Publications by authors named "Catharina Svanborg"

98 Publications

Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex.

Nat Commun 2021 06 8;12(1):3427. Epub 2021 Jun 8.

Division of Microbiology, Immunology and Glycobiology, Department of Laboratory Medicine, Faculty of Medicine, Lund University, Lund, Sweden.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.
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http://dx.doi.org/10.1038/s41467-021-23748-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8187399PMC
June 2021

A bacterial protease depletes c-MYC and increases survival in mouse models of bladder and colon cancer.

Nat Biotechnol 2021 Jun 11;39(6):754-764. Epub 2021 Feb 11.

Department of Laboratory Medicine, Division of Microbiology, Immunology and Glycobiology, Lund University, Lund, Sweden.

Is the oncogene MYC upregulated or hyperactive? In the majority of human cancers, finding agents that target c-MYC has proved difficult. Here we report specific bacterial effector molecules that inhibit cellular MYC (c-MYC) in human cells. We show that uropathogenic Escherichia coli (UPEC) degrade the c-MYC protein and attenuate MYC expression in both human cells and animal tissues. c-MYC protein was rapidly degraded by both cell-free bacterial lysates and the purified bacterial protease Lon. In mice, intravesical or peroral delivery of Lon protease delayed tumor progression and increased survival in MYC-dependent bladder and colon cancer models, respectively. These results suggest that bacteria have evolved strategies to control c-MYC tissue levels in the host and that the Lon protease shows promise for therapeutic targeting of c-MYC in cancer.
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http://dx.doi.org/10.1038/s41587-020-00805-3DOI Listing
June 2021

Active bacterial modification of the host environment through RNA polymerase II inhibition.

J Clin Invest 2021 Feb;131(4)

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

Unlike pathogens, which attack the host, commensal bacteria create a state of friendly coexistence. Here, we identified a mechanism of bacterial adaptation to the host niche, where they reside. Asymptomatic carrier strains were shown to inhibit RNA polymerase II (Pol II) in host cells by targeting Ser2 phosphorylation, a step required for productive mRNA elongation. Assisted by a rare, spontaneous loss-of-function mutant from a human carrier, the bacterial NlpD protein was identified as a Pol II inhibitor. After internalization by host cells, NlpD was shown to target constituents of the Pol II phosphorylation complex (RPB1 and PAF1C), attenuating host gene expression. Therapeutic efficacy of a recombinant NlpD protein was demonstrated in a urinary tract infection model, by reduced tissue pathology, accelerated bacterial clearance, and attenuated Pol II-dependent gene expression. The findings suggest an intriguing, evolutionarily conserved mechanism for bacterial modulation of host gene expression, with a remarkable therapeutic potential.
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http://dx.doi.org/10.1172/JCI140333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880420PMC
February 2021

Peptide-Oleate Complexes Create Novel Membrane-Bound Compartments.

Mol Biol Evol 2020 11;37(11):3083-3093

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

A challenging question in evolutionary theory is the origin of cell division and plausible molecular mechanisms involved. Here, we made the surprising observation that complexes formed by short alpha-helical peptides and oleic acid can create multiple membrane-enclosed spaces from a single lipid vesicle. The findings suggest that such complexes may contain the molecular information necessary to initiate and sustain this process. Based on these observations, we propose a new molecular model to understand protocell division.
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http://dx.doi.org/10.1093/molbev/msaa138DOI Listing
November 2020

Bladder cancer therapy without toxicity-A dose-escalation study of alpha1-oleate.

Int J Cancer 2020 11 11;147(9):2479-2492. Epub 2020 May 11.

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

Potent chemotherapeutic agents are required to counteract the aggressive behavior of cancer cells and patients often experience severe side effects, due to tissue toxicity. Our study addresses if a better balance between efficacy and toxicity can be attained using the tumoricidal complex alpha1-oleate, formed by a synthetic, alpha-helical peptide comprising the N-terminal 39 amino acids of alpha-lactalbumin and the fatty acid oleic acid. Bladder cancer was established, by intravesical instillation of MB49 cells on day 0 and the treatment group received five instillations of alpha1-oleate (1.7-17 mM) on days 3 to 11. A dose-dependent reduction in tumor size, bladder size and bladder weight was recorded in the alpha1-oleate treated group, compared to sham-treated mice. Tumor markers Ki-67, Cyclin D1 and VEGF were inhibited in a dose-dependent manner, as was the expression of cancer-related genes. Remarkably, toxicity for healthy tissue was not detected in alpha1-oleate-treated, tumor-bearing mice or healthy mice or rabbits, challenged with increasing doses of the active complex. The results define a dose-dependent therapeutic effect of alpha1-oleate in a murine bladder cancer model.
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http://dx.doi.org/10.1002/ijc.33019DOI Listing
November 2020

Fimbriae reprogram host gene expression - Divergent effects of P and type 1 fimbriae.

PLoS Pathog 2019 06 10;15(6):e1007671. Epub 2019 Jun 10.

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Klinikgatan, Lund, Sweden.

Pathogens rely on a complex virulence gene repertoire to successfully attack their hosts. We were therefore surprised to find that a single fimbrial gene reconstitution can return the virulence-attenuated commensal strain Escherichia coli 83972 to virulence, defined by a disease phenotype in human hosts. E. coli 83972pap stably reprogrammed host gene expression, by activating an acute pyelonephritis-associated, IRF7-dependent gene network. The PapG protein was internalized by human kidney cells and served as a transcriptional agonist of IRF-7, IFN-β and MYC, suggesting direct involvement of the fimbrial adhesin in this process. IRF-7 was further identified as a potent upstream regulator (-log (p-value) = 61), consistent with the effects in inoculated patients. In contrast, E. coli 83972fim transiently attenuated overall gene expression in human hosts, enhancing the effects of E. coli 83972. The inhibition of RNA processing and ribosomal assembly indicated a homeostatic rather than a pathogenic end-point. In parallel, the expression of specific ion channels and neuropeptide gene networks was transiently enhanced, in a FimH-dependent manner. The studies were performed to establish protective asymptomatic bacteriuria in human hosts and the reconstituted E. coli 83972 variants were developed to improve bacterial fitness for the human urinary tract. Unexpectedly, P fimbriae were able to drive a disease response, suggesting that like oncogene addiction in cancer, pathogens may be addicted to single super-virulence factors.
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http://dx.doi.org/10.1371/journal.ppat.1007671DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557620PMC
June 2019

Beta-sheet-specific interactions with heat shock proteins define a mechanism of delayed tumor cell death in response to HAMLET.

J Mol Biol 2019 06 11;431(14):2612-2627. Epub 2019 May 11.

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, S-223 62 Lund, Sweden. Electronic address:

As chaperones, heat shock proteins (HSPs) protect host cells against misfolded proteins that constitute a by-product of protein synthesis. Certain HSPs are also expressed on the surface of tumor cells, possibly to scavenge extracellular unfolded protein ligands and prevent them from becoming cytotoxic. HAMLET-a complex of partially unfolded alpha-lactalbumin and oleic acid-is relying on its N-terminal alpha-helical domain to perturb tumor cell membranes, and the cells die as a consequence of this interaction. Here we show that in parallel, cell surface HSPs bind the beta-sheet domain of alpha-lactalbumin and activate a temporarily protective loop, involving vesicular uptake and lysosomal accumulation. Later, HAMLET destroys lysosomal membrane integrity, and HAMLET release kills the remaining tumor cells. HSPs were identified as HAMLET targets in a proteomic screen and Hsp70-specific antibodies or shRNAs inhibited HAMLET uptake by tumor cells, which showed increased Hsp70 surface expression compared to differentiated cells. The results suggest that HAMLET engages tumor cells by two parallel recognition mechanisms, defined by alpha-helical- or beta-sheet domains of alpha-lactalbumin and resulting in an immediate death response, or a delay due to transient accumulation of the complex in the lysosomes. This dual response pattern was conserved among tumor cells but not seen in normal, differentiated cells. By two different mechanisms, HAMLET thus achieves a remarkably efficient elimination of tumor cells.
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http://dx.doi.org/10.1016/j.jmb.2019.05.007DOI Listing
June 2019

Neuroepithelial control of mucosal inflammation in acute cystitis.

Sci Rep 2018 07 20;8(1):11015. Epub 2018 Jul 20.

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, 223 62, Lund, Sweden.

The nervous system is engaged by infection, indirectly through inflammatory cascades or directly, by bacterial attack on nerve cells. Here we identify a neuro-epithelial activation loop that participates in the control of mucosal inflammation and pain in acute cystitis. We show that infection activates Neurokinin-1 receptor (NK1R) and Substance P (SP) expression in nerve cells and bladder epithelial cells in vitro and in vivo in the urinary bladder mucosa. Specific innate immune response genes regulated this mucosal response, and single gene deletions resulted either in protection (Tlr4 and Il1b mice) or in accentuated bladder pathology (Asc and Nlrp3 mice), compared to controls. NK1R/SP expression was lower in Tlr4 and Il1b mice than in C56BL/6WT controls but in Asc and Nlrp3 mice, NK1R over-activation accompanied the exaggerated disease phenotype, due, in part to transcriptional de-repression of Tacr1. Pharmacologic NK1R inhibitors attenuated acute cystitis in susceptible mice, supporting a role in disease pathogenesis. Clinical relevance was suggested by elevated urine SP levels in patients with acute cystitis, compared to patients with asymptomatic bacteriuria identifying NK1R/SP as potential therapeutic targets. We propose that NK1R and SP influence the severity of acute cystitis through a neuro-epithelial activation loop that controls pain and mucosal inflammation.
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http://dx.doi.org/10.1038/s41598-018-28634-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6054610PMC
July 2018

HAMLET - A protein-lipid complex with broad tumoricidal activity.

Biochem Biophys Res Commun 2017 01 3;482(3):454-458. Epub 2017 Feb 3.

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, S-223 62 Lund, Sweden. Electronic address:

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.
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http://dx.doi.org/10.1016/j.bbrc.2016.10.092DOI Listing
January 2017

Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets.

PLoS Pathog 2016 10 12;12(10):e1005848. Epub 2016 Oct 12.

Division of Microbiology, Immunology and Glycobiology, Department of Laboratory Medicine, Lund University, Lund, Sweden.

Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1β)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1β processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1β processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc-/- and Nlrp3-/- mice. The resulting IL-1β hyper-activation loop included a large number of IL-1β-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc-/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1β and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man.

Trial Registration: The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov).
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http://dx.doi.org/10.1371/journal.ppat.1005848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061333PMC
October 2016

Protein-dependent Membrane Interaction of A Partially Disordered Protein Complex with Oleic Acid: Implications for Cancer Lipidomics.

Sci Rep 2016 10 12;6:35015. Epub 2016 Oct 12.

CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India.

Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.
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http://dx.doi.org/10.1038/srep35015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5059734PMC
October 2016

Deliberate Establishment of Asymptomatic Bacteriuria-A Novel Strategy to Prevent Recurrent UTI.

Pathogens 2016 Jul 29;5(3). Epub 2016 Jul 29.

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, 221 00 Lund, Sweden.

We have established a novel strategy to reduce the risk for recurrent urinary tract infection (UTI), where rapidly increasing antibiotic resistance poses a major threat. Epidemiologic studies have demonstrated that asymptomatic bacteriuria (ABU) protects the host against symptomatic infections with more virulent strains. To mimic this protective effect, we deliberately establish ABU in UTI-prone patients, who are refractory to conventional therapy. The patients are inoculated with Escherichia coli (E. coli) 83972, now widely used as a prototype ABU strain. Therapeutic efficacy has been demonstrated in a placebo-controlled trial, supporting the feasibility of using E. coli 83972 as a tool to prevent recurrent UTI and, potentially, to outcompete antibiotic-resistant strains from the human urinary tract. In addition, the human inoculation protocol offers unique opportunities to study host-parasite interaction in vivo in the human urinary tract. Here, we review the clinical evidence for protection using this approach as well as some molecular insights into the pathogenesis of UTI that have been gained during these studies.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039432PMC
http://dx.doi.org/10.3390/pathogens5030052DOI Listing
July 2016

Bacterial Suppression of RNA Polymerase II-Dependent Host Gene Expression.

Pathogens 2016 Jul 13;5(3). Epub 2016 Jul 13.

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, 22184 Lund, Sweden.

Asymptomatic bacteriuria (ABU) is a bacterial carrier state in the urinary tract that resembles commensalism at other mucosal sites. ABU strains often lack the virulence factors that characterize uropathogenic Escherichia coli (E. coli) strains and therefore elicit weak innate immune responses in the urinary tract. In addition, ABU strains are active modifiers of the host environment, which they influence by suppressing RNA polymerase II (Pol II)-dependent host gene expression. In patients inoculated with the ABU strain E. coli 83972, gene expression was markedly reduced after 24 h (>60% of all regulated genes). Specific repressors and activators of Pol II-dependent transcription were modified, and Pol II Serine 2 phosphorylation was significantly inhibited, indicating reduced activity of the polymerase. This active inhibition included disease-associated innate immune response pathways, defined by TLR4, IRF-3 and IRF-7, suggesting that ABU strains persist in human hosts by active suppression of the antibacterial defense. In a search for the mechanism of inhibition, we compared the whole genome sequences of E. coli 83972 and the uropathogenic strain E. coli CFT073. In addition to the known loss of virulence genes, we observed that the ABU strain has acquired several phages and identified the lytic Prophage 3 as a candidate Pol II inhibitor. Intact phage particles were released by ABU during in vitro growth in human urine. To address if Prophage 3 affects Pol II activity, we constructed a Prophage 3 negative deletion mutant in E. coli 83972 and compared the effect on Pol II phosphorylation between the mutant and the E. coli 83972 wild type (WT) strains. No difference was detected, suggesting that the Pol II inhibitor is not encoded by the phage. The review summarizes the evidence that the ABU strain E. coli 83972 modifies host gene expression by inhibition of Pol II phosphorylation, and discusses the ability of ABU strains to actively create an environment that enhances their persistence.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039429PMC
http://dx.doi.org/10.3390/pathogens5030049DOI Listing
July 2016

Susceptibility to Urinary Tract Infection: Benefits and Hazards of the Antibacterial Host Response.

Microbiol Spectr 2016 06;4(3)

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, S-223 62, Sweden.

A paradigm shift is needed to improve and personalize the diagnosis of infectious disease and to select appropriate therapies. For many years, only the most severe and complicated bacterial infections received more detailed diagnostic and therapeutic attention as the efficiency of antibiotic therapy has guaranteed efficient treatment of patients suffering from the most common infections. Indeed, treatability almost became a rationale not to analyze bacterial and host parameters in these larger patient groups. Due to the rapid spread of antibiotic resistance, common infections like respiratory tract- or urinary-tract infections (UTIs) now pose new and significant therapeutic challenges. It is fortunate and timely that infectious disease research can offer such a wealth of new molecular information that is ready to use for the identification of susceptible patients and design of new suitable therapies. Paradoxically, the threat of antibiotic resistance may become a window of opportunity, by encouraging the implementation of new diagnostic and therapeutic approaches. The frequency of antibiotic resistance is rising rapidly in uropathogenic organisms and the molecular and genetic understanding of UTI susceptibility is quite advanced. More bold translation of the new molecular diagnostic and therapeutic tools would not just be possible but of great potential benefit in this patient group. This chapter reviews the molecular basis for susceptibility to UTI, including recent advances in genetics, and discusses the consequences for diagnosis and therapy. By dissecting the increasingly well-defined molecular interactions between bacteria and host and the molecular features of excessive bacterial virulence or host-response malfunction, it is becoming possible to isolate the defensive from the damaging aspects of the host response. Distinguishing "good" from "bad" inflammation has been a long-term quest of biomedical science and in UTI, patients need the "good" aspects of the inflammatory response to resist infection while avoiding the "bad" aspects, causing chronicity and tissue damage.
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http://dx.doi.org/10.1128/microbiolspec.UTI-0019-2014DOI Listing
June 2016

Uropathogenic Escherichia coli strain CFT073 disrupts NLRP3 inflammasome activation.

J Clin Invest 2016 07 23;126(7):2425-36. Epub 2016 May 23.

Successful bacterial pathogens produce an array of virulence factors that allow subversion of the immune system and persistence within the host. For example, uropathogenic Escherichia coli strains, such as CFT073, express Toll/IL-1 receptor-containing (TIR-containing) protein C (TcpC), which impairs TLR signaling, thereby suppressing innate immunity in the urinary tract and enhancing persistence in the kidneys. Here, we have reported that TcpC also reduces secretion of IL-1β by directly interacting with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome, which is crucial for recognition of pathogens within the cytosol. At a low MOI, IL-1β secretion was minimal in CFT073-infected macrophages; however, IL-1β release was markedly increased in macrophages infected with CFT073 lacking tcpC. Induction of IL-1β secretion by CFT073 and tcpC-deficient CFT073 required the NLRP3 inflammasome. TcpC attenuated activation of the NLRP3 inflammasome by binding both NLRP3 and caspase-1 and thereby preventing processing and activation of caspase-1. Moreover, in a murine urinary tract infection model, CFT073 infection rapidly induced expression of the NLRP3 inflammasome in the bladder mucosa; however, the presence of TcpC in WT CFT073 reduced IL-1β levels in the urine of infected mice. Together, these findings illustrate how uropathogenic E. coli use the multifunctional virulence factor TcpC to attenuate innate immune responses in the urinary tract.
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http://dx.doi.org/10.1172/JCI81916DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922710PMC
July 2016

IRF7 inhibition prevents destructive innate immunity-A target for nonantibiotic therapy of bacterial infections.

Sci Transl Med 2016 04;8(336):336ra59

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, 223 62 Lund, Sweden.

Boosting innate immunity represents an important therapeutic alternative to antibiotics. However, the molecular selectivity of this approach is a major concern because innate immune responses often cause collateral tissue damage. We identify the transcription factor interferon regulatory factor 7 (IRF-7), a heterodimer partner of IRF-3, as a target for non-antibiotics-based therapy of bacterial infections. We found that the efficient and self-limiting innate immune response to bacterial infection relies on a tight balance between IRF-3 and IRF-7. Deletion of Irf3 resulted in overexpression of Irf7 and led to an IRF-7-driven hyperinflammatory phenotype, which was entirely prevented if Irf7 was deleted. We then identified a network of strongly up-regulated, IRF-7-dependent genes in Irf3(-/-) mice with kidney pathology, which was absent in Irf7(-/-) mice. IRF-3 and IRF-7 from infected kidney cell nuclear extracts were shown to bind OAS1, CCL5, and IFNB1 promoter oligonucleotides. These data are consistent in children with low IRF7 expression in the blood: attenuating IRF7 promoter polymorphisms (rs3758650-T and rs10902179-G) negatively associated with recurrent pyelonephritis. Finally, we identified IRF-7 as a target for immunomodulatory therapy. Administering liposomal Irf7 siRNA to Irf3(-/-) mice suppressed mucosal IRF-7 expression, and the mice were protected against infection and renal tissue damage. These findings offer a response to the classical but unresolved question of "good versus bad inflammation" and identify IRF7 as a therapeutic target for protection against bacterial infection.
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http://dx.doi.org/10.1126/scitranslmed.aaf1156DOI Listing
April 2016

A Comparative Analysis of the Mechanism of Toll-Like Receptor-Disruption by TIR-Containing Protein C from Uropathogenic Escherichia coli.

Pathogens 2016 Feb 29;5(1). Epub 2016 Feb 29.

Institute of Medical Microbiology and Hygiene, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, Mannheim 68167, Germany.

The TIR-containing protein C (TcpC) of uropathogenic Escherichia coli strains is a powerful virulence factor by impairing the signaling cascade of Toll-like receptors (TLRs). Several other bacterial pathogens like Salmonella, Yersinia, Staphylococcus aureus but also non-pathogens express similar proteins. We discuss here the pathogenic potential of TcpC and its interaction with TLRs and TLR-adapter proteins on the molecular level and compare its activity with the activity of other bacterial TIR-containing proteins. Finally, we analyze and compare the structure of bacterial TIR-domains with the TIR-domains of TLRs and TLR-adapters.
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http://dx.doi.org/10.3390/pathogens5010025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810146PMC
February 2016

Urinary Tract Infection Molecular Mechanisms and Clinical Translation.

Pathogens 2016 Feb 24;5(1). Epub 2016 Feb 24.

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, 221 00 Lund, Sweden.

Rapid developments in infection biology create new and exciting options for individualized diagnostics and therapy. Such new practices are needed to improve patient survival and reduce morbidity. Molecular determinants of host resistance to infection are being characterized, making it possible to identify susceptible individuals and to predict their risk for future morbidity. Immunotherapy is emerging as a new strategy to treat infections worldwide and controlled boosting of the host immune defense represents an important therapeutic alternative to antibiotics. In proof of concept studies, we have demonstrated that this approach is feasible. The long-term goal is not just to remove the pathogens but to also develop technologies that restore resistance to infection in disease-prone patients and devise personalized therapeutic interventions. Here, we discuss some approaches to reaching these goals, in patients with urinary tract infection (UTI). We describe critical host signaling pathways that define symptoms and pathology and the genetic control of innate immune responses that balance protection against tissue damage. For some of these genes, human relevance has been documented in clinical studies, identifying them as potential targets for immune-modulatory therapies, as a complement to antibiotics.
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http://dx.doi.org/10.3390/pathogens5010024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810145PMC
February 2016

Asymtomatic Bacteriuria as a Model to Study the Coevolution of Hosts and Bacteria.

Pathogens 2016 Feb 15;5(1). Epub 2016 Feb 15.

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden.

During asymptomatic bacteriuria (ABU), bacteria colonize the urinary tract for extended periods of time without causing symptoms of urinary tract infection. Previous studies indicate that many Escherichia coli (E. coli) strains that cause ABU have evolved from uropathogenic E. coli (UPEC) by reductive evolution and loss of the ability to express functional virulence factors. For instance, the prototype ABU strain 83972 has a smaller genome than UPEC strains with deletions or point mutations in several virulence genes. To understand the mechanisms of bacterial adaptation and to find out whether the bacteria adapt in a host-specific manner, we compared the complete genome sequences of consecutive reisolates of ABU strain 83972 from different inoculated individuals and compared them with the genome of the parent strain. Reisolates from different hosts exhibited individual patterns of genomic alterations. Non-synonymous SNPs predominantly occurred in coding regions and often affected the amino acid sequence of proteins with global or pleiotropic regulatory function. These gene products are involved in different bacterial stress protection strategies, and metabolic and signaling pathways. Our data indicate that adaptation of E. coli 83972 to prolonged growth in the urinary tract involves responses to specific growth conditions and stresses present in the individual hosts. Accordingly, modulation of gene expression required for survival and growth under stress conditions seems to be most critical for long-term growth of E. coli 83972 in the urinary tract.
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http://dx.doi.org/10.3390/pathogens5010021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4810142PMC
February 2016

Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

Sci Rep 2015 Nov 12;5:16432. Epub 2015 Nov 12.

Department of Microbiology, Immunology and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, S-223 62 Lund, Sweden.

A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.
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http://dx.doi.org/10.1038/srep16432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642337PMC
November 2015

The molecular motor F-ATP synthase is targeted by the tumoricidal protein HAMLET.

J Mol Biol 2015 May 11;427(10):1866-74. Epub 2015 Feb 11.

School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Republic of Singapore. Electronic address:

HAMLET (human alpha-lactalbumin made lethal to tumor cells) interacts with multiple tumor cell compartments, affecting cell morphology, metabolism, proteasome function, chromatin structure and viability. This study investigated if these diverse effects of HAMLET might be caused, in part, by a direct effect on the ATP synthase and a resulting reduction in cellular ATP levels. A dose-dependent reduction in cellular ATP levels was detected in A549 lung carcinoma cells, and by confocal microscopy, co-localization of HAMLET with the nucleotide-binding subunits α (non-catalytic) and β (catalytic) of the energy converting F1F0 ATP synthase was detected. As shown by fluorescence correlation spectroscopy, HAMLET binds to the F1 domain of the F1F0 ATP synthase with a dissociation constant (KD) of 20.5μM. Increasing concentrations of the tumoricidal protein HAMLET added to the enzymatically active α3β3γ complex of the F-ATP synthase lowered its ATPase activity, demonstrating that HAMLET binding to the F-ATP synthase effects the catalysis of this molecular motor. Single-molecule analysis was applied to study HAMLET-α3β3γ complex interaction. Whereas the α3β3γ complex of the F-ATP synthase rotated in a counterclockwise direction with a mean rotational rate of 3.8±0.7s(-1), no rotation could be observed in the presence of bound HAMLET. Our findings suggest that direct effects of HAMLET on the F-ATP synthase may inhibit ATP-dependent cellular processes.
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http://dx.doi.org/10.1016/j.jmb.2015.01.024DOI Listing
May 2015

Innate immunity and genetic determinants of urinary tract infection susceptibility.

Curr Opin Infect Dis 2015 Feb;28(1):88-96

Department of Microbiology, Immunology and Glycobiology, Institute for Laboratory Medicine, Lund University, Lund, Sweden.

Purpose Of Review: Urinary tract infections (UTIs) are common, dangerous and interesting. Susceptible individuals experience multiple, often clustered episodes, and in a subset of patients, infections progress to acute pyelonephritis (APN), sometimes accompanied by uro-sepsis. Others develop asymptomatic bacteriuria (ABU). Here, we review the molecular basis for these differences, with the intention to distinguish exaggerated host responses that drive disease from attenuated responses that favour protection and to highlight the genetic basis for these extremes, based on knock-out mice and clinical studies.

Recent Findings: The susceptibility to UTI is controlled by specific innate immune signalling and by promoter polymorphisms and transcription factors that modulate the expression of genes controlling these pathways. Gene deletions that disturb innate immune activation either favour asymptomatic bacteriuria or create acute morbidity and disease. Promoter polymorphisms and transcription factor variants affecting those genes are associated with susceptibility in UTI-prone patients.

Summary: It is time to start using genetics in UTI-prone patients, to improve diagnosis and to assess the risk for chronic sequels such as renal malfunction, hypertension, spontaneous abortions, dialysis and transplantation. Furthermore, the majority of UTI patients do not need follow-up, but for lack of molecular markers, they are unnecessarily investigated.
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http://dx.doi.org/10.1097/QCO.0000000000000127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4286230PMC
February 2015

Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens.

Int Immunol 2014 Oct 20;26(10):531-8. Epub 2014 May 20.

Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX13RE, UK Present address: Crucell Holland B.V., Archimedesweg 4-6, 2333CN Leiden, The Netherlands

Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.
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http://dx.doi.org/10.1093/intimm/dxu055DOI Listing
October 2014

The humoral pattern recognition molecule PTX3 is a key component of innate immunity against urinary tract infection.

Immunity 2014 Apr;40(4):621-32

Humanitas Clinical Research Center, via Manzoni 56, 20089 Rozzano (Milano), Italy; Department of Biotechnology and Translational Medicine, University of Milan, 20089 Rozzano (Milano), Italy. Electronic address:

Immunity in the urinary tract has distinct and poorly understood pathophysiological characteristics and urinary tract infections (UTIs) are important causes of morbidity and mortality. We investigated the role of the soluble pattern recognition molecule pentraxin 3 (PTX3), a key component of the humoral arm of innate immunity, in UTIs. PTX3-deficient mice showed defective control of UTIs and exacerbated inflammation. Expression of PTX3 was induced in uroepithelial cells by uropathogenic Escherichia coli (UPEC) in a Toll-like receptor 4 (TLR4)- and MyD88-dependent manner. PTX3 enhanced UPEC phagocytosis and phagosome maturation by neutrophils. PTX3 was detected in urine of UTI patients and amounts correlated with disease severity. In cohorts of UTI-prone patients, PTX3 gene polymorphisms correlated with susceptibility to acute pyelonephritis and cystitis. These results suggest that PTX3 is an essential component of innate resistance against UTIs. Thus, the cellular and humoral arms of innate immunity exert complementary functions in mediating resistance against UTIs.
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http://dx.doi.org/10.1016/j.immuni.2014.02.015DOI Listing
April 2014

Rare emergence of symptoms during long-term asymptomatic Escherichia coli 83972 carriage without an altered virulence factor repertoire.

J Urol 2014 Feb 30;191(2):519-28. Epub 2013 Jul 30.

Institute for Molecular Biology of Infectious Diseases, University of Würzburg, Würzburg, Germany; Institute for Hygiene, University of Münster, Münster, Germany. Electronic address:

Purpose: Asymptomatic bacteriuria established by intravesical inoculation of Escherichia coli 83972 is protective in patients with recurrent urinary tract infections. In this randomized, controlled crossover study a total of 3 symptomatic urinary tract infection episodes developed in 2 patients while they carried E. coli 83972. We examined whether virulence reacquisition by symptom isolates may account for the switch from asymptomatic bacteriuria to symptomatic urinary tract infection.

Materials And Methods: We used E. coli 83972 re-isolates from 2 patients in a prospective study and from another 2 in whom symptoms developed after study completion. We phylogenetically classified the re-isolates, and identified the genomic restriction patterns and gene expression profiles as well as virulence gene structure and phenotypes. In vivo virulence was examined in the murine urinary tract infection model.

Results: The fim, pap, foc, hlyA, fyuA, iuc, iroN, kpsMT K5 and malX genotypes of the symptomatic re-isolates remained unchanged. Bacterial gene expression profiles of flagellated symptomatic re-isolates were unique to each host, providing no evidence of common deregulation. Symptomatic isolates did not differ in virulence from the wild-type strain, as defined in the murine urinary tract infection model by persistence, symptoms or innate immune activation.

Conclusions: The switch from asymptomatic E. coli 83972 carriage to symptomatic urinary tract infection was not explained by reversion to a functional virulence gene repertoire.
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http://dx.doi.org/10.1016/j.juro.2013.07.060DOI Listing
February 2014

Bacterial control of host gene expression through RNA polymerase II.

J Clin Invest 2013 Jun;123(6):2366-79

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coli develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II–dependent (Pol II–dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coli 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II–dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur.
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http://dx.doi.org/10.1172/JCI66451DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668848PMC
June 2013

Urinary tract infections in children: microbial virulence versus host susceptibility.

Adv Exp Med Biol 2013 ;764:205-10

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

Urinary tract infections (UTI) are common, dangerous and interesting. This review includes a general background on UTIs and molecular mechanisms of pathogenesis. In addition, we discuss UTI susceptibility and especially the effect of genetic variation on innate immunity. The symptoms of acute pyelonephritis are caused by the innate immune response and inflammation in the urinary tract decreases renal tubular function and may give rise to renal scarring, especially in childhood. The disease severity is explained by pathogens and their virulence factors triggering signaling through Toll-like receptors (TLRs), interferon regulatory factors (IRFs) and type 1 interferons, and the activation of a host response mediating disease or pathology or clearance of infection. In children with asymptomatic bacteriuria (ABU), in contrast, bacteria persist without causing symptoms or pathology. ABU strains mostly lack virulence factors, and the lack of symptoms has largely been attributed to their lack of virulence. Recently, rapid progress has been made in the understanding of host susceptibility mechanisms. For example, genetic alterations that reduce TLR4 function are associated with ABU while polymorphisms reducing IRF3 or CXCR1 expression are associated with acute pyelonephritis and an increased risk for renal scarring. Understanding bacterial virulence and host resistance promises new tools to improve the diagnostic accuracy in children with UTI. By combining information on bacterial virulence and the host response, it should be possible to start individualizing diagnosis and therapy. Finally, we propose that the prediction of future disease risk and decisions on prophylaxis and invasive diagnostic procedures might be improved by genetic analysis.
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http://dx.doi.org/10.1007/978-1-4614-4726-9_17DOI Listing
June 2013

Lipids as tumoricidal components of human α-lactalbumin made lethal to tumor cells (HAMLET): unique and shared effects on signaling and death.

J Biol Chem 2013 Jun 29;288(24):17460-71. Epub 2013 Apr 29.

Department of Microbiology, Immunology, and Glycobiology (MIG), Institute of Laboratory Medicine, Lund University, Sölvegatan 23, S-223 62 Lund, Sweden.

Long-chain fatty acids are internalized by receptor-mediated mechanisms or receptor-independent diffusion across cytoplasmic membranes and are utilized as nutrients, building blocks, and signaling intermediates. Here we describe how the association of long-chain fatty acids to a partially unfolded, extracellular protein can alter the presentation to target cells and cellular effects. HAMLET (human α-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded α-lactalbumin and oleic acid (OA). As OA lacks independent tumoricidal activity at concentrations equimolar to HAMLET, the contribution of the lipid has been debated. We show by natural abundance (13)C NMR that the lipid in HAMLET is deprotonated and by chromatography that oleate rather than oleic acid is the relevant HAMLET constituent. Compared with HAMLET, oleate (175 μm) showed weak effects on ion fluxes and gene expression. Unlike HAMLET, which causes metabolic paralysis, fatty acid metabolites were less strongly altered. The functional overlap increased with higher oleate concentrations (500 μm). Cellular responses to OA were weak or absent, suggesting that deprotonation favors cellular interactions of fatty acids. Fatty acids may thus exert some of their essential effects on host cells when in the deprotonated state and when presented in the context of a partially unfolded protein.
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http://dx.doi.org/10.1074/jbc.M113.468405DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682546PMC
June 2013

A unifying mechanism for cancer cell death through ion channel activation by HAMLET.

PLoS One 2013 7;8(3):e58578. Epub 2013 Mar 7.

Department of Microbiology, Immunology and Glycobiology, Institute of Laboratory Medicine, Lund University, Lund, Sweden.

Ion channels and ion fluxes control many aspects of tissue homeostasis. During oncogenic transformation, critical ion channel functions may be perturbed but conserved tumor specific ion fluxes remain to be defined. Here we used the tumoricidal protein-lipid complex HAMLET as a probe to identify ion fluxes involved in tumor cell death. We show that HAMLET activates a non-selective cation current, which reached a magnitude of 2.74±0.88 nA within 1.43±0.13 min from HAMLET application. Rapid ion fluxes were essential for HAMLET-induced carcinoma cell death as inhibitors (amiloride, BaCl2), preventing the changes in free cellular Na(+) and K(+) concentrations also prevented essential steps accompanying carcinoma cell death, including changes in morphology, uptake, global transcription, and MAP kinase activation. Through global transcriptional analysis and phosphorylation arrays, a strong ion flux dependent p38 MAPK response was detected and inhibition of p38 signaling delayed HAMLET-induced death. Healthy, differentiated cells were resistant to HAMLET challenge, which was accompanied by innate immunity rather than p38-activation. The results suggest, for the first time, a unifying mechanism for the initiation of HAMLET's broad and rapid lethal effect on tumor cells. These findings are particularly significant in view of HAMLET's documented therapeutic efficacy in human studies and animal models. The results also suggest that HAMLET offers a two-tiered therapeutic approach, killing cancer cells while stimulating an innate immune response in surrounding healthy tissues.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0058578PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3591364PMC
September 2013