Publications by authors named "Diana Karpman"

83 Publications

Annexin Induces Cellular Uptake of Extracellular Vesicles and Delays Disease in O157:H7 Infection.

Microorganisms 2021 May 26;9(6). Epub 2021 May 26.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 221 85 Lund, Sweden.

Enterohemorrhagic secrete Shiga toxin and lead to hemolytic uremic syndrome. 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 investigated 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 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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http://dx.doi.org/10.3390/microorganisms9061143DOI Listing
May 2021

Extracellular vesicles in renal inflammatory and infectious diseases.

Free Radic Biol Med 2021 Aug 29;171:42-54. Epub 2021 Apr 29.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185, Lund, Sweden.

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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http://dx.doi.org/10.1016/j.freeradbiomed.2021.04.032DOI Listing
August 2021

Isolation and Characterization of Shiga Toxin-Associated Microvesicles.

Methods Mol Biol 2021 ;2291:207-228

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

Microvesicles are shed from cell surfaces during infectious or inflammatory conditions and may contribute to the pathogenesis of disease. During Shiga toxin-producing Escherichia coli (STEC) infection, microvesicles are released from blood cells. These microvesicles play a part in inflammation, thrombosis, hemolysis, and the transfer of the main virulence factor of STEC strains, Shiga toxin, to target organ cells. This chapter describes how to isolate blood cell- and cell culture-derived microvesicles from plasma or cell culture medium, respectively, and how to characterize these microvesicles by various methods, with special focus on Shiga toxin-associated microvesicles.
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http://dx.doi.org/10.1007/978-1-0716-1339-9_9DOI Listing
April 2021

Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit.

Toxins (Basel) 2020 07 10;12(7). Epub 2020 Jul 10.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185 Lund, Sweden.

Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.
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http://dx.doi.org/10.3390/toxins12070449DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7404996PMC
July 2020

Shiga Toxin-Bearing Microvesicles Exert a Cytotoxic Effect on Recipient Cells Only When the Cells Express the Toxin Receptor.

Front Cell Infect Microbiol 2020 25;10:212. Epub 2020 May 25.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

Shiga toxin is the main virulence factor of non-invasive enterohemorrhagic 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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http://dx.doi.org/10.3389/fcimb.2020.00212DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261856PMC
June 2021

Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism.

Sci Rep 2019 10 7;9(1):14362. Epub 2019 Oct 7.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

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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http://dx.doi.org/10.1038/s41598-019-50692-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6779916PMC
October 2019

Blockade of the kallikrein-kinin system reduces endothelial complement activation in vascular inflammation.

EBioMedicine 2019 Sep 20;47:319-328. Epub 2019 Aug 20.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden. Electronic address:

Background: The complement and kallikrein-kinin systems (KKS) are activated during vascular inflammation. The aim of this study was to investigate if blockade of the KKS can affect complement activation on the endothelium during inflammation.

Methods: Complement deposition on endothelial microvesicles was assayed in vasculitis patient plasma samples and controls. Plasma was perfused over glomerular endothelial cells and complement deposition assayed by flow cytometry. The effect of the kinin system was assessed using kinin receptor antagonists and C1-inhibitor. The in vivo effect was assessed in kidney sections from mice with nephrotoxic serum-induced glomerulonephritis treated with a kinin receptor antagonist.

Findings: Vasculitis patient plasma had significantly more C3- and C9-positive endothelial microvesicles than controls. Perfusion of patient acute-phase plasma samples over glomerular endothelial cells induced the release of significantly more complement-positive microvesicles, in comparison to remission or control plasma. Complement activation on endothelial microvesicles was reduced by kinin B1- and B2-receptor antagonists or by C1-inhibitor (the main inhibitor of the classical pathway and the KKS). Likewise, perfusion of glomerular endothelial cells with C1-inhibitor-depleted plasma induced the release of complement-positive microvesicles, which was significantly reduced by kinin-receptor antagonists or C1-inhibitor. Mice with nephrotoxic serum-induced glomerulonephritis exhibited significantly reduced glomerular C3 deposition when treated with a B1-receptor antagonist.

Interpretation: Excessive complement deposition on the endothelium will promote endothelial injury and the release of endothelial microvesicles. This study demonstrates that blockade of the KKS can reduce complement activation and thereby the inflammatory response on the endothelium.

Funding: Full details are provided in the Acknowledgements/Funding section.
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http://dx.doi.org/10.1016/j.ebiom.2019.08.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796560PMC
September 2019

[A case of quetiapine-induced congenital thrombotic thrombocytopenic purpura, atypical phenotype diagnosed in adulthood].

Lakartidningen 2019 Feb 12;116. Epub 2019 Feb 12.

Institution för kliniska vetenskaper, Lunds Universitet, Avdelning för pediatrik Lund, Sweden Institution för kliniska vetenskaper, Lunds Universitet, Avdelning för pediatrik Lund, Sweden.

Congenital thrombocytopenic purpura (TTP) is a rare but serious condition. We present a case of a 29-year-old woman, diagnosed with this disease in adulthood. The episode that led to diagnosis was triggered by quetiapine. She presented with neurological symptoms and laboratory findings including low platelets and elevated creatinine. Interestingly, the signs of hemolysis were very subtle. Her symptoms were relieved by withdrawal of the medicine. The diagnosis was confirmed by very low ADAMTS13 activity, lack of antibodies against ADAMTS13 and the presence of a compound heterozygous ADAMTS13 mutation. Despite prophylactic plasma infusions, the patient developed a second episode of microangiopathy, leading to an extensive cerebral infarction. It is possible that even the latter episode was triggered by drugs. We suggest that the diagnosis of TTP should be considered in patients with neurological symptoms and unexplained thrombocytopenia.
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February 2019

Clinical and Complement Long-Term Follow-Up of a Pediatric Patient with C3 Mutation-Related Atypical Hemolytic Uremic Syndrome.

Case Rep Nephrol 2018 18;2018:3810249. Epub 2018 Dec 18.

Research Laboratory, Nordland Hospital, Bodø, Norway.

We report a pediatric patient with atypical hemolytic uremic syndrome due to a C3 gain-of-function mutation diagnosed in infancy. She was treated from the start with a constant dose of 300 mg eculizumab every second week from the onset and followed by routine complement analyses for six years. Her complement system was completely inhibited and the dose interval was prolonged from 2 to 3 weeks without alteration of the dose and the complement activity continued to be completely inhibited. Blood samples taken immediately before, immediately after, and between eculizumab doses were analyzed for eculizumab-C5 complexes and percentage of total complement activity, using the Wieslab® test, and compared to a pool of sera from 20 healthy controls. The patient exhibited complete complement inhibition at all three time-points and had no free circulating C5 suggesting there was complete binding to eculizumab. She has now been treated for six years with full complement blockade. We suggest therefore that analysis of complement activity using the Wieslab® test is useful for evaluating the effect of eculizumab when dose intervals are prolonged.
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http://dx.doi.org/10.1155/2018/3810249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312603PMC
December 2018

Aliskiren inhibits renin-mediated complement activation.

Kidney Int 2018 10 5;94(4):689-700. Epub 2018 Jun 5.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden. Electronic address:

Certain kidney diseases are associated with complement activation although a renal triggering factor has not been identified. Here we demonstrated that renin, a kidney-specific enzyme, cleaves C3 into C3b and C3a, in a manner identical to the C3 convertase. Cleavage was specifically blocked by the renin inhibitor aliskiren. Renin-mediated C3 cleavage and its inhibition by aliskiren also occurred in serum. Generation of C3 cleavage products was demonstrated by immunoblotting, detecting the cleavage product C3b, by N-terminal sequencing of the cleavage product, and by ELISA for C3a release. Functional assays showed mast cell chemotaxis towards the cleavage product C3a and release of factor Ba when the cleavage product C3b was combined with factor B and factor D. The renin-mediated C3 cleavage product bound to factor B. In the presence of aliskiren this did not occur, and less C3 deposited on renin-producing cells. The effect of aliskiren was studied in three patients with dense deposit disease and this demonstrated decreased systemic and renal complement activation (increased C3, decreased C3a and C5a, decreased renal C3 and C5b-9 deposition and/or decreased glomerular basement membrane thickness) over a follow-up period of four to seven years. Thus, renin can trigger complement activation, an effect inhibited by aliskiren. Since renin concentrations are higher in renal tissue than systemically, this may explain the renal propensity of complement-mediated disease in the presence of complement mutations or auto-antibodies.
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http://dx.doi.org/10.1016/j.kint.2018.04.004DOI Listing
October 2018

Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases.

Pediatr Nephrol 2019 01 27;34(1):11-30. Epub 2017 Nov 27.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185, Lund, Sweden.

Extracellular vesicles are cell-derived membrane particles ranging from 30 to 5,000 nm in size, including exosomes, microvesicles, and apoptotic bodies. They are released under physiological conditions, but also upon cellular activation, senescence, and apoptosis. They play an important role in intercellular communication. Their release may also maintain cellular integrity by ridding the cell of damaging substances. This review describes the biogenesis, uptake, and detection of extracellular vesicles in addition to the impact that they have on recipient cells, focusing on mechanisms important in the pathophysiology of kidney diseases, such as thrombosis, angiogenesis, tissue regeneration, immune modulation, and inflammation. In kidney diseases, extracellular vesicles may be utilized as biomarkers, as they are detected in both blood and urine. Furthermore, they may contribute to the pathophysiology of renal disease while also having beneficial effects associated with tissue repair. Because of their role in the promotion of thrombosis, inflammation, and immune-mediated disease, they could be the target of drug therapy, whereas their favorable effects could be utilized therapeutically in acute and chronic kidney injury.
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http://dx.doi.org/10.1007/s00467-017-3816-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244861PMC
January 2019

Microvesicle Involvement in Shiga Toxin-Associated Infection.

Toxins (Basel) 2017 11 19;9(11). Epub 2017 Nov 19.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22184 Lund, Sweden.

Shiga toxin is the main virulence factor of enterohemorrhagic , a non-invasive pathogen that releases virulence factors in the intestine, causing hemorrhagic colitis and, in severe cases, hemolytic uremic syndrome (HUS). HUS manifests with acute renal failure, hemolytic anemia and thrombocytopenia. Shiga toxin induces endothelial cell damage leading to platelet deposition in thrombi within the microvasculature and the development of thrombotic microangiopathy, mostly affecting the kidney. Red blood cells are destroyed in the occlusive capillary lesions. This review focuses on the importance of microvesicles shed from blood cells and their participation in the prothrombotic lesion, in hemolysis and in the transfer of toxin from the circulation into the kidney. Shiga toxin binds to blood cells and may undergo endocytosis and be released within microvesicles. Microvesicles normally contribute to intracellular communication and remove unwanted components from cells. Many microvesicles are prothrombotic as they are tissue factor- and phosphatidylserine-positive. Shiga toxin induces complement-mediated hemolysis and the release of complement-coated red blood cell-derived microvesicles. Toxin was demonstrated within blood cell-derived microvesicles that transported it to renal cells, where microvesicles were taken up and released their contents. Microvesicles are thereby involved in all cardinal aspects of Shiga toxin-associated HUS, thrombosis, hemolysis and renal failure.
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http://dx.doi.org/10.3390/toxins9110376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5705991PMC
November 2017

Extracellular vesicles in renal disease.

Nat Rev Nephrol 2017 Sep 24;13(9):545-562. Epub 2017 Jul 24.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Klinikgatan 28, 22184 Lund, Sweden.

Extracellular vesicles, such as exosomes and microvesicles, are host cell-derived packages of information that allow cell-cell communication and enable cells to rid themselves of unwanted substances. The release and uptake of extracellular vesicles has important physiological functions and may also contribute to the development and propagation of inflammatory, vascular, malignant, infectious and neurodegenerative diseases. This Review describes the different types of extracellular vesicles, how they are detected and the mechanisms by which they communicate with cells and transfer information. We also describe their physiological functions in cellular interactions, such as in thrombosis, immune modulation, cell proliferation, tissue regeneration and matrix modulation, with an emphasis on renal processes. We discuss how the detection of extracellular vesicles could be utilized as biomarkers of renal disease and how they might contribute to disease processes in the kidney, such as in acute kidney injury, chronic kidney disease, renal transplantation, thrombotic microangiopathies, vasculitides, IgA nephropathy, nephrotic syndrome, urinary tract infection, cystic kidney disease and tubulopathies. Finally, we consider how the release or uptake of extracellular vesicles can be blocked, as well as the associated benefits and risks, and how extracellular vesicles might be used to treat renal diseases by delivering therapeutics to specific cells.
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http://dx.doi.org/10.1038/nrneph.2017.98DOI Listing
September 2017

C1-Inhibitor Decreases the Release of Vasculitis-Like Chemotactic Endothelial Microvesicles.

J Am Soc Nephrol 2017 Aug 13;28(8):2472-2481. Epub 2017 Mar 13.

Departments of Pediatrics and

The kinin system is activated during vasculitis and may contribute to chronic inflammation. C1-inhibitor is the main inhibitor of the kinin system. In this study, we investigated the presence of the kinin B1 receptor on endothelial microvesicles and its contribution to the inflammatory process. Compared with controls (=15), patients with acute vasculitis (=12) had markedly higher levels of circulating endothelial microvesicles, identified by flow cytometry analysis, and significantly more microvesicles that were positive for the kinin B1 receptor (<0.001). Compared with microvesicles from wild-type cells, B1 receptor-positive microvesicles derived from transfected human embryonic kidney cells induced a significant neutrophil chemotactic effect, and a B1 receptor antagonist blocked this effect. Likewise, patient plasma induced neutrophil chemotaxis, an effect decreased by reduction of microvesicle levels and by blocking the B1 receptor. We used a perfusion system to study the effect of patient plasma (=6) and control plasma (=6) on the release of microvesicles from glomerular endothelial cells. Patient samples induced the release of significantly more B1 receptor-positive endothelial microvesicles than control samples, an effect abrogated by reduction of the microvesicles in the perfused samples. Perfusion of C1-inhibitor-depleted plasma over glomerular endothelial cells promoted excessive release of B1 receptor-positive endothelial microvesicles compared with normal plasma, an effect significantly decreased by addition of C1-inhibitor or B1 receptor-antagonist. Thus, B1 receptor-positive endothelial microvesicles may contribute to chronic inflammation by inducing neutrophil chemotaxis, and the reduction of these microvesicles by C1-inhibitor should be explored as a potential treatment for neutrophil-induced inflammation.
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http://dx.doi.org/10.1681/ASN.2016060637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533224PMC
August 2017

Thrombotic microangiopathy mimicking membranoproliferative glomerulonephritis.

Nephrol Dial Transplant 2017 03;32(3):584

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

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http://dx.doi.org/10.1093/ndt/gfx013DOI Listing
March 2017

Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli - An Anti-thrombotic Mechanism in the Kidney.

EBioMedicine 2017 Feb 24;16:302-311. Epub 2017 Jan 24.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden. Electronic address:

Adequate cleavage of von Willebrand factor (VWF) prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM) was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3), cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.
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http://dx.doi.org/10.1016/j.ebiom.2017.01.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474509PMC
February 2017

Microvesicle transfer of kinin B1-receptors is a novel inflammatory mechanism in vasculitis.

Kidney Int 2017 01 1;91(1):96-105. Epub 2016 Dec 1.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden. Electronic address:

During vasculitis, activation of the kinin system induces inflammation, whereby the kinin B1-receptor is expressed and activated after ligand binding. Additionally, activated blood cells release microvesicles into the circulation. Here we determined whether leukocyte-derived microvesicles bear B1-kinin receptors during vasculitis, and if microvesicles transfer functional B1-receptors to recipient cells, thus promoting inflammation. By flow cytometry, plasma from patients with vasculitis were found to contain high levels of leukocyte-derived microvesicles bearing B1-receptors. Importantly, renal biopsies from two patients with vasculitis showed leukocyte-derived microvesicles bearing B1-receptors docking on glomerular endothelial cells providing in vivo relevance. Microvesicles derived from B1-receptor-transfected human embryonic kidney cells transferred B1-receptors to wild-type human embryonic kidney cells, lacking the receptor, and to glomerular endothelial cells. The transferred B1-receptors induced calcium influx after B1-receptor agonist stimulation: a response abrogated by a specific B1-receptor antagonist. Microvesicles derived from neutrophils also transferred B1-receptors to wild-type human embryonic kidney cells and induced calcium influx after stimulation. Thus, we found a novel mechanism by which microvesicles transfer functional receptors and promote kinin-associated inflammation.
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http://dx.doi.org/10.1016/j.kint.2016.09.023DOI Listing
January 2017

Orphan drug policies and use in pediatric nephrology.

Pediatr Nephrol 2017 01 13;32(1):1-6. Epub 2016 Oct 13.

Department of Clinical Chemistry and Pharmacology, Laboratory Medicine Lund, Lund University, Lund, Sweden.

Orphan drugs designed to treat rare diseases are often overpriced per patient. Novel treatments are sometimes even more expensive for patients with ultra-rare diseases, in part due to the limited number of patients. Pharmaceutical companies that develop a patented life-saving drug are in a position to charge a very high price, which, at best, may enable these companies to further develop drugs for use in rare disease. However, is there a limit to how much a life-saving drug should cost annually per patient? Government interventions and regulations may opt to withhold a life-saving drug solely due to its high price and cost-effectiveness. Processes related to drug pricing, reimbursement, and thereby availability, vary between countries, thus having implications on patient care. These processes are discussed, with specific focus on three drugs used in pediatric nephrology: agalsidase beta (for Fabry disease), eculizumab (for atypical hemolytic uremic syndrome), and cysteamine bitartrate (for cystinosis). Access to and costs of orphan drugs have most profound implications for patients, but also for their physicians, hospitals, insurance policies, and society at large, particularly from financial and ethical standpoints.
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http://dx.doi.org/10.1007/s00467-016-3520-4DOI Listing
January 2017

Haemolytic uraemic syndrome.

J Intern Med 2017 02 10;281(2):123-148. Epub 2016 Oct 10.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

Haemolytic uraemic syndrome (HUS) is defined by the simultaneous occurrence of nonimmune haemolytic anaemia, thrombocytopenia and acute renal failure. This leads to the pathological lesion termed thrombotic microangiopathy, which mainly affects the kidney, as well as other organs. HUS is associated with endothelial cell injury and platelet activation, although the underlying cause may differ. Most cases of HUS are associated with gastrointestinal infection with Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) strains. Atypical HUS (aHUS) is associated with complement dysregulation due to mutations or autoantibodies. In this review, we will describe the causes of HUS. In addition, we will review the clinical, pathological, haematological and biochemical features, epidemiology and pathogenetic mechanisms as well as the biochemical, microbiological, immunological and genetic investigations leading to diagnosis. Understanding the underlying mechanisms of the different subtypes of HUS enables tailoring of appropriate treatment and management. To date, there is no specific treatment for EHEC-associated HUS but patients benefit from supportive care, whereas patients with aHUS are effectively treated with anti-C5 antibody to prevent recurrences, both before and after renal transplantation.
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http://dx.doi.org/10.1111/joim.12546DOI Listing
February 2017

Complement contributes to the pathogenesis of Shiga toxin-associated hemolytic uremic syndrome.

Kidney Int 2016 10;90(4):726-9

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

Complement is activated during Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome (STEC-HUS). There is evidence of complement activation via the alternative pathway in STEC-HUS patients as well as from in vivo and in vitro models. Ozaki et al. demonstrate activation of the mannose-binding lectin (MBL) pathway in Shiga toxin-treated mice expressing human MBL2, but lacking murine Mbls. Treatment with anti-human MBL2 antibody was protective, suggesting that MBL pathway activation also contributes to Shiga toxin-mediated renal injury.
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http://dx.doi.org/10.1016/j.kint.2016.07.002DOI Listing
October 2016

Early Terminal Complement Blockade and C6 Deficiency Are Protective in Enterohemorrhagic Escherichia coli-Infected Mice.

J Immunol 2016 08 15;197(4):1276-86. Epub 2016 Jul 15.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185 Lund, Sweden;

Complement activation occurs during enterohemorrhagic Escherichia coli (EHEC) infection and may exacerbate renal manifestations. In this study, we show glomerular C5b-9 deposits in the renal biopsy of a child with EHEC-associated hemolytic uremic syndrome. The role of the terminal complement complex, and its blockade as a therapeutic modality, was investigated in a mouse model of E. coli O157:H7 infection. BALB/c mice were treated with monoclonal anti-C5 i.p. on day 3 or 6 after intragastric inoculation and monitored for clinical signs of disease and weight loss for 14 d. All infected untreated mice (15 of 15) or those treated with an irrelevant Ab (8 of 8) developed severe illness. In contrast, only few infected mice treated with anti-C5 on day 3 developed symptoms (three of eight, p < 0.01 compared with mice treated with the irrelevant Ab on day 3) whereas most mice treated with anti-C5 on day 6 developed symptoms (six of eight). C6-deficient C57BL/6 mice were also inoculated with E. coli O157:H7 and only 1 of 14 developed disease, whereas 10 of 16 wild-type mice developed weight loss and severe disease (p < 0.01). Complement activation via the terminal pathway is thus involved in the development of disease in murine EHEC infection. Early blockade of the terminal complement pathway, before the development of symptoms, was largely protective, whereas late blockade was not. Likewise, lack of C6, and thereby deficient terminal complement complex, was protective in murine E. coli O157:H7 infection.
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http://dx.doi.org/10.4049/jimmunol.1502377DOI Listing
August 2016

Complement Interactions with Blood Cells, Endothelial Cells and Microvesicles in Thrombotic and Inflammatory Conditions.

Adv Exp Med Biol 2015 ;865:19-42

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden,

The complement system is activated in the vasculature during thrombotic and inflammatory conditions. Activation may be associated with chronic inflammation on the endothelial surface leading to complement deposition. Complement mutations allow uninhibited complement activation to occur on platelets, neutrophils, monocytes, and aggregates thereof, as well as on red blood cells and endothelial cells. Furthermore, complement activation on the cells leads to the shedding of cell derived-microvesicles that may express complement and tissue factor thus promoting inflammation and thrombosis. Complement deposition on red blood cells triggers hemolysis and the release of red blood cell-derived microvesicles that are prothrombotic. Microvesicles are small membrane vesicles ranging from 0.1 to 1 μm, shed by cells during activation, injury and/or apoptosis that express components of the parent cell. Microvesicles are released during inflammatory and vascular conditions. The repertoire of inflammatory markers on endothelial cell-derived microvesicles shed during inflammation is large and includes complement. These circulating microvesicles may reflect the ongoing inflammatory process but may also contribute to its propagation. This overview will describe complement activation on blood and endothelial cells and the release of microvesicles from these cells during hemolytic uremic syndrome, thrombotic thrombocytopenic purpura and vasculitis, clinical conditions associated with enhanced thrombosis and inflammation.
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http://dx.doi.org/10.1007/978-3-319-18603-0_2DOI Listing
December 2015

Enterohemorrhagic Escherichia coli Pathogenesis and the Host Response.

Microbiol Spectr 2014 Oct;2(5)

Department of Pediatrics, Clinical Sciences, Lund University, Lund, Sweden.

Enterohemorrhagic Escherichia coli (EHEC) is a highly pathogenic bacterial strain capable of causing watery or bloody diarrhea, the latter termed hemorrhagic colitis, and hemolytic-uremic syndrome (HUS). HUS is defined as the simultaneous development of non-immune hemolytic anemia, thrombocytopenia, and acute renal failure. The mechanism by which EHEC bacteria colonize and cause severe colitis, followed by renal failure with activated blood cells, as well as neurological symptoms, involves the interaction of bacterial virulence factors and specific pathogen-associated molecular patterns with host cells as well as the host response. The innate immune host response comprises the release of antimicrobial peptides as well as cytokines and chemokines in addition to activation and/or injury to leukocytes, platelets, and erythrocytes and activation of the complement system. Some of the bacterial interactions with the host may be protective in nature, but, when excessive, contribute to extensive tissue injury, inflammation, and thrombosis, effects that may worsen the clinical outcome of EHEC infection. This article describes aspects of the host response occurring during EHEC infection and their effects on specific organs.
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http://dx.doi.org/10.1128/microbiolspec.EHEC-0009-2013DOI Listing
October 2014

An international consensus approach to the management of atypical hemolytic uremic syndrome in children.

Pediatr Nephrol 2016 Jan 11;31(1):15-39. Epub 2015 Apr 11.

Assistance Publique-Hôpitaux de Paris, Hôpital Européen Georges Pompidou, Laboratory of Immunology, INSERM UMRS 1138, Paris, France.

Atypical hemolytic uremic syndrome (aHUS) emerged during the last decade as a disease largely of complement dysregulation. This advance facilitated the development of novel, rational treatment options targeting terminal complement activation, e.g., using an anti-C5 antibody (eculizumab). We review treatment and patient management issues related to this therapeutic approach. We present consensus clinical practice recommendations generated by HUS International, an international expert group of clinicians and basic scientists with a focused interest in HUS. We aim to address the following questions of high relevance to daily clinical practice: Which complement investigations should be done and when? What is the importance of anti-factor H antibody detection? Who should be treated with eculizumab? Is plasma exchange therapy still needed? When should eculizumab therapy be initiated? How and when should complement blockade be monitored? Can the approved treatment schedule be modified? What approach should be taken to kidney and/or combined liver-kidney transplantation? How should we limit the risk of meningococcal infection under complement blockade therapy? A pressing question today regards the treatment duration. We discuss the need for prospective studies to establish evidence-based criteria for the continuation or cessation of anticomplement therapy in patients with and without identified complement mutations.
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http://dx.doi.org/10.1007/s00467-015-3076-8DOI Listing
January 2016

A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles.

PLoS Pathog 2015 Feb 26;11(2):e1004619. Epub 2015 Feb 26.

Department of Pediatrics, Clinical Sciences Lund, Lund University, Lund, Sweden.

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli, which are non-invasive strains that can lead to hemolytic uremic syndrome (HUS), associated with renal failure and death. Although bacteremia does not occur, bacterial virulence factors gain access to the circulation and are thereafter presumed to cause target organ damage. Stx was previously shown to circulate bound to blood cells but the mechanism by which it would potentially transfer to target organ cells has not been elucidated. Here we show that blood cell-derived microvesicles, shed during HUS, contain Stx and are found within patient renal cortical cells. The finding was reproduced in mice infected with Stx-producing Escherichia coli exhibiting Stx-containing blood cell-derived microvesicles in the circulation that reached the kidney where they were transferred into glomerular and peritubular capillary endothelial cells and further through their basement membranes followed by podocytes and tubular epithelial cells, respectively. In vitro studies demonstrated that blood cell-derived microvesicles containing Stx undergo endocytosis in glomerular endothelial cells leading to cell death secondary to inhibited protein synthesis. This study demonstrates a novel virulence mechanism whereby bacterial toxin is transferred within host blood cell-derived microvesicles in which it may evade the host immune system.
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http://dx.doi.org/10.1371/journal.ppat.1004619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4342247PMC
February 2015

Shiga toxin-induced complement-mediated hemolysis and release of complement-coated red blood cell-derived microvesicles in hemolytic uremic syndrome.

J Immunol 2015 Mar 30;194(5):2309-18. Epub 2015 Jan 30.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22184 Lund, Sweden;

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemolytic uremic syndrome (HUS). This study investigated whether Stx2 induces hemolysis and whether complement is involved in the hemolytic process. RBCs and/or RBC-derived microvesicles from patients with STEC-HUS (n = 25) were investigated for the presence of C3 and C9 by flow cytometry. Patients exhibited increased C3 deposition on RBCs compared with controls (p < 0.001), as well as high levels of C3- and C9-bearing RBC-derived microvesicles during the acute phase, which decreased after recovery. Stx2 bound to P1 (k) and P2 (k) phenotype RBCs, expressing high levels of the P(k) Ag (globotriaosylceramide), the known Stx receptor. Stx2 induced the release of hemoglobin and lactate dehydrogenase in whole blood, indicating hemolysis. Stx2-induced hemolysis was not demonstrated in the absence of plasma and was inhibited by heat inactivation, as well as by the terminal complement pathway Ab eculizumab, the purinergic P2 receptor antagonist suramin, and EDTA. In the presence of whole blood or plasma/serum, Stx2 induced the release of RBC-derived microvesicles coated with C5b-9, a process that was inhibited by EDTA, in the absence of factor B, and by purinergic P2 receptor antagonists. Thus, complement-coated RBC-derived microvesicles are elevated in HUS patients and induced in vitro by incubation of RBCs with Stx2, which also induced hemolysis. The role of complement in Stx2-mediated hemolysis was demonstrated by its occurrence only in the presence of plasma and its abrogation by heat inactivation, EDTA, and eculizumab. Complement activation on RBCs could play a role in the hemolytic process occurring during STEC-HUS.
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http://dx.doi.org/10.4049/jimmunol.1402470DOI Listing
March 2015

Eculizumab treatment for rescue of renal function in IgA nephropathy.

Pediatr Nephrol 2014 Nov 13;29(11):2225-8. Epub 2014 Jun 13.

Section of Pediatric Nephrology, Department of Pediatric and Adolescent Medicine, Skåne University Hospital, Lund, Sweden.

Background: Immunoglobulin A (IgA) nephropathy is a chronic glomerulonephritis with excessive glomerular deposition of IgA1, C3 and C5b-9, which may lead to renal failure.

Case Diagnosis/treatment: We describe the clinical course of an adolescent with rapidly progressive disease leading to renal failure in spite of immunosuppressive treatment. Due to refractory disease the patient was treated with eculizumab (anti-C5) for 3 months in an attempt to rescue renal function. Treatment led to clinical improvement with stabilization of the glomerular filtration rate and reduced proteinuria. Discontinuation of treatment led to a rapid deterioration of renal function. This was followed by a single dose of eculizumab, which again reduced creatinine levels temporarily.

Conclusions: Early initiation of eculizumab therapy in patients with progressive IgA nephropathy may have a beneficial effect by blocking complement-mediated renal inflammation.
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http://dx.doi.org/10.1007/s00467-014-2863-yDOI Listing
November 2014

The combined role of galactose-deficient IgA1 and streptococcal IgA-binding M Protein in inducing IL-6 and C3 secretion from human mesangial cells: implications for IgA nephropathy.

J Immunol 2014 Jul 21;193(1):317-26. Epub 2014 May 21.

Department of Pediatrics, Clinical Sciences Lund, Lund University, 22185 Lund, Sweden;

IgA nephropathy (IgAN) is characterized by mesangial cell proliferation and extracellular matrix expansion associated with immune deposits consisting of galactose-deficient polymeric IgA1 and C3. We have previously shown that IgA-binding regions of streptococcal M proteins colocalize with IgA in mesangial immune deposits in patients with IgAN. In the present study, the IgA-binding M4 protein from group A Streptococcus was found to bind to galactose-deficient polymeric IgA1 with higher affinity than to other forms of IgA1, as shown by surface plasmon resonance and solid-phase immunoassay. The M4 protein was demonstrated to bind to mesangial cells not via the IgA-binding region but rather via the C-terminal region, as demonstrated by flow cytometry. IgA1 enhanced binding of M4 to mesangial cells, but not vice versa. Costimulation of human mesangial cells with M4 and galactose-deficient polymeric IgA1 resulted in a significant increase in IL-6 secretion compared with each stimulant alone. Galactose-deficient polymeric IgA1 alone, but not M4, induced C3 secretion from the cells, and costimulation enhanced this effect. Additionally, costimulation enhanced mesangial cell proliferation compared with each stimulant alone. These results indicate that IgA-binding M4 protein binds preferentially to galactose-deficient polymeric IgA1 and that these proteins together induce excessive proinflammatory responses and proliferation of human mesangial cells. Thus, tissue deposition of streptococcal IgA-binding M proteins may contribute to the pathogenesis of IgAN.
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http://dx.doi.org/10.4049/jimmunol.1302249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4065838PMC
July 2014