. In HL-60 cells, all three compounds precluded formation of the phosphorylated intermediate of SERCA-type Ca2+-ATPases. They also decreased, in parallel, ATP-dependent Ca2+ accumulation and the amount of Ins(1,4,5)P3-releasable Ca2+. Immunoblotting with subtype-directed antibodies demonstrated that HL-60 cells contain the Ca2+ pump SERCA2 (subtype b), and the Ca2+-release-channel type-1 Ins(1,4,5)P3 receptor. In subcellular fractionation studies, SERCA2 and type-1 Ins(1,4,5)P3 receptor co-purified. Immunofluorescence studies demonstrated that both type-1 Ins(1,4,5)P3 receptor and SERCA2 were evenly distributed throughout the cell in moving neutrophils. During phagocytosis both proteins translocated to the periphagosomal space. Taken together, our results suggest that in myeloid cells (i) SERCA-type Ca2+-ATPases function as Ca2+ pumps of Ins(1,4,5)P3-sensitive Ca2+ stores, and (ii) SERCA2 and type-1 Ins(1,4,5)P3 receptor reside either in the same or two tightly associated subcellular compartments.
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28.
  • Forsberg, Maria, 1973-, et al. (författare)
  • Activation of Rac2 and Cdc42 on Fc and complement receptor ligation in human neutrophils
  • 2003
  • Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 0741-5400 .- 1938-3673. ; 74:4, s. 611-619
  • Tidskriftsartikel (refereegranskat)abstract
    • Phagocytosis is a complex process engaging a concerted action of signal-transduction cascades that leads to ingestion, subsequent phagolysosome fusion, and oxidative activation. We have previously shown that in human neutrophils, C3bi-mediated phagocytosis elicits a significant oxidative response, suggesting that activation of the small GTPase Rac is involved in this process. This is contradictory to macrophages, where only Fc receptor for immunoglobulin G (FcγR)-mediated activation is Rac-dependent. The present study shows that engagement of the complement receptor 3 (CR3) and FcγR and CR3- and FcγR-mediated phagocytosis activates Rac, as well as Cdc42. Furthermore, following receptor-engagement of the CR3 or FcγRs, a downstream target of these small GTPases, p21-activated kinase, becomes phosphorylated, and Rac2 is translocated to the membrane fraction. Using the methyltransferase inhibitors N-acetyl-S-farnesyl-L-cysteine and N-acetyl-S-geranylgeranyl-L-cysteine, we found that the phagocytic uptake of bacteria was not Rac2- or Cdc42-dependent, whereas the oxidative activation was decreased. In conclusion, our results indicate that in neutrophils, Rac2 and Cdc42 are involved in FcR- and CR3-induced activation and for properly functioning signal transduction involved in the generation of oxygen radicals.
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29.
  • Forsberg, Maria, et al. (författare)
  • Differential effects of invasion by and phagocytosis of Salmonella typhimurium on apoptosis in human macrophages : potential role of Rho–GTPases and Akt
  • 2003
  • Ingår i: Journal of Leukocyte Biology. - : Oxford University Press (OUP). - 0741-5400 .- 1938-3673. ; 74:4, s. 620-629
  • Tidskriftsartikel (refereegranskat)abstract
    • In addition to direct activation of caspase-1 and induction of apoptosis by SipB, invasive Salmonella stimulates multiple signaling pathways that are key regulators of host cell survival. Nevertheless, little is known about the relative contributions of these pathways to Salmonella-mediated death of macrophages. We studied human monocytic U937 cells and found that apoptosis was induced by invading wild-type Salmonella typhimurium but not by phagocytosed, serum-opsonized, noninvasive Salmonella mutants. Pretreating U937 cells with inhibitors of tyrosine kinases or phosphatidylinositol-3 kinase (PI-3K) completely blocked phagocytosis of opsonized Salmonella mutants but did not affect invasion by wild-type Salmonella or the apoptosis caused by invasion. However, pretreatment with GGTI-298, a geranylgeranyltransferase-1 inhibitor that prevents prenylation of Cdc42 and Rac1, suppressed Salmonella-induced apoptosis by ∼70%. Transduction of Tat fusion constructs containing dominant-negative Cdc42 or Rac1 significantly inhibited Salmonella-induced cell death, indicating that the cytotoxicity of Salmonella requires activation of Cdc42 and Rac. In contrast to phagocytosis of opsonized bacteria, invasion by S. typhimurium stimulated Cdc42 and Rac1, regardless of the activities of tyrosine- or PI-3K. Moreover, Salmonella infection activated Akt protein in a tyrosine-kinase or PI-3K-dependent manner, and a reduced expression of Akt by antisense transfection rendered the cells more sensitive to apoptosis induced by opsonized Salmonella. These results indicate that direct activation of Cdc42 and Rac1 by invasive Salmonella is a prerequisite of Salmonella-mediated death of U937 cells, whereas the simultaneous activation of Akt by tyrosine kinase and PI-3K during receptor-mediated phagocytosis protects cells from apoptosis.
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30.
  • Forsberg, Maria, 1973-, et al. (författare)
  • Tumour necrosis factor-α potentiates CR3-induced respiratory burst by activating p38 MAP kinase in human neutrophils
  • 2001
  • Ingår i: Immunology. - : Wiley. - 0019-2805 .- 1365-2567. ; 103:4, s. 465-472
  • Tidskriftsartikel (refereegranskat)abstract
    • CR3 and FcγRs are the main receptors involved in the phagocytic process leading to engulfment and killing of microbes by production of reactive oxygen intermediates (ROI) and degranulation. Various inflammatory mediators, such as tumour necrosis factor-α (TNF-α) and lipopolysaccharide (LPS), are known to prime neutrophils leading to increased bactericidal responses, but the underlying mechanism of priming has only been partially elucidated. The purpose of this study was to investigate how TNF-α primes neutrophils for subsequent stimuli via either CR3 or FcγR. The receptors were specifically activated with pansorbins (protein-A-positive Staphylococcus aureus) coated with anti-CR3, anti-FcγRIIa, or anti-FcγRIIIb monoclonal antibody. Activation of neutrophils with these particles resulted in ROI production as measured by chemiluminescence. Anti-CR3 pansorbins induced the most prominent ROI production in neutrophils. TNF-α potentiated the CR3-mediated respiratory burst but had little effect on that mediated by FcγRs. The priming effect of TNF-α on CR3-mediated ROI production is associated with an increased activation of p38 MAPK as well as tyrosine phosphorylation of p72syk. Pretreatment of neutrophils with the inhibitors for p38 MAPK and p72syk markedly suppressed the respiratory burst induced by CR3. Furthermore, TNF-α induced about a three-fold increase in the expression of CR3 in neutrophils, an effect which is blocked by the p38 MAPK inhibitor. Taken together, these results showed that TNF-α potentiates the CR3-mediated respiratory burst in neutrophils not only by triggering a p38 MAPK-dependent up-regulation of CD11b/CD18 but also by modulating the signalling pathways.
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31.
  • Hedlund, Sebastian, et al. (författare)
  • Dendritic cell activation by sensing Mycobacterium tuberculosis-induced apoptotic neutrophils via DC-SIGN
  • 2010
  • Ingår i: HUMAN IMMUNOLOGY. - : Elsevier Science B.V., Amsterdam.. - 0198-8859 .- 1879-1166. ; 71:6, s. 535-540
  • Tidskriftsartikel (refereegranskat)abstract
    • Mycobacterium tuberculosis (Mtb) manipulates cells of the innate immune system to provide the bacteria with a sustainable intracellular niche. Mtb spread through aerosol carrying them deep into the lungs, where they are internalized by phagocytic cells, such as neutrophils (PMNs), dendritic cells (DCs), and macrophages. PMNs undergo accelerated apoptosis after interaction with the bacterium, and apoptotic cells are sequestered by neighboring phagocytes. Removal of aged apoptotic cells because of natural tissue turnover is described as an immunologically silent process facilitating resolution of inflammation and inhibition of DC maturation. Silencing of immune cells could be favorable for intracellular bacteria. The aim of this study was to clarify the interaction between Mtb-induced apoptotic PMNs and DCs, and evaluate whether this interaction follows the proposed anti-inflammatory pathway. In contrast to aged apoptotic cells, Mtb-induced apoptotic PMNs induced functional DC maturation. We found that the cell fraction from Mtb-induced apoptotic PMNs contained almost all stimulatory capacity, suggesting that cell-cell interaction is crucial for DC activation. Inhibitory studies showed that this cell contact-dependent activation required binding of the PMN Mac-1 (CD11b/CD18) to the DC via DC-SIGN and endocytic activity involving the alpha(v)beta(5) but did not involve the scavenger receptor CD36. Taken together, this study demonstrates that the DCs can distinguish between normal and infected apoptotic PMNs via cellular crosstalk, where the DCs can sense the presence of danger on the Mtb-infected PMNs and modulate their response accordingly.
  •  
32.
  • Hedlund, Sebastian R., et al. (författare)
  • Dendritic cell activation by sensing Mycobacterium tuberculosis-induced apoptotic neutrophils via DC-SIGN
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • In Mycobacterium tuberculosis (Mtb)-infected individuals cells of the innate immune system accumulate in the spleen and in granulomas, but how this relates to the protection against Mtb or in the pathogenesis is unknown. Mtb is internalized in the lung by phagocytic cells, such as neutrophils (PMNs), dendritic cells (DCs) and macrophages. PMNs undergo accelerated apoptosis after internalization of the bacterium and are subsequently sequestered by neighbouring phagocytes. Removal of aged apoptotic cells is an immunologically silent process and the aim of this study was to clarify the interaction between Mtb-induced apoptotic PMNs and DCs, and evaluate if this interaction induced functional maturation of the DCs. In fact, Mtb-induced apoptotic PMNs induced DC maturation, whereas exposure to spontaneous apoptotic PMNs had no effect on DCs maturation status. We found that the cell fraction contained almost all stimulatory capacity, suggesting that the cell-cell interaction is crucial for DC activation. Inhibitory studies showed that this cell contact-dependent activation required binding of the PMN Mac-1 (CD11b/CD18) to the DC via DC-SIGN and endocytic activity. Taken together, this study proves that the DCs can distinguish between normal and infected apoptotic PMNs via cellular cross talk, where the DCs can sense the presence of danger on the Mtb-infected PMNs and modulate their response accordingly.
  •  
33.
  • Idh, Jonna, et al. (författare)
  • Kinetics of the QuantiFERON((R))-TB Gold In-Tube test during treatment of patients with sputum smear-positive tuberculosis in relation to initial TST result and severity of disease
  • 2010
  • Ingår i: Scandinavian journal of infectious diseases. - : Informa UK Limited. - 1651-1980 .- 0036-5548. ; 42:9, s. 650-657
  • Tidskriftsartikel (refereegranskat)abstract
    • Abstract The QuantiFERON((R))-TB Gold In-Tube test (QFN) measures interferon-gamma production in response to Mycobacterium tuberculosis antigens. Our aim was to assess the kinetics of the QFN and initial tuberculin skin test (TST) result in relation to severity of disease in a tuberculosis (TB) endemic area. Smear-positive TB patients (n = 71) were recruited at Gondar University Hospital, Ethiopia. The TST, QFN, CD4+ cell count and clinical symptoms (TB score) were assessed and followed up during treatment. From baseline to 7 months after treatment, there was a significant decrease in QFN reactivity (93.8% to 62.5% in HIV-negative/TB; 70.3% to 33.3% in HIV-positive/TB patients) down to a level comparable to a control group of blood donors (51.2%). The agreement between TST and QFN was poor in TB patients compared to healthy controls. A negative TST correlated to more advanced TB in contrast to a negative QFN test. We conclude that the QFN reactivity is significantly reduced at the end of treatment against active TB to the background level of healthy blood donors, and that the agreement between TST and QFN is poor including correlation to the severity of disease.
  •  
34.
  • Idh, Jonna, et al. (författare)
  • Nitric oxide production in the exhaled air of patients with pulmonary tuberculosis in relation to HIV co-infection
  • 2008
  • Ingår i: BMC Infectious Diseases. - : Springer Science and Business Media LLC. - 1471-2334. ; 8:146
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: Nitric oxide (NO) is essential for host defense in rodents, but the role of NO during tuberculosis (TB) in man remains controversial. However, earlier observations that arginine supplementation facilitates anti-TB treatment, supports the hypothesis that NO is important in the host defense against TB. Local production of NO measured in fractional exhaled air (FeNO) in TB patients with and without HIV co-infection has not been reported previously. Thus, our aim was to investigate levels of FeNO in relation to clinical symptoms and urinary NO metabolites (uNO). Methods: In a cross sectional study, FeNO and uNO were measured and clinical symptoms, chest x-ray, together with serum levels of arginine, tumor necrosis factor alpha (TNF-alpha) and interleukin 12 (IL-12) were evaluated in sputum smear positive TB patients (HIV+/TB, n = 36, HIV-/TB, n = 59), their household contacts (n = 17) and blood donors (n = 46) from Gondar University Hospital, Ethiopia. Results: The proportion of HIV-/TB patients with an increased FeNO level (> 25 ppb) was significantly higher as compared to HIV+/TB patients, but HIV+/TB patients had significantly higher uNO than HIV-/TB patients. HIV+ and HIV-/TB patients both had lower levels of FeNO compared to blood donors and household contacts. The highest levels of both uNO and FeNO were found in household contacts. Less advanced findings on chest x-ray, as well as higher sedimentation rate were observed in HIV+/TB patients as compared to HIV-/TB patients. However, no significant correlation was found between FeNO and uNO, chest x-ray grading, clinical symptoms, TNF-alpha, IL-12, arginine levels or sedimentation rate. Conclusion: In both HIV negative and HIV co infected TB patients, low levels of exhaled NO compared to blood donors and household were observed. Future studies are needed to confirm whether low levels of exhaled NO could be a risk factor in acquiring TB and the relative importance of NO in human TB.
  •  
35.
  • Idh, Jonna, et al. (författare)
  • Reduced susceptibility of clinical strains of Mycobacterium tuberculosis to reactive nitrogen species promotes survival in activated macrophages
  • 2017
  • Ingår i: PLOS ONE. - : PLOS. - 1932-6203. ; 12:7
  • Tidskriftsartikel (refereegranskat)abstract
    • Background Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. Method Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA. Results When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO. Conclusion In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.
  •  
36.
  • Idh, Jonna, et al. (författare)
  • Resistance to First-Line Anti-TB Drugs is Associated with Reduced Nitric Oxide Susceptibility in Mycobacterium tuberculosis
  • 2012
  • Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 7:1, s. e39891-
  • Tidskriftsartikel (refereegranskat)abstract
    • Background and objective: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug resistance and clinical outcome is not known.Methods: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO.Results: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters, but a tendency towards lower rate of weight gain after two months of treatment.Conclusion: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions.
  •  
37.
  • Idh, Jonna, et al. (författare)
  • Susceptibility of Clinical Strains of Mycobacterium tuberculosis to Reactive Nitrogen Species in Activated Macrophages
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Background: Nitric oxide (NO) is produced in macrophages by the inducible NO synthase (iNOS) upon activation by pro-inflammatory cytokines. NO has been shown to be essential for the control of Mycobacterium tuberculosis infection in murine models whereas its importance in man is not as clear. There is a lack of studies regarding the susceptibility to reactive nitrogen species (RNS) in clinical strains of M. tuberculosis and the relation to first-line drug resistance, such as to isoniazid (INH). The aim of this study was to explore susceptibility to RNS and intracellular survival of clinical strains of M. tuberculosis, with or without INH resistance. Method: Seven clinical strains of M. tuberculosis were transformed with the pSMT1-plasmid encoding Vibrio harveyi luciferase. Survival was analysed by luminometry following exposure to the NO donor DETA/NO or peroxynitrite (SIN-1). Intracellular killing was studied in murine macrophages (RAW 264.7) activated with interferon gamma (IFN-γ) and lipopolysaccharide (LPS). Results: There was a significant effect on growth control of M. tuberculosis strains upon macrophage activation, which showed variability among clinical isolates. In the cell-free system, all strains showed a dose-dependent susceptibility to DETA/NO and SIN-1, and clinical strains were in general more resistant than H37Rv to DETA/NO. INH-resistant strains with an inhA mutation were significantly more tolerant to DETA/NO than inhA wild type. Conclusion: Reactive nitrogen species inhibited growth of clinical M. tuberculosis isolates both in an intra- and extracellular model with significant difference between strains. Increased tolerance to NO was associated with isoniazid resistance mediated by inhA.
  •  
38.
  • Idh, Jonna, 1979- (författare)
  • The Role of Nitric Oxide in Host Defence Against Mycobacterium tuberculosis : Clinical and Experimental Studies
  • 2012
  • 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.
  •  
39.
  • Janols, Helena, et al. (författare)
  • Early treatment response evaluated by a clinical scoring system correlates with the prognosis of pulmonary tuberculosis patients in Ethiopia : A prospective follow-up study.
  • 2012
  • Ingår i: Scandinavian journal of infectious diseases. - : Informa UK Limited. - 1651-1980 .- 0036-5548. ; 44:11, s. 828-834
  • Tidskriftsartikel (refereegranskat)abstract
    • Background: In resource-limited settings the monitoring of tuberculosis (TB) patients is challenging, and early identification of TB patients with a high mortality risk is important. The aim of this study was to investigate prospectively whether early changes in a clinical scoring system (TB score) can predict treatment outcome in Ethiopian patients with pulmonary tuberculosis. Method: TB patients (n = 250) and blood donors (n = 82) were recruited prospectively at Gondar University Hospital, Ethiopia. Clinical scoring was performed using an interview-based questionnaire and clinical examination. Results: Among TB patients (53.6% of whom were HIV co-infected) the median TB score declined from week 0 to week 2 (8 (interquartile range (IQR) 6-9) vs 4 (IQR 2-6)) and dropped to a low level at week 8, which was still significantly higher than that found in blood donors (2 (IQR 1-4) vs 0 (IQR 0-1), p < 0.0001). Patients who died had a significantly higher TB score at week 0, week 2, and week 8 than survivors. Mortality was associated with a failure to achieve a decrease greater than 25% in the TB score at 2 weeks. Baseline CD4 + cell counts (< 200 cells/mm(3)) were associated with mortality but not with initial TB score results. Conclusions: The TB score was increased during the first 2 months of treatment among patients who died. Failure to achieve a greater than 25% decrease in TB score after 2 weeks of treatment was associated with increased mortality. Repeated clinical scoring during the intensive phase of TB treatment could be useful to identify high-risk patients.
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40.
  • Jerström Skarman, Petra, et al. (författare)
  • Subcellular distribution of annexins, gelsolin and filamentous actin in adherent human neutrophils during phagocytosis
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Subcellular elevations of cytosolic free calcium concentration ([ea2+];), are critical for certain functional responses within the neutrophil, such asfilamentous actin (F-actin) reorganization and phagolysosome fusion (PLF). During this event, an accumulation of phospholipid- and calciumbinding proteins, annexins, can be seen in the periphagosomal area. A prerequisite for phagolysosome fusion is the elimination of F-actin around the phagosomes to facilitate the membrane contact between lysosomes and phagosomes. Gelsolin is a protein that severs F:actin by binding to the barbed ends, and thereby affect further polymerization. In this study, we used immunofluorescence staining and immunogold technique to analyse the distribution of annexin I, annexin III and gelsolin, in relation to the rearrangement ofF-actin during phagocytosos of complement-opsonized yeast particles by adherent human neutrophils. Iu unchallenged cells, both the aunexins and gelsolin were evenly distributed throughout the cells, whereas F-actin was found mostly in the protruding pseudopodia. Upon phagocytosis an accumulation of both. annexin I and annexin III, and gelsolin could be seen w1thm the vicimty of the phagocytic cups and phagosomes where they colocalized with Factin around the ingested particle.In calcium-depleted cells, the subcellular distributions of annexins and gelsolin were unaffected. On the other hand, there was a total increase inF-actin polymerization.Our data may indicate that gelsolin is important for the rearrangement of F-actin and that annexin I and annexin III, which are present in high concentrations in neutrophils, may participate in the following calciumdependent PLF in human neutrophils.
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41.
  • Jirström, Karin, et al. (författare)
  • Adverse effect of adjuvant tamoxifen in premenopausal breast cancer with cyclin D1 gene amplification
  • 2005
  • Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 65:17, s. 8009-8016
  • Tidskriftsartikel (refereegranskat)abstract
    • Cyclins D1 and A2 are cell cycle regulators that also have the ability to interact with the estrogen receptor (ER) and consequently interfere with antiestrogen treatment in breast cancer. Experimental data support this concept, but the clinical relevance needs to be further established. In this study, we evaluated cyclin D1 and A2 protein expression by immunohistochemistry and cyclin D1 gene (CCND1) amplification by fluorescence in situ hybridization in 500 primary breast cancers arranged in tissue microarrays. Patients had been randomized to 2 years of adjuvant tamoxifen or no treatment with a median follow-up of 14 years, allowing for subgroup analysis of treatment response defined by cyclin status. We found that both cyclin D1 and A2 protein overexpression was associated with an impaired tamoxifen response, although not significant in multivariate interaction analyses, whereas tamoxifen-treated patients with CCND1-amplified tumors had a substantially increased risk for disease recurrence after tamoxifen treatment in univariate analyses [relative risk (RR), 2.22; 95% confidence interval (95% CI), 0.94-5.26; P = 0.06] in contrast to nonamplified tumors (RR, 0.39; 95% CI, 0.23-0.65; P < 0.0001). Consequently, a highly significant interaction between tamoxifen treatment and CCND1 amplification could be shown regarding both recurrence-free survival (RR, 6.38; 95% CI, 2.29-17.78; P < 0.001) and overall survival (RR, 5.34; 95% CI, 1.84-15.51; P = 0.002), suggesting an agonistic effect of tamoxifen in ER-positive tumors. In node-positive patients, the disparate outcome according to gene amplification status was even more accentuated. In summary, our data implicate that despite a significant correlation to cyclin D1 protein expression, amplification status of the CCND1 gene seems a strong independent predictor of tamoxifen response, and possibly agonism, in premenopausal breast cancer.
  •  
42.
  • Larsson, Jenny, 1974-, et al. (författare)
  • Involvement of the ß2-integrin CD18 in apoptosis signal transduction in human neutrophils
  • 2000
  • Ingår i: Inflammation Research. - : Springer Science and Business Media LLC. - 1023-3830 .- 1420-908X. ; 49:9, s. 452-459
  • Tidskriftsartikel (refereegranskat)abstract
    • Objective and design: To examine the hypothesis that an accelerated rate of neutrophil apoptosis occurs following β2-integrin activation, and further investigate the signal transduction pathways involved.Material: Human polymorphonuclear neutrophils.Treatment: Neutrophils were challenged with pansorbins coated with antibodies towards the β2-integrin subunit CD18 in a proportion of 1:100 with or without the inhibitors diphenylene iodonium (10 M), cytochalasin B (5 μg/ml), genistein (10 nM), herbimycin A (10 M) and Z-VAD-FMK (10 μM).Methods: Measurement of phosphatidylserine exposure and DNA fragmentation in flow cytometry and assessment of H2O2-production through spectrofluorometry. The results were analysed using Mann Whitney U test and Kruskal Wallis.Results: Pansorbins coated with antibodies to CD18 induce apoptosis in neutrophils (p < 0.01), and activate the production of reactive oxygen species (p < 0.01). Pre-treatment with the inhibitors have no effect on anti-CD18 induced apoptosis.Conclusion: Anti-CD18 pansorbins induce apoptosis in neutrophils through an alternative pathway not involving reactive oxygen species and independent of tyrosine phosphorylation, cytoskeletal reorganisation and caspases.
  •  
43.
  • Lerm, Maria, et al. (författare)
  • Inactivation of Cdc42 is nessecary for depolymerization of phagosomal F-actin and subsequent phagosomal maturation
  • 2007
  • Ingår i: Journal of Immunology. - 0022-1767 .- 1550-6606. ; 178:11, s. 7357-7365
  • Tidskriftsartikel (refereegranskat)abstract
    • Phagocytosis is a complex process involving the activation of various signaling pathways, such as the Rho GTPases, and the subsequent reorganization of the actin cytoskeleton. In neutrophils, Rac and Cdc42 are activated during phagocytosis but less is known about the involvement of these GTPases during the different stages of the phagocytic process. The aim of this study was to elucidate the role of Cdc42 in phagocytosis and the subsequent phagosomal maturation. Using a TAT-based protein transduction technique, we introduced dominant negative and constitutively active forms of Cdc42 into neutrophil-like HL60 (human leukemia) cells that were allowed to phagocytose IgG-opsonized yeast particles. Staining of cellular F-actin in cells transduced with constitutively active Cdc42 revealed that the activation of Cdc42 induced sustained accumulation of periphagosomal actin. Moreover, the fusion of azurophilic granules with the phagosomal membrane was prevented by the accumulated F-actin. In contrast, introducing dominant negative Cdc42 impaired the translocation per se of azurophilic granules to the periphagosomal area. These results show that efficient phagosomal maturation and the subsequent eradication of ingested microbes in human neutrophils is dependent on a strictly regulated Cdc42. To induce granule translocation, Cdc42 must be in its active state but has to be inactivated to allow depolymerization of the F-actin cage around the phagosome, a process essential for phagolysosome formation.
  •  
44.
  • Lindmark, Maria, et al. (författare)
  • Complement- and IgG-mediated phagocytosis in human macrophages in calcium dependent and involves synoptotagmin IV
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Calcium regulates membrane fusion events during phagolysosome formation in neutrophil granulocytes and vesicle fusion at the neural synapse. Calcium is also required for uptake of IgG-opsonised particles by human neutrophils. The role of calcium during macrophage phagocytosis is less clear. Here we show that phagocytosis of IgG- or serum-opsonised prey is strictly calcium dependent in human monocyte-derived macrophages. We also show the presence and involvement of synaptotagmin II and IV in human macrophages and in the murine macrophage cell line J774. Synaptotagmin IV displayed a granular distribution in resting human macrophages with some translocation to the plasma membrane. Synaptotagrnin IV did not eo localise with the nucleus, the endoplasmic reticulum or the Golgi apparatus. During phagocytosis of IgG- or serum-opsonised prey we observed a distinct, transient translocation of synaptotagmin IV to the phagosome. The kinnetics of synaptotagmin IV translocation was similar to Rab5. LAMP-1, a marker of late endosomes and mature phagolysosomes fused with the phagosome at a later time point. Our results show that complement- and IgG-mediated phagocytosis are dependent on calcium in human macrophages and indicate a role for synaptotagmin IV in the calcium dependent fusion of the phagosome with components of the early endocytic pathway.
  •  
45.
  • Lindmark, Maria, et al. (författare)
  • Lipophosphoglycan (LPG) from Leishmania donovani inhibits phagosomal maturation and oxygen redical production in human neutrophils
  • Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
    • Lipophosphoglycan (LPG) is the major surface glycoconjugate on Leishmania donovani promastigotes. LPG inhibits phagosome maturation and is crucial for parasite survival in macrophages. Fusion of vesicles with the phagosome is essential for the formation of a mature phagolysosorne and depolymerization of periphagosomal F-actin is likely a prerequisite for vesicle fusion. In macrophages LPG induces an accumulation of periphagosomal F-actin which is correlated to inhibition of vesicle fusion to the phagosome. In this work we investigated the effects of LPG on phagosome maturation in human neutrophils. We found that ingestion of serum-opsonised, LPG-coated yeast particles induced increased levels of periphagosomal Factin in neutrophils. Phagosome maturation was studied using antibodies to CD63 (azurophil granules), synaptotagmin II (specific granules) and LAMP-1 (specific granules, secretory vesicles, multivesicular bodies/multilaminar compartments). Results showed impaired translocation of all these three markers to phagosomes containing LPG-coated prey. The translocation of the early endosome marker Rab5A to the phagosome was not affected by LPG. The late endosomal marker Rab7 was not found in human neutrophils. Chemiluminescence studies revealed that serum-opsonised, LPG-coated yeast induced less production of reactive oxygen metabolites (ROM) compared to controls and that the production was mainly intracellular.
  •  
46.
  • Lindmark, Maria, 1972-, et al. (författare)
  • Synaptotagmin II could confer Ca(2+) sensitivity to phagocytosis in human neutrophils.
  • 2002
  • Ingår i: Biochimica et biophysica acta. - 0006-3002. ; 1590:1-3, s. 159-166
  • Tidskriftsartikel (refereegranskat)abstract
    • Phagolysosome fusion and granule exocytosis in neutrophils are calcium-dependent processes. The calcium requirements vary between granule types, suggesting the presence of different calcium sensors. The synaptotagmins, a family of calcium-binding proteins, previously shown to participate in vesicle fusion and vesicle recycling in excitable cells, are putative calcium-sensors of exocytosis in excitable cells. In this study, we show that synaptotagmin II is present in human neutrophils and may participate in phagocytic and in exocytotic processes. In protein extracts from human neutrophils, we identified synaptotagmin II by Western blot as an 80 kDa protein. Subcellular fractionation revealed that synaptotagmin II was associated with the specific granules. In fMLP-stimulated cells, synaptotagmin II translocated to the plasma membrane. This correlated with the upregulation of complement receptor 3 (CR 3), reflecting the translocation of specific granules to the cell surface. Synaptotagmin II also translocated to the phagosome after complement-mediated phagocytosis in the presence of calcium. LAMP-1 translocated in parallel but probably was located to another subcellular compartment than synaptotagmin II. Under calcium-reduced conditions, neither synaptotagmin II nor LAMP-1 translocated to the phagosome. We therefore suggest a role for synaptotagmin II as calcium-sensor during phagocytosis and secretion in neutrophils.
  •  
47.
  • Löfgren, Ragnhild, et al. (författare)
  • CR3, FcγRIIA and FcγRIIIB induce activation of the respiratory burst in human neutrophils : the role of intracellular Ca2+, phospholipase D and tyrosine phosphorylation
  • 1999
  • Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - 0167-4889 .- 1879-2596. ; 1452:1, s. 46-59
  • Tidskriftsartikel (refereegranskat)abstract
    • Human neutrophils express two different types of phagocytic receptors, complement receptors (CR) and Fc receptors. In order to characterize the different signaling properties of each receptor we have used non-adherent human neutrophils and investigated CR3, FcγRIIA and FcγRIIIB for their signaling capacity. Selective activation of each receptor was achieved by coupling specific antibodies to heat-killed Staphylococcus aureus particles, Pansorbins, through their Fc moiety. Despite the fact that these particles are not phagocytosed, we show that addition of Pansorbins with anti-CD18 antibodies recognizing CR3 induced prominent signals leading to a respiratory burst. Stimulation with anti-FcγRIIIB Pansorbins induced about half of the response induced by anti-CR3 Pansorbins, whereas anti-FcγRIIA Pansorbins induced an even weaker signal. However, FcγRIIA induced strong phosphorylation of p72syk whereas FcγRIIIB induced only a very weak p72syk phosphorylation. During CR3 stimulation no tyrosine phosphorylation of p72syk was seen. Both phospholipase D and NADPH oxidase activities were dependent on intracellular calcium. This is in contrast to tyrosine phosphorylation of p72syk that occurred even in calcium-depleted cells, indicating that oxygen metabolism does not affect p72syk phosphorylation. Inhibitors of tyrosine phosphorylation blocked the respiratory burst induced by both FcγRIIA and FcγRIIIB as well as CR3. This shows that tyrosine phosphorylation of p72syk is an early signal in the cascade induced by FcγRIIA but not by CR3.
  •  
48.
  •  
49.
  • Majeed, Meytham, et al. (författare)
  • Roles of Ca2+ and F-actin in intracellular aggregation of Chlamydia trachomatis in eucaryotic cells
  • 1993
  • Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 61:4, s. 1406-1414
  • Tidskriftsartikel (refereegranskat)abstract
    • The effect of intracellular free Ca2+ ([Ca2+]i) on the intracellular aggregation of Chlamydia trachomatis serovars L2 and E in McCoy and HeLa cells is investigated. Loading the cells with the Ca2+ chelator MAPT/AM (1,2-bis-5-methyl-amino-phenoxylethane-N,N-n'-tetra-acetoxymethyl acetate), thereby decreasing the [Ca2+]i from 67 to 19 nM, decreased the number of cells with a local aggregation of chlamydiae in a dose-dependent manner. Neither the attachment nor the uptake of elementary bodies (EBs) was, however, affected after depletion of Ca2+ from the cells. There was no significant difference in the level of measured [Ca2+]i between infected and uninfected cells. Reducing the [Ca2+]i also significantly inhibited chlamydial inclusion formation. Differences in the organization of the actin filament network were observed in response to [Ca2+]i depletion. In Ca(2+)-depleted cells, where few EB aggregates were formed, few local accumulations of F-actin were observed in the cytosol. These results suggest that the aggregation of EBs in eucaryotic cells requires a normal homeostasis of intracellular Ca2+. By affecting F-actin reorganization and putatively certain Ca(2+)-binding proteins, [Ca2+]i plays a vital role in the infectious process of chlamydiae.
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50.
  • Majeed, Meytham, et al. (författare)
  • Roles of calcium and annexins in phagocytosis and elimination of an attenuated strain of Mycobacterium tuberculosisin human neutrophils
  • 1998
  • Ingår i: Microbial Pathogenesis. - : Elsevier BV. - 0882-4010 .- 1096-1208. ; 24:5, s. 309-320
  • Tidskriftsartikel (refereegranskat)abstract
    • The phagocytic function of neutrophils is a crucial element in the host defence against invading microorganisms. We investigated phagocytosis and intracellular killing of an attenuated strain of Mycobacterium tuberculosis(H37Ra) by human neutrophils focusing on the role of the cytosolic free calcium concentration [Ca2+]iand certain cytosolic calcium-dependent membrane-binding proteins annexins. Phagocytic uptake did not trigger a calcium rise and occurred independently of different calcium conditions, and in a serum-dependent manner. Changes in the viability of H37Ra were determined by agar plate colony count and a radiometric assay. Neutrophils showed a capacity to kill ingested mycobacteria and this occurred without a rise in [Ca2+]i. The ability to kill H37Ra decreased in the absence of extracellular calcium and when intra-extracellular calcium was reduced. Immunofluorescence staining revealed that during phagocytosis of H37Ra, annexins III, IV and VI translocated from cytoplasm to the proximity of the H37Ra-containing phagosomes, whereas the localization of annexin I and V remained unchanged. The translocation of annexin IV occurred even when Ca2+-depleted neutrophils ingested H37Ra in the absence of extracellular calcium. We concluded that neutrophil-mediated killing of mycobacteria is a Ca2+-dependent process. The fact that the association of certain annexins to the membrane vesicle containing H37Ra differ from other phagosomes suggests a selective regulatory mechanism during phagocytosis of mycobacteria by neutrophils.
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