Infection of Immunocompetent Conventional Mice with Shiga Toxin-Producing E. coli: The DSS + STEC Model.

Previous methods of infecting mice with Shiga toxin-producing E. coli (STEC) required suppression of host immune function or ablation of the gut microbiota to induce susceptibility to gastrointestinal colonization. Consequently, many pathogen-host interactions occurring in immunocompetent hosts during STEC infection and Shiga toxicosis have remained unclear. The following protocol describes the use of dextran sulfate sodium (DSS) to induce a mild colitis in immunocompetent conventional C57BL/6 mice to facilitate susceptibility to STEC infection by oral gavage. STEC colonization in infected mice was confirmed by recovery of live STEC via fecal cultures and quantified via quantitative polymerase chain reaction of fecal DNA for the STEC-specific gene eae. DSS colitis is well established, broadly reproducible, and does not require specialized equipment or high levels of technical proficiency to be a useful method of inducing susceptibility to gastrointestinal STEC colonization. The DSS + STEC mouse model provides a novel and useful tool for the exploration of local STEC-host interactions in the gut environment and the pathogenesis of Shiga toxicosis.

A computational solution to improve biomarker reproducibility during long-term projects.

Biomarkers are fundamental to basic and clinical research outcomes by reporting host responses and providing insight into disease pathophysiology. Measuring biomarkers with research-use ELISA kits is universal, yet lack of kit standardization and unexpected lot-to-lot variability presents analytic challenges for long-term projects. During an ongoing two-year project measuring plasma biomarkers in cancer patients, control concentrations for one biomarker (PF) decreased significantly after changes in ELISA kit lots. A comprehensive operations review pointed to standard curve shifts with the new kits, an analytic variable that jeopardized data already collected on hundreds of patient samples. After excluding other reasonable contributors to data variability, a computational solution was developed to provide a uniform platform for data analysis across multiple ELISA kit lots. The solution (ELISAtools) was developed within open-access R software in which variability between kits is treated as a batch effect. A defined best-fit Reference standard curve is modelled, a unique Shift factor "S" is calculated for every standard curve and data adjusted accordingly. The averaged S factors for PF ELISA kit lots #1-5 ranged from -0.086 to 0.735, and reduced control inter-assay variability from 62.4% to <9%, within quality control limits. S factors calculated for four other biomarkers provided a quantitative metric to monitor ELISAs over the 10 month study period for quality control purposes. Reproducible biomarker measurements are essential, particularly for long-term projects with valuable patient samples. Use of research-use ELISA kits is ubiquitous and judicious use of this computational solution maximizes biomarker reproducibility.

Dextran Sulfate Sodium Colitis Facilitates Colonization with Shiga Toxin-Producing Escherichia coli: a Novel Murine Model for the Study of Shiga Toxicosis.

Shiga toxin-producing Escherichia coli (STEC) bacteria are globally important gastrointestinal pathogens causing hemorrhagic gastroenteritis with variable progression to potentially fatal Shiga toxicosis. Little is known about the potential effects of E. coli-derived Shiga-like toxins (STXs) on host gastrointestinal immune responses during infection, in part due to the lack of a reproducible immunocompetent-animal model of STEC infection without depleting the commensal microbiota. Here, we describe a novel and reproducible murine model utilizing dextran sulfate sodium (DSS) colitis to induce susceptibility to colonization with clinical-isolate STEC strains. After exposure to DSS and subsequent oral STEC challenge, all the mice were colonized, and 66% of STEC-infected mice required early euthanasia. Morbidity during STEC infection, but not infection with an isogenic STEC mutant with toxin deleted, was associated with increased renal transcripts of the injury markers KIM1 and NGAL, histological evidence of renal tubular injury, and increased renal interleukin 6 gene (IL-6) and CXCL1 inflammatory transcripts. Interestingly, the intestinal burden of STEC during infection was increased compared to its isogenic Shiga toxin deletion strain. Increased bacterial burdens during Shiga toxin production coincided with decreased induction of colonic IL-23 axis transcripts known to be critical for clearance of similar gastrointestinal pathogens in mice, suggesting a previously undescribed role for STEC Shiga toxins in suppressing host immune responses during STEC infection and survival. The DSS+STEC model establishes infection with clinical-isolate strains of STEC in immunocompetent mice without depleting the gastrointestinal microbiota, enabling characterization of the effects of STXs on the IL-23 axis and other gastrointestinal pathogen-host interactions.

Quiescent complement in nonhuman primates during E coli Shiga toxin-induced hemolytic uremic syndrome and thrombotic microangiopathy.

Enterohemorrhagic Escherichia coli (EHEC) produce ribosome-inactivating Shiga toxins (Stx1, Stx2) responsible for development of hemolytic uremic syndrome (HUS) and acute kidney injury (AKI). Some patients show complement activation during EHEC infection, raising the possibility of therapeutic targeting of complement for relief. Our juvenile nonhuman primate (Papio baboons) models of endotoxin-free Stx challenge exhibit full spectrum HUS, including thrombocytopenia, hemolytic anemia, and AKI with glomerular thrombotic microangiopathy. There were no significant increases in soluble terminal complement complex (C5b-9) levels after challenge with lethal Stx1 (n = 6) or Stx2 (n = 5) in plasma samples from T0 to euthanasia at 49.5 to 128 hours post-challenge. d-dimer and cell injury markers (HMGB1, histones) confirmed coagulopathy and cell injury. Thus, complement activation is not required for the development of thrombotic microangiopathy and HUS induced by EHEC Shiga toxins in these preclinical models, and benefits or risks of complement inhibition should be studied further for this infection.

Distinct physiologic and inflammatory responses elicited in baboons after challenge with Shiga toxin type 1 or 2 from enterohemorrhagic Escherichia coli.

Shiga toxin-producing Escherichia coli is a principal source of regional outbreaks of bloody diarrhea and hemolytic-uremic syndrome in the United States and worldwide. Primary bacterial virulence factors are Shiga toxin types 1 and 2 (Stx1 and Stx2), and we performed parallel analyses of the pathophysiologies elicited by the toxins in nonhuman primate models to identify shared and unique consequences of the toxemias. After a single intravenous challenge with purified Stx1 or Stx2, baboons (Papio) developed thrombocytopenia, anemia, and acute renal failure with loss of glomerular function, in a dose-dependent manner. Differences in the timing and magnitude of physiologic responses were observed between the toxins. The animals were more sensitive to Stx2, with mortality at lower doses, but Stx2-induced renal injury and mortality were delayed 2 to 3 days compared to those after Stx1 challenge. Multiplex analyses of plasma inflammatory cytokines revealed similarities (macrophage chemoattractant protein 1 [MCP-1] and tumor necrosis factor alpha [TNF-alpha]) and differences (interleukin-6 [IL-6] and granulocyte colony-stimulating factor [G-CSF]) elicited by the toxins with respect to the mediator induced and timing of the responses. Neither toxin induced detectable levels of plasma TNF-alpha. To our knowledge, this is the first time that the in vivo consequences of the toxins have been compared in a parallel and reproducible manner in nonhuman primates, and the data show similarities to patient observations. The availability of experimental nonhuman primate models for Stx toxemias provides a reproducible platform for testing antitoxin compounds and immunotherapeutics with outcome criteria that have clinical meaning.

PROC, PROCR and PROS1 polymorphisms, plasma anticoagulant phenotypes, and risk of cardiovascular disease and mortality in older adults: the Cardiovascular Health Study.

BACKGROUND AND OBJECTIVES: Genes encoding protein C anticoagulant pathways are candidates for atherothrombotic and other aging-related disorders. METHODS: Using a tagSNP approach, and data from the Cardiovascular Health Study (CHS), we assessed associations of common polymorphisms of PROC, PROS1 and PROCR with: (i) plasma protein C, soluble protein C endothelial receptor (sEPCR) and protein S levels measured in a subsample of 336 participants at study entry; and (ii) risk of incident clinical outcomes [coronary heart disease (CHD), stroke, and mortality] in 4547 participants during follow-up. Secondarily, we explored associations between plasma protein C, protein S and sEPCR levels and other candidate genes involved in thrombosis, inflammation, and aging. RESULTS: The PROCR Ser219Gly polymorphism (rs867186) was strongly associated with higher sEPCR levels, explaining 75% of the phenotypic variation. The PROCR Ser219Gly variant was also associated with higher levels of circulating protein C antigen. An IL10 polymorphism was associated with higher free protein S levels. The minor alleles of PROC rs2069901 and PROS1 rs4857343 were weakly associated with lower protein C and free protein S levels, respectively. There was no association between PROCR Ser219Gly and risk of CHD, stroke, or mortality. The minor allele of another common PROCR tagSNP, rs2069948, was associated with lymphoid PROCR mRNA expression and with increased risk of incident stroke and all-cause mortality, and decreased healthy survival during follow-up. CONCLUSIONS: A common PROCR variant may be associated with decreased healthy survival in older adults. Additional studies are warranted to establish the role of PROCR variants in ischemic and aging-related disorders.

EPCR Ser219Gly: elevated sEPCR, prothrombin F1+2, risk for coronary heart disease, and increased sEPCR shedding in vitro.

We have progressively analysed three studies of coronary heart disease (CHD) for a variant in EPCR (Ser219Gly). Initially, in a prospective study, NPHSII, while no overall CHD-risk was identified in heterozygotes, homozygotes for 219Gly exhibited a three-fold elevated risk (HR 3.3, CI 1.22-8.96). In diabetics within NPHSII, there was a suggestion that 219Gly+ was associated with elevated CHD-risk (HR 1.89, CI 0.39-9.06) although numbers were small. To further assess the effect of the variant in diabetes, a case-control study of MI, HIFMECH, was used, in which previous analysis had defined a group with metabolic syndrome, by factor analysis. A significant CHD-risk interaction was identified between genotype and the 'metabolic syndrome' factor (interaction p=0.009). To further assess CHD-risk for this variant in type-2 diabetes and to assess the effect of the variant upon thrombin generation and plasma levels of soluble EPCR, a cross-sectional study of type-2 diabetes was used. A significant CHD-risk was identified for European Whites (OR 2.84, CI 1.38-5.85) and Indian Asians in this study (OR 1.6, CI 1.00-2.57) and the frequency of 219Gly was two-fold higher in Indian Asians. Soluble EPCR levels were strongly associated with genotype, with homozygotes for 219Gly having four-fold higher levels (p<0.0001). In vitro studies of EPCR-transfected cells suggested increased basal release of sEPCR from cells expressing the 219Gly EPCR phenotype. Furthermore, in base-line samples from NPHSII and in the diabetic study, a significant increase in prothrombin F1+2 level was observed for 219Gly. The increased CHD-risk and thrombin generation appears to be acting through increased shedding of the Gly allele from the cell surface.

Reduced neutrophil CD10 expression in nonhuman primates and humans after in vivo challenge with E. coli or lipopolysaccharide.

CD10, also known as neutral endopeptidase or CALLA, is a major metalloproteinase that regulates levels of biologically active peptides that initiate inflammatory, cardiovascular, and neurogenic responses. Relative tissue expression levels of CD10, its peptide substrates, and their receptors constitute the basic regulatory mechanism. Neutrophils contain abundant CD10 and are rapid responders to an inflammatory septic challenge. Expression of neutrophil surface antigens in response to inflammation was studied in the primate model of Escherichia coli-mediated sepsis and in human volunteers injected with lipopolysaccharide (LPS). There was a rapid and profound (up to 95%) reduced baboon neutrophil CD10 expression in response to E. coli injections of 5.71 x 106 CFU/kg to 2.45 x 109 CFU/kg that gradually resolved to preinjection levels. The reduction was both dose and time dependent. Reduced CD10 antigen on mature baboon neutrophils and bands was observed by immunohistochemistry. Human volunteers challenged with 4ng/kg LPS experienced transient chills, nausea, fever, and myalgia. Up to approximately 20% of their neutrophils had reduced CD10 expression, peaking at 2 to 8 h after injection. By 24 h, neutrophil CD10 expression resolved to preinjection levels. In contrast, in both the baboon and human studies, other neutrophil surface antigens were only slightly decreased (CD11a) or increased (CD11b, CD18, CD35, CD66b, and CD63). These data present the novel observation that neutrophil CD10 expression decreases significantly in response to in vivo inflammatory challenge. This decrease appears to be unique to CD10 and may contribute to a reduced regulation of bioactive peptides released in response to inflammatory challenge.

Plasma levels of soluble endothelial cell protein C receptor in patients with Wegener's granulomatosis.

Elevated soluble thrombomodulin (sTM) levels are an accepted marker of endothelial damage. The physiological significance of plasma endothelial protein C receptor (sEPCR) levels is not known. To assess the relevance of this plasma protein in Wegener's granulomatosis (WG), sEPCR levels were measured in sera obtained from WG patients and related to disease activity, sTM levels, and other known markers of disease activity. In total, 129 sera (37 at active disease, 92 during follow-up) from 31 WG patients were tested. During active disease, eight (22%) and 17 (46%) out of 37 active sera had elevated levels of sEPCR and sTM, respectively (NS); sEPCR (r = 0.39; P = 0.02) and sTM (r = 0.53; P <0.01) levels correlated with disease activity (Birmingham Vasculitis Activity Score). Analysis of longitudinal sera revealed a significant increase in sEPCR (P = 0.01) and sTM (P = 0.04) levels prior to the moment of a relapse. Corrected for renal function, the increase in sEPCR remained significant (P =0.04) whereas sTM did not (NS). Levels of sEPCR correlated with sTM levels (r = 0.32; P < 0.001). Plasma levels of sEPCR respond to changes in the disease in patients with WG.

The soluble endothelial protein C receptor binds to activated neutrophils: involvement of proteinase-3 and CD11b/CD18.

The protein C pathway is a primary regulator of blood coagulation and a critical component of the host response to inflammatory stimuli. The most recent member of this pathway is the endothelial protein C receptor (EPCR), a type I transmembrane protein with homology to CD1d/MHC class I proteins. EPCR accelerates formation of activated protein C, a potent anticoagulant and antiinflammatory agent. The current study demonstrates that soluble EPCR binds to PMA-activated neutrophils. Using affinity chromatography, binding studies with purified components, and/or blockade with specific Abs, it was found that soluble EPCR binds to proteinase-3 (PR3), a neutrophil granule proteinase. Furthermore, soluble EPCR binding to neutrophils was partially dependent on Mac-1 (CD11b/CD18), a beta(2) integrin involved in neutrophil signaling, and cell-cell adhesion events. PR3 is involved in multiple diverse processes, including hemopoietic proliferation, antibacterial activity, and autoimmune-mediated vasculitis. The observation that soluble EPCR binds to activated neutrophils via PR3 and a beta(2) integrin suggests that there may be a link between the protein C anticoagulant pathway and neutrophil functions.

The endothelial cell protein C receptor aids in host defense against Escherichia coli sepsis.

The influence of the endothelial protein C receptor (EPCR) on the host response to Escherichia coli was studied. Animals were treated with 4 separate protocols for survival studies and analysis of physiologic and biochemical parameters: (1) monoclonal antibody (mAb) that blocks protein C/activated protein C binding to EPCR plus sublethal numbers of E coli (SLEC) (n = 4); (2) mAb to EPCR that does not block binding plus SLEC (n = 3); (3) SLEC alone (n = 4); and (4) blocking mAB alone (n = 1). Those animals receiving blocking mAb to EPCR plus sublethal E coli died 7 to 54 hours after challenge, whereas all animals treated with the other protocols were permanent survivors. Histopathologic studies of tissues from animals receiving blocking mAb plus SLEC removed at postmortem were compared with those animals receiving SLEC alone killed at T+24 hours. The animals receiving the blocking mAb exhibited consumption of fibrinogen, microvascular thrombosis with hemorrhage of both the adrenal and renal cortex, and an intense influx of neutrophils into the adrenal, renal, and hepatic microvasculature, whereas the tissues from animals receiving only sublethal E coli exhibited none of these abnormal histopathologic changes. Compared with the control animals, the animals receiving the blocking mAb exhibited significantly elevated serum glutamic pyruvic transaminase, anion gap, thrombin-antithrombin complex, IL-6, IL-8, and soluble thrombomodulin. The levels of circulating activated protein C varied too widely to allow a clear determination of whether the extent of protein C activation was altered in vivo by blocking protein C binding to EPCR. We conclude that protein C/activated protein C binding to EPCR contributes to the negative regulation of the coagulopathic and inflammatory response to E coli and that EPCR provides an additional critical step in the host defense against E coli. (Blood. 2000;95:1680-1686)

Regulation and functions of the protein C anticoagulant pathway.

The protein C pathway plays a critical role in the negative regulation of the blood clotting process. We recently identified an endothelial cell receptor for protein C/activated protein C (APC). The receptor is localized almost exclusively on endothelial cells of large vessels and is present at only trace levels or indeed absent from capillaries in most tissues. Patients with sepsis or lupus erythematosus exhibit elevated levels of plasma EPCR which migrates on gels as a single band and is fully capable of binding protein C/APC. There is no correlation with thrombomodulin levels, probably due to different vascular localizations and/or cellular release mechanisms. To understand the mechanisms by which EPCR plasma levels are elevated, we examined EPCR mRNA expression in a rat endotoxin shock model. The EPCR mRNA gene exhibited an early immediate gene response to endotoxin with the mRNA levels increasing nearly 4 fold in the first 3-6 hrs, before returning toward baseline. Plasma levels of EPCR also rose about 4 fold with little change in tissue EPCR levels. Both processes were markedly attenuated by hirudin suggesting that thrombin was responsible for increases in mRNA and plasma EPCR levels. At the level of mRNA, the induction is mediated by a thrombin response element in the 5' flanking region of the gene. Direct thrombin infusion and cell culture experiments support this contention. On endothelium, thrombin is capable of releasing cell surface EPCR and this process is blocked by the metalloproteinase inhibitor orthophenanthroline. Taken together these studies indicate that elevation in soluble plasma EPCR reflects endothelial cell activation in the larger vessels and is likely to be an indication of local thrombin generation near these vessel surfaces.

Inflammation, sepsis, and coagulation.

The molecular links between inflammation and coagulation are unquestioned. Inflammation promotes coagulation by leading to intravascular tissue factor expression, eliciting the expression of leukocyte adhesion molecules on the intravascular cell surfaces, and down regulating the fibrinolytic and protein C anticoagulant pathways. Thrombin, in turn, can promote inflammatory responses. This creates a cycle that logically progresses to vascular injury as occurs in septic shock. Most complex systems are regulated by product inhibition. This inflammation-coagulation cycle seems to follow this same principle with the protein C pathway serving as the regulatory mechanism. The molecular basis by which the protein C pathway functions as an anticoagulant is relatively well established compared to the mechanisms involved in regulating inflammation. As one approach to identifying the mechanisms involved in regulating inflammation, we set out to identify novel receptors that could modulate the specificity of APC in a manner analogous to the mechanisms by which thrombomodulin modulates thrombin specificity. This approach led to the identification of an endothelial cell protein C receptor (EPCR). To understand the mechanism, we obtained a crystal structure of APC (lacking the Gla domain). The crystal structure reveals a deep groove in a location analogous to anion binding exosite 1 of thrombin, the location of interaction for thrombomodulin, platelet thrombin receptor and fibrinogen. Thrombomodulin blocks the activation of platelets and fibrinogen without blocking reactivity with chromogenic substrates or inhibitors. Similarly, in solution, EPCR blocks factor Va inactivation without modulating reactivity with protease inhibitors. Thus, these endothelial cell receptors for the protein C system share many properties in common including the ability to be modulated by inflammatory cytokines. Current studies seek to identify the substrate for the APC-EPCR complex as the next step in elucidating the mechanisms by which the protein C pathway modulates the response to injury and inflammation.

A chimeric protein C containing the prothrombin Gla domain exhibits increased anticoagulant activity and altered phospholipid specificity.

To determine the structural basis of phosphatidylethanolamine (PE)-dependent activated protein C (APC) activity, we prepared a chimeric molecule in which the Gla domain and hydrophobic stack of protein C were replaced with the corresponding region of prothrombin. APC inactivation of factor Va was enhanced 10-20-fold by PE. Protein S enhanced inactivation 2-fold and independently of PE. PE and protein S had little effect on the activity of the chimera. Factor Va inactivation by APC was approximately 5-fold less efficient than with the chimera on vesicles lacking PE and slightly more efficient on vesicles containing PE. The cleavage patterns of factor Va by APC and the chimera were similar, and PE enhanced the rate of Arg506 and Arg306 cleavage by APC but not the chimera. APC and the chimera bound to phosphatidylserine:phosphatidylcholine vesicles with similar affinity (Kd approximately 500 nM), and PE increased affinity 2-3-fold. Factor Va and protein S synergistically increased the affinity of APC on vesicles without PE to 140 nM and with PE to 14 nM, but they were less effective in enhancing chimera binding to either vesicle. In a factor Xa one-stage plasma clotting assay, the chimera had approximately 5 times more anticoagulant activity than APC on PE-containing vesicles. Unlike APC, which showed a 10 fold dependence on protein S, the chimera was insensitive to protein S. To map the site of the PE and protein S dependence further, we prepared a chimera in which residues 1-22 were derived from prothrombin and the remainder were derived from protein C. This protein exhibited PE and protein S dependence. Thus, these special properties of the protein C Gla domain are resident outside of the region normally hypothesized to be critical for membrane interaction. We conclude that the protein C Gla domain possesses unique properties allowing synergistic interaction with factor Va and protein S on PE-containing membranes.

Identification of functional endothelial protein C receptor in human plasma.

The endothelial cell protein C receptor (EPCR) binds protein C and facilitates activation by the thrombin-thrombomodulin complex. EPCR also binds activated protein C (APC) and inhibits APC anticoagulant activity. In this study, we detected a soluble form of EPCR in normal human plasma. Plasma EPCR appears to be approximately 43, 000 D, and circulates at approximately 100 ng/ml (98.4+/-27.8 ng/ml, n = 22). Plasma EPCR was purified from human citrated plasma using ion exchange, immunoaffinity, and protein C affinity chromatography. Flow cytometry experiments demonstrated that plasma EPCR bound APC with an affinity similar to that previously determined for recombinant soluble EPCR (Kdapp = 30 nM). Furthermore, plasma EPCR inhibited both protein C activation on an endothelial cell line and APC anticoagulant activity in a one-stage Factor Xa clotting assay. The physiological function of plasma EPCR is uncertain, but if the local concentrations are sufficiently high, particularly in disease states, the present data suggest that the soluble plasma EPCR could attenuate the membrane-bound EPCR augmentation of protein C activation and the anticoagulant function of APC.

The endothelial cell protein C receptor augments protein C activation by the thrombin-thrombomodulin complex.

Protein C activation on the surface of the endothelium is critical to the negative regulation of blood coagulation. We now demonstrate that monoclonal antibodies that block protein C binding to the endothelial cell protein C receptor (EPCR) reduce protein C activation rates by the thrombin-thrombomodulin complex on endothelium, but that antibodies that bind to EPCR without blocking protein C binding have no effect. The kinetic result of blocking the EPCR-protein C interaction is an increased apparent Km for the activation without altering the affinity of thrombin for thrombomodulin. Activation rates of the protein C derivative lacking the gamma-carboxyglutamic acid domain, which is required for binding to EPCR, are not altered by the anti-EPCR antibodies. These data indicate that the protein C activation complex involves protein C, thrombin, thrombomodulin, and EPCR. These observations open new questions about the control of coagulation reactions on vascular endothelium.