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- Karimi, Shokoufeh
(författare)
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Exploring probiotics-host interactions : intestinal immune and defence responses to Lactobacillus reuteri in health and disease
- 2017
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Lactobacillus reuteri, a ubiquitous inhabitant of the mammalian gastrointestinal (GI) tract has known health-promoting effects and various strains are commercially available as probiotics. Several probiosis mechanisms have been suggested in L. reuteri’s mode of action, but the mediators and factors involved are not well understood. This thesis examined the function of probiotics, particularly L. reuteri, in the GI tract by equipping L. reuteri ATCC PTA 6475 and R2LC with reporter genes (luminescence and fluorescence) and a mutant of strain 6475 was generated by inactivation of chaperon dnaK. Different in vitro and in vivo applications of fluorescent and luminescent strains were evaluated, and it was demonstrated that flow cytometry can be a powerful method for determination of plasmid persistence. Biophotonic imaging (BPI) enabled low doses (~1x10^5) of luminescent bacteria to be monitored in the GI tract and revealed retention of large numbers of bacteria in the stomach up to 3 hours post-gavage. The effect of four strains of L. reuteri (6475, R2LC, DSM 17938, 1563F) was examined in an epithelial infection model using IPEC-J2 cells induced by enterotoxigenic E. coli. By analysing transepithelial electrical resistance (TEER) and leakage of FITC-dextran, it was shown that L. reuteri pre-treatment prevented damage by ETEC to epithelial monolayer integrity. The strains also reduced expression of pro-inflammatory cytokines (TNF-alfa and IL-6) and maintained expression of adherens junction(E-cadherin) and upregulated tight junction (ZO-1) proteins. To further explore the L. reuteri mode of action, five mutants were evaluated in DSS-induced acute colitis and IPEC-J2 models. It was found that dnaK-, pduC-, cmbA-, amidase- and srtA- may not play major roles in the mechanisms by which 6475 maintains mucosal integrity and counteracts inflammation. However, mutants 6475 pduC- and 6475 cmbA- had a tendency to weaken the protective effect of 6475 in the colitis model. Studies on the effects of L. reuteri strains on mast cell activation and inflammatory response revealed no inhibition of degranulation mediated by IgE-antigen activation, but down-regulated expression of the pro-inflammatory cytokines IL-6 and IL-13, irrespective of degranulation. Thus pre-treatment with L. reuteri strains can protect against intestinal barrier dysfunction and mucosal inflammation, partly through altering junctional complex proteins, mediators of immunity and mast cells.
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| 2. |
- Karimi, Shokoufeh, et al.
(författare)
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In Vivo and In Vitro Detection of Luminescent and Fluorescent Lactobacillus reuteri and Application of Red Fluorescent mCherry for Assessing Plasmid Persistence
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:3
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Tidskriftsartikel (refereegranskat)abstract
- Lactobacillus reuteri is a symbiont that inhabits the gastrointestinal (GI) tract of mammals, and several strains are used as probiotics. After introduction of probiotic strains in a complex ecosystem like the GI tract, keeping track of them is a challenge. The main objectives of this study were to introduce reporter proteins that would enable in vivo and in vitro detection of L. reuteri and increase knowledge about its interactions with the host. We describe for the first time cloning of codon-optimized reporter genes encoding click beetle red luciferase (CBRluc) and red fluorescent protein mCherry in L. reuteri strains ATCC PTA 6475 and R2LC. The plasmid persistence of mCherry-expressing lactobacilli was evaluated by both flow cytometry (FCM) and conventional plate count (PC), and the plasmid loss rates measured by FCM were lower overall than those determined by PC. Neutralization of pH and longer induction duration significantly improved the mCherry signal. The persistency, dose-dependent signal intensity and localization of the recombinant bacteria in the GI tract of mice were studied with an in vivo imaging system (IVIS), which allowed us to detect fluorescence from 6475-CBRluc-mCherry given at a dose of 1x10(10) CFU and luminescence signals at doses ranging from 1x10(5) to 1x10(10) CFU. Both 6475-CBRluc-mCherry and R2LC-CBRluc were localized in the colon 1 and 2 h after ingestion, but the majority of the latter were still found in the stomach, possibly reflecting niche specificity for R2LC. Finally, an in vitro experiment showed that mCherry-producing R2LC adhered efficiently to the intra cellular junctions of cultured IPEC-J2 cells. In conclusion, the two reporter genes CBRluc and mCherry were shown to be suitable markers for biophotonic imaging (BPI) of L. reuteri and may provide useful tools for future studies of in vivo and in vitro interactions between the bacteria and the host.
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| 4. |
- Simonds, Erin F., et al.
(författare)
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Deep immune profiling reveals targetable mechanisms of immune evasion in immune checkpoint inhibitor-refractory glioblastoma
- 2021
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Ingår i: Journal for ImmunoTherapy of Cancer. - : BMJ. - 2051-1426. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- Background Glioblastoma (GBM) is refractory to immune checkpoint inhibitor (ICI) therapy. We sought to determine to what extent this immune evasion is due to intrinsic properties of the tumor cells versus the specialized immune context of the brain, and if it can be reversed.Methods We used CyTOF mass cytometry to compare the tumor immune microenvironments (TIME) of human tumors that are generally ICI-refractory (GBM and sarcoma) or ICI-responsive (renal cell carcinoma), as well as mouse models of GBM that are ICI-responsive (GL261) or ICI-refractory (SB28). We further compared SB28 tumors grown intracerebrally versus subcutaneously to determine how tumor site affects TIME and responsiveness to dual CTLA-4/PD-1 blockade. Informed by these data, we explored rational immunotherapeutic combinations.Results ICI-sensitivity in human and mouse tumors was associated with increased T cells and dendritic cells (DCs), and fewer myeloid cells, in particular PD-L1+ tumor-associated macrophages. The SB28 mouse model of GBM responded to ICI when grown subcutaneously but not intracerebrally, providing a system to explore mechanisms underlying ICI resistance in GBM. The response to ICI in the subcutaneous SB28 model required CD4 T cells and NK cells, but not CD8 T cells. Recombinant FLT3L expanded DCs, improved antigen-specific T cell priming, and prolonged survival of mice with intracerebral SB28 tumors, but at the cost of increased Tregs. Targeting PD-L1 also prolonged survival, especially when combined with stereotactic radiation.Conclusions Our data suggest that a major obstacle for effective immunotherapy of GBM is poor antigen presentation in the brain, rather than intrinsic immunosuppressive properties of GBM tumor cells. Deep immune profiling identified DCs and PD-L1+ tumor-associated macrophages as promising targetable cell populations, which was confirmed using therapeutic interventions in vivo.
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