1. |
- Arvidsson, Ida, et al.
(författare)
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Apyrase decreases phage induction and Shiga toxin release from E. coli O157:H7 and has a protective effect during infection
- 2022
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Ingår i: Gut microbes. - : Informa UK Limited. - 1949-0976 .- 1949-0984. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) cause gastrointestinal infection and, in severe cases, hemolytic uremic syndrome which may lead to death. There is, to-date, no therapy for this infection. Stx induces ATP release from host cells and ATP signaling mediates its cytotoxic effects. Apyrase cleaves and neutralizes ATP and its effect on Stx and EHEC infection was therefore investigated. Apyrase decreased bacterial RecA and dose-dependently decreased toxin release from E. coli O157:H7 in vitro, demonstrated by reduced phage DNA and protein levels. The effect was investigated in a mouse model of E. coli O157:H7 infection. BALB/c mice infected with Stx2-producing E. coli O157:H7 were treated with apyrase intraperitoneally, on days 0 and 2 post-infection, and monitored for 11 days. Apyrase-treated mice developed disease two days later than untreated mice. Untreated infected mice lost significantly more weight than those treated with apyrase. Apyrase-treated mice exhibited less colonic goblet cell depletion and apoptotic cells, as well as lower fecal ATP and Stx2, compared to untreated mice. Apyrase also decreased platelet aggregation induced by co-incubation of human platelet-rich-plasma with Stx2 and E. coli O157 lipopolysaccharide in the presence of collagen. Thus, apyrase had multiple protective effects, reducing RecA levels, stx2 and toxin release from EHEC, reducing fecal Stx2 and protecting mouse intestinal cells, as well as decreasing platelet activation, and could thereby delay the development of disease.
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2. |
- Friberg Hed, Niklas, et al.
(författare)
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Arginase release in hemolytic uremic syndrome affects the vasculature
- 2023
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Hemolysis is a cardinal feature of hemolytic uremic syndrome (HUS) and during hemolysis excess arginase 1 is released from erythrocytes. Increased arginase activity leads to reduced arginine, as it is converted to urea and ornithine, and thereby reduced nitric oxide bioavailability, with secondary vascular injury. In this study we investigated arginase release in HUS patients and laboratory models. Two separate cohorts of patients (n=47 in total) with HUS associated with Shiga toxin-producing enterohemorrhagic E. coli (EHEC) and pediatric controls (n=35) were investigated. HUS patients had excessively high arginase 1 levels and activity (conversion to urea and ornithine) in plasma/serum during the acute phase, compared to remission and controls. Arginase 1 levels correlated with lactate dehydrogenase activity, indicating hemolysis, as well as the need for dialysis treatment. Patients also exhibited high levels of plasma alpha-1-microglobulin, a heme scavenger. Two mouse models were used, mice were challenged intragastrically with E. coli O157:H7 or injected intraperitoneally with Shiga toxin 2. Both models exhibited significantly elevated plasma arginase 1 levels and activity. Arginase 1 levels correlated with lactate dehydrogenase activity and alpha-1-microglobulin in the plasma of EHEC-infected mice. In vitro perfusion of Shiga toxin 2- and E. coli O157-lipopolysaccharide-stimulated human blood cells combined with ADAMTS13-deficient plasma over glomerular endothelial cells induced hemolysis and the release of bioactive arginase 1. The high levels of arginase released during HUS could thereby contribute to microvascular injury during HUS.
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3. |
- Friberg, Niklas, et al.
(författare)
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Red blood cell-derived arginase release in hemolytic uremic syndrome
- 2024
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Ingår i: Journal of Translational Medicine. - 1479-5876. ; 22:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundHemolysis is a cardinal feature of hemolytic uremic syndrome (HUS) and during hemolysis excess arginase 1 is released from red blood cells. Increased arginase activity leads to reduced L-arginine, as it is converted to urea and L-ornithine, and thereby reduced nitric oxide bioavailability, with secondary vascular injury. The objective of this study was to investigate arginase release in HUS patients and laboratory models and correlate arginase levels to hemolysis and kidney injury.MethodsTwo separate cohorts of patients (n = 47 in total) with HUS associated with Shiga toxin-producing enterohemorrhagic E. coli (EHEC) and pediatric controls (n = 35) were investigated. Two mouse models were used, in which mice were either challenged intragastrically with E. coli O157:H7 or injected intraperitoneally with Shiga toxin 2. An in vitro model of thrombotic microangiopathy was developed in which Shiga toxin 2- and E. coli O157 lipopolysaccharide-stimulated human blood cells combined with ADAMTS13-deficient plasma were perfused over glomerular endothelial cells. Two group statistical comparisons were performed using the Mann–Whitney test, multiple groups were compared using the Kruskal–Wallis test followed by Dunn’s procedure, the Wilcoxon signed rank test was used for paired data, or linear regression for continuous variables.ResultsHUS patients had excessively high plasma arginase 1 levels and activity (conversion of L-arginine to urea and L-ornithine) during the acute phase, compared to remission and controls. Arginase 1 levels correlated with lactate dehydrogenase activity, indicating hemolysis, as well as the need for dialysis treatment. Patients also exhibited high levels of plasma alpha-1-microglobulin, a heme scavenger. Both mouse models exhibited significantly elevated plasma arginase 1 levels and activity. Plasma arginase 1 levels correlated with lactate dehydrogenase activity, alpha-1-microglobulin and urea levels, the latter indicative of kidney dysfunction. In the in vitro model of thrombotic microangiopathy, bioactive arginase 1 was released and levels correlated to the degree of hemolysis.ConclusionsElevated red blood cell-derived arginase was demonstrated in HUS patients and in relevant in vivo and in vitro models. The excessively high arginase levels correlated to the degree of hemolysis and kidney dysfunction. Thus, arginase inhibition should be investigated in HUS.
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4. |
- Jimenez, Itzia, et al.
(författare)
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Allelic difference in Mhc2ta confers altered microglial activation and susceptibility to α-synuclein-induced dopaminergic neurodegeneration
- 2017
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Ingår i: Neurobiology of Disease. - : Elsevier BV. - 0969-9961. ; 106, s. 279-290
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Tidskriftsartikel (refereegranskat)abstract
- Parkinson's Disease (PD) is a complex and heterogeneous neurodegenerative disease characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta and pathological intracellular accumulation of alpha-synuclein (α-syn). In the vast majority of PD patients, the disease has a complex etiology, defined by multiple genetic and environmental risk factors. Common genetic variants in the human leukocyte-antigen (HLA) region have been associated to PD risk and the carriage of these can double the risk to develop PD. Among these common genetic variants are the ones that modulate the expression of MHCII genes. MHCII molecules encoded in the HLA-region are responsible for antigen presentation to the adaptive immune system and have a key role in inflammatory processes. In addition to cis‑variants affecting MHCII expression, a transactivator encoded by the Mhc2ta gene is the major regulator of MHCII expression. We have previously identified variations in the promoter region of Mhc2ta, encoded in the VRA4 region, to regulate MHCII expression in rats. The expression of MHCII is known to be required in the response to α-syn. However, how the expression of MHCII affects the activation of microglial or the impact of physiological, differential Mhc2ta expression on degeneration of dopaminergic neurons has not previously been addressed. Here we addressed the implications of common genetic allelic variants of the major regulator of MHCII expression on α-syn-induced microglia activation and the severity of the dopaminergic neurodegeneration. We used a viral vector technology to overexpress α-syn in two rat strains; Dark agouti (DA) wild type and DA.VRA4-congenic rats. The congenic strain carries PVG alleles in the VRA4 locus and therefore displays lower Mhc2ta expression levels compared to DA rats. We analyzed the impact of this physiological differential Mhc2ta expression on gliosis, inflammation, degeneration of the nigro-striatal dopamine system and behavioral deficits after α-syn overexpression. We report that allelic variants of Mhc2ta differently modified the microglial activation in response to overexpression of human α-syn in rats. Overexpression of α-syn led to a larger denervation of the nigro-striatal system and significant behavioral deficits in DA.VRA4 congenic rats with lower Mhc2ta expression compared to DA rats. These results indicate that Mhc2ta is a key upstream regulator of the inflammatory response in PD pathology.
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5. |
- Johansson, Karl E., et al.
(författare)
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Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism
- 2019
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 9:1
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Tidskriftsartikel (refereegranskat)abstract
- Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.
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6. |
- Johansson, Karl, et al.
(författare)
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Shiga Toxin-Bearing Microvesicles Exert a Cytotoxic Effect on Recipient Cells Only When the Cells Express the Toxin Receptor
- 2020
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Ingår i: Frontiers in cellular and infection microbiology. - : Frontiers Media SA. - 2235-2988. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic Escherichia coli strains capable of causing hemolytic uremic syndrome. Our group has previously shown that the toxin can reach the kidney within microvesicles where it is taken up by renal cells and the vesicles release their cargo intracellularly, leading to toxin-mediated inhibition of protein synthesis and cell death. The aim of this study was to examine if recipient cells must express the globotriaosylceramide (Gb3) toxin receptor for this to occur, or if Gb3-negative cells are also susceptible after uptake of Gb3-positive and toxin-positive microvesicles. To this end we generated Gb3-positive A4GALT–transfected CHO cells, and a vector control lacking Gb3 (CHO-control cells), and decreased Gb3 synthesis in native HeLa cells by exposing them to the glycosylceramide synthase inhibitor PPMP. We used these cells, and human intestinal DLD-1 cells lacking Gb3, and exposed them to Shiga toxin 2-bearing Gb3-positive microvesicles derived from human blood cells. Results showed that only recipient cells that possessed endogenous Gb3 (CHO-Gb3 transfected and native HeLa cells) exhibited cellular injury, reduced cell metabolism and protein synthesis, after uptake of toxin-positive microvesicles. In Gb3-positive cells the toxin introduced via vesicles followed the retrograde pathway and was inhibited by the retrograde transport blocker Retro-2.1. CHO-control cells, HeLa cells treated with PPMP and DLD-1 cells remained unaffected by toxin-positive microvesicles. We conclude that Shiga toxin-containing microvesicles can be taken up by Gb3-negative cells but the recipient cell must express endogenous Gb3 for the cell to be susceptible to the toxin.
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7. |
- Karpman, Diana, et al.
(författare)
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Extracellular vesicles in renal inflammatory and infectious diseases
- 2021
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Ingår i: Free Radical Biology and Medicine. - : Elsevier BV. - 0891-5849. ; 171, s. 42-54
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Forskningsöversikt (refereegranskat)abstract
- Extracellular vesicles can mediate cell-to-cell communication, or relieve the parent cell of harmful substances, in order to maintain cellular integrity. The content of extracellular vesicles includes miRNAs, mRNAs, growth factors, complement factors, cytokines, chemokines and receptors. These may contribute to inflammatory and infectious diseases by the exposure or transfer of potent effectors that induce vascular inflammation by leukocyte recruitment and thrombosis. Furthermore, vesicles release cytokines and induce their release from cells. Extracellular vesicles possess immune modulatory and anti-microbial properties, and induce receptor signaling in the recipient cell, not least by the transfer of pro-inflammatory receptors. Additionally, the vesicles may carry virulence factors systemically. Extracellular vesicles in blood and urine can contribute to the development of kidney diseases or exhibit protective effects. In this review we will describe the role of EVs in inflammation, thrombosis, immune modulation, angiogenesis, oxidative stress, renal tubular regeneration and infection. Furthermore, we will delineate their contribution to renal ischemia/reperfusion, vasculitis, glomerulonephritis, lupus nephritis, thrombotic microangiopathies, IgA nephropathy, acute kidney injury, urinary tract infections and renal transplantation. Due to their content of miRNAs and growth factors, or when loaded with nephroprotective modulators, extracellular vesicles have the potential to be used as therapeutics for renal regeneration.
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8. |
- Tontanahal, Ashmita, et al.
(författare)
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Annexin induces cellular uptake of extracellular vesicles and delays disease in Escherichia coli O157:H7 infection
- 2021
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Ingår i: Microorganisms. - : MDPI AG. - 2076-2607. ; 9:6
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Tidskriftsartikel (refereegranskat)abstract
- Enterohemorrhagic Escherichia coli secrete Shiga toxin and lead to hemolytic uremic syn-drome. Patients have high levels of circulating prothrombotic extracellular vesicles (EVs) that expose phosphatidylserine and tissue factor and transfer Shiga toxin from the circulation into the kidney. Annexin A5 (AnxA5) binds to phosphatidylserine, affecting membrane dynamics. This study investi-gated the effect of anxA5 on EV uptake by human and murine phagocytes and used a mouse model of EHEC infection to study the effect of anxA5 on disease and systemic EV levels. EVs derived from human whole blood or HeLa cells were more readily taken up by THP-1 cells or RAW264.7 cells when the EVs were coated with anxA5. EVs from HeLa cells incubated with RAW264.7 cells induced phosphatidylserine exposure on the cells, suggesting a mechanism by which anxA5-coated EVs can bind to phagocytes before uptake. Mice treated with anxA5 for six days after inoculation with E. coli O157:H7 showed a dose-dependent delay in the development of clinical disease. Treated mice had lower levels of EVs in the circulation. In the presence of anxA5, EVs are taken up by phagocytes and their systemic levels are lower, and, as EVs transfer Shiga toxin to the kidney, this could postpone disease development.
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9. |
- Tontanahal, Ashmita, et al.
(författare)
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IgG Binds Escherichia coli Serine Protease EspP and Protects Mice From E. coli O157:H7 Infection
- 2022
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Ingår i: Frontiers in Immunology. - : Frontiers Media SA. - 1664-3224. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Shiga toxin-producing Escherichia coli O157:H7 is a virulent strain causing severe gastrointestinal infection, hemolytic uremic syndrome and death. To date there are no specific therapies to reduce progression of disease. Here we investigated the effect of pooled immunoglobulins (IgG) on the course of disease in a mouse model of intragastric E. coli O157:H7 inoculation. Intraperitoneal administration of murine IgG on day 3, or both on day 3 and 6, post-inoculation improved survival and decreased intestinal and renal pathology. When given on both day 3 and 6 post-inoculation IgG treatment also improved kidney function in infected mice. Murine and human commercially available IgG preparations bound to proteins in culture filtrates from E. coli O157:H7. Bound proteins were extracted from membranes and peptide sequences were identified by mass spectrometry. The findings showed that murine and human IgG bound to E. coli extracellular serine protease P (EspP) in the culture filtrate, via the IgG Fc domain. These results were confirmed using purified recombinant EspP and comparing culture filtrates from the wild-type E. coli O157:H7 strain to a deletion mutant lacking espP. Culture filtrates from wild-type E. coli O157:H7 exhibited enzymatic activity, specifically associated with the presence of EspP and demonstrated as pepsin cleavage, which was reduced in the presence of murine and human IgG. EspP is a virulence factor previously shown to promote colonic cell injury and the uptake of Shiga toxin by intestinal cells. The results presented here suggest that IgG binds to EspP, blocks its enzymatic activity, and protects the host from E. coli O157:H7 infection, even when given post-inoculation.
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10. |
- Tontanahal, Ashmita
(författare)
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Novel in vivo therapeutic approaches to Escherichia coli O157:H7 infection
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Shiga toxin (Stx), the unique virulence factor released by enterohemorrhagic Escherichia coli (EHEC), associated with gastrointestinal infection and in severe cases hemolytic uremic syndrome (HUS). Up until now, no effective therapies have been developed to control disease progression. In this thesis, four novel treatment strategies have been investigated targeting different aspects of EHEC pathogenesis. An established mouse model was used in which mice were infected with EHEC intragastrically. During EHEC infection, extracellular vesicles (EVs) are involved in the transport of Stx from the gut to the kidney. High levels of prothrombotic EVs that expose phosphatidylserine and tissue factor have been detected in patients with EHEC-associated HUS.In the first study, treatment with annexin A5 (anxA5) induced an increase in phagocytic uptake of EVs in vitro. Administration of anxA5 to EHEC-infected mice resulted in lower levels of circulating platelet-derived EVs and delayed disease development.In the second study, Stx triggered adenosine triphosphate (ATP) release from HeLa cells and similar results were obtained in a mouse model. ATP signals via purinergic receptors. Inhibition of purinergic P2X receptors with NF449 or suramin inhibited Stx-induced calcium influx and EV release. NF449 protected cells from Stx-induced toxicity and suramin decreased EV release even in vivo in mice.The third study targeted extracellular ATP by using apyrase in EHEC-infected mice. Apyrase cleaves extracellular ATP and adenosine diphosphate (ADP). Mice were injected with apyrase intraperitoneally. Treatment protected the mouse intestines from damage and delayed the onset of disease symptoms. Apyrase decreased bacterial release of Stx2 by reducing RecA involved in the SOS response and bacteriophage activation. In addition, apyrase lowered platelet aggregation when platelets were co-incubated with Stx2 and E. coli O157:H7 lipopolysaccharide in the presence of collagen. Thus, apyrase had a dual protective effect on both the bacterial release of toxin and host cell activation and injury.The fourth study focused on the effect of immunoglobulin G (IgG) on EHEC infection. Intraperitoneal administration of murine IgG to EHEC-infected mice had a clear beneficial effect on bacterial colonization, survival and intestinal and renal pathology. In vitro studies utilized both mouse and human IgG and showed that the Fc domain bound to the EHEC virulence factor, EspP, E. coli secreted serine protease. EspP is involved in bacterial adherence to the intestine, and intestinal injury during EHEC infection. It has potent enzymatic activity that was inhibited by the interaction with IgG. This indicates that the protective effects of IgG administration in EHEC-infected mice could be due to the interaction between IgG and EspP.In summary, this thesis investigated novel treatment strategies targeting different aspects of EHEC pathogenesis such as bacterial colonization, release of Stx from EHEC, phagocytic clearance of Stx-containing and prothrombotic EVs in the circulation and protection against the cytotoxic and prothrombotic effects of Stx using both in vitro and in vivo experimental set-ups. The results show that treatments such as anxA5, apyrase and IgG, when administered at an early stage of infection, may have therapeutic potential in EHEC infection.
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