| 1. |
- Bestas, Burcu, et al.
(författare)
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Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model
- 2014
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Ingår i: Journal of Clinical Investigation. - 0021-9738 .- 1558-8238. ; 124:9, s. 4067-4081
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Tidskriftsartikel (refereegranskat)abstract
- X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency that results from mutations within the gene encoding Bruton's tyrosine kinase (BTK). Many XLA-associated mutations affect splicing of BTK pre-mRNA and severely impair B cell development. Here, we assessed the potential of antisense, splice-correcting oligonucleotides (SCOs) targeting mutated BTKtranscripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we engineered a transgenic mouse that harbors a BAC with an authentic, mutated, splice-defective human BTK gene. BTK transgenic mice were bred onto a Btk knockout background to avoid interference of the orthologous mouse protein. Using this model, we determined that BTK-specific SCOs are able to correct aberrantly spliced BTK in B lymphocytes, including pro-B cells. Correction of BTK mRNA restored expression of functional protein, as shown both by enhanced lymphocyte survival and reestablished BTK activation upon B cell receptor stimulation. Furthermore, SCO treatment corrected splicing and restored BTK expression in primary cells from patients with XLA. Together, our data demonstrate that SCOs can restore BTK function and that BTK-targeting SCOs have potential as personalized medicine in patients with XLA.
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| 2. |
- Ali, Dina, et al.
(författare)
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Anti-leukaemic effects induced by APR-246 are dependent on induction of oxidative stress and the NFE2L2/HMOX1 axis that can be targeted by PI3K and mTOR inhibitors in acute myeloid leukaemia cells
- 2016
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Ingår i: British Journal of Haematology. - : Wiley. - 0007-1048 .- 1365-2141. ; 174:1, s. 117-126
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Tidskriftsartikel (refereegranskat)abstract
- The small molecule APR-246 (PRIMA-1(MET)) is a novel drug that restores the activity of mutated and unfolded TP53 protein. However, the mechanisms of action and potential off-target effects are not fully understood. Gene expression profiling in TP53 mutant KMB3 acute myeloid leukaemia (AML) cells showed that genes which protected cells from oxidative stress to be the most up-regulated. APR-246 exposure also induced reactive oxygen species (ROS) formation and depleted glutathione in AML cells. The genes most up-regulated by APR-246, confirmed by quantitative real time polymerase chain reaction, were heme oxygenase-1 (HMOX1, also termed HO-1), SLC7A11 and RIT1. Up-regulation of HMOX1, a key regulator of cellular response to ROS, was independent of TP53 mutational status. NFE2L2 (also termed Nrf2), a master regulator of HMOX1 expression, showed transcriptional up-regulation and nuclear translocation by APR-246. Down-regulation of NFE2L2 by siRNA in AML cells significantly increased the antitumoural effects of APR-246. The PI3K inhibitor wortmannin and the mTOR inhibitor rapamycin inhibited APR-246-induced nuclear translocation of NFE2L2 and counteracted the protective cellular responses to APR-246, resulting in synergistic cell killing together with APR-246. In conclusion, ROS induction is important for antileukaemic activities of APR-246 and inhibiting the protective response of the Nrf-2/HMOX1 axis using PI3K inhibitors, enhances the antileukaemic effects.
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| 3. |
- Amin, Risul, et al.
(författare)
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The kidney injury caused by the onset of acute graft-versus-host disease is associated with down-regulation of alpha Klotho
- 2020
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Ingår i: International Immunopharmacology. - : Elsevier BV. - 1567-5769 .- 1878-1705. ; 78
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Tidskriftsartikel (refereegranskat)abstract
- Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in alpha Klotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of alpha Klotho, a sight that may inspire novel therapeutic approaches.
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| 4. |
- Gupta, Dhanu, et al.
(författare)
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Amelioration of systemic inflammation via the display of two different decoy protein receptors on extracellular vesicles
- 2021
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Ingår i: Nature Biomedical Engineering. - Stockholm : Karolinska Institutet, Dept of Laboratory Medicine. - 2157-846X.
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular vesicles (EVs) can be functionalized to display specific protein receptors on their surface. However, surface-display technology typically labels only a small fraction of the EV population. Here, we show that the joint display of two different therapeutically relevant protein receptors on EVs can be optimized by systematically screening EV-loading protein moieties. We used cytokine-binding domains derived from tumour necrosis factor receptor 1 (TNFR1) and interleukin-6 signal transducer (IL-6ST), which can act as decoy receptors for the pro-inflammatory cytokines tumour necrosis factor alpha (TNF-α) and IL-6, respectively. We found that the genetic engineering of EV-producing cells to express oligomerized exosomal sorting domains and the N-terminal fragment of syntenin (a cytosolic adaptor of the single transmembrane domain protein syndecan) increased the display efficiency and inhibitory activity of TNFR1 and IL-6ST and facilitated their joint display on EVs. In mouse models of systemic inflammation, neuroinflammation and intestinal inflammation, EVs displaying the cytokine decoys ameliorated the disease phenotypes with higher efficacy as compared with clinically approved biopharmaceutical agents targeting the TNF-α and IL-6 pathways.
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| 5. |
- Mohammad, Dara K., et al.
(författare)
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B Cell Receptor Activation Predominantly Regulates AKT-mTORC1/2 Substrates Functionally Related to RNA Processing
- 2016
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 11:8
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Tidskriftsartikel (refereegranskat)abstract
- Protein kinase B (AKT) phosphorylates numerous substrates on the consensus motif RXRXXpS/T, a docking site for 14-3-3 interactions. To identify novel AKT-induced phosphorylation events following B cell receptor (BCR) activation, we performed proteomics, biochemical and bioinformatics analyses. Phosphorylated consensus motif-specific antibody enrichment, followed by tandem mass spectrometry, identified 446 proteins, containing 186 novel phosphorylation events. Moreover, we found 85 proteins with up regulated phosphorylation, while in 277 it was down regulated following stimulation. Up regulation was mainly in proteins involved in ribosomal and translational regulation, DNA binding and transcription regulation. Conversely, down regulation was preferentially in RNA binding, mRNA splicing and mRNP export proteins. Immunoblotting of two identified RNA regulatory proteins, RBM25 and MEF-2D, confirmed the proteomics data. Consistent with these findings, the AKT-inhibitor (MK-2206) dramatically reduced, while the mTORC-inhibitor PP242 totally blocked phosphorylation on the RXRXXpS/T motif. This demonstrates that this motif, previously suggested as an AKT target sequence, also is a substrate for mTORC1/2. Proteins with PDZ, PH and/or SH3 domains contained the consensus motif, whereas in those with an HMG-box, H15 domains and/or NF-X1-zinc-fingers, the motif was absent. Proteins carrying the consensus motif were found in all eukaryotic clades indicating that they regulate a phylogenetically conserved set of proteins.
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