Complexes between C-Reactive Protein and Very Low Density Lipoprotein Delay Bacterial Clearance in Sepsis.

C-reactive protein (CRP) can increase up to 1000-fold in blood and form complexes with very low density lipoproteins (VLDL). These complexes are associated with worse outcomes for septic patients, and this suggests a potential pathological role in sepsis. Complex formation is heightened when CRP is over 200 mg/l and levels are associated with the severity of sepsis and blood bacterial culture positivity. Using a mouse bacteremia model, blood bacterial clearance can be delayed by i.v. injection of CRP-VLDL complexes. Complexes are more efficiently taken up by activated U937 cells in vitro and Kupffer cells in vivo than VLDL alone. Both in vitro-generated and naturally occurring CRP-VLDL complexes reduce phagocytosis of bacteria by activated U937 cells. Fcγ and scavenger receptors are involved and a competitive mechanism for clearance of CRP-VLDL complexes and bacteria is demonstrated. Interaction of phosphocholine groups on VLDL with CRP is the major driver for complex formation and phosphocholine can disrupt the complexes to reverse their inhibitory effects on phagocytosis and bacterial clearance. Increased formation of CRP-VLDL complexes is therefore harmful and could be a novel target for therapy in sepsis.

Procalcitonin and Other Common Biomarkers Do Not Reliably Identify Patients at Risk for Bacterial Infection After Congenital Heart Surgery.

OBJECTIVES: Following surgery, it is difficult to distinguish a postoperative inflammatory reaction from infection. This study examined the predictive value of the biomarkers; procalcitonin, C-reactive protein, lactate, neutrophils, lymphocytes, platelets, and the biphasic activated partial thromboplastin time waveform in diagnosing bacterial infection following cardiac surgery. DESIGN: Prospective, observational study. SETTING: A regional, PICU in the United Kingdom. PATIENTS: Three-hundred sixty-eight children under the age of 16 admitted to the PICU for elective cardiac surgery were enrolled in the study. INTERVENTIONS: All biomarker measurements were determined daily until postoperative day 7. Children were assessed for postoperative infection until day 28 and divided into four groups: bacterial infection, culture-negative sepsis, viral infection, and no infection. We used the Kruskal-Wallis test, chi-square test, analysis of variance, and area under the curve in our analysis. MEASUREMENTS AND MAIN RESULTS: In total, 71 of 368 children (19%) developed bacterial infection postoperatively, the majority being surgical site infections. In those with bacterial infection, procalcitonin was elevated on postoperative days 1-3 and the last measurement prior to event compared with those without bacterial infection. The most significant difference was the last measurement prior to event; 0.72 ng/mL in the bacterial infection group versus 0.13 ng/mL in the no infection group (for all groups; p < 0.001). Longitudinal profiles of all biomarkers were indistinct in the bacterial infection and nonbacterial infection groups except in those with culture-negative infections who had distinct procalcitonin kinetics on postoperative days 1-4. Children with culture-negative sepsis required longer ventilatory support and PICU stay and were more likely to develop complications than the other groups. CONCLUSIONS: None of the biomarkers studied within 3 days of infection distinguished between infection and postoperative inflammatory reaction. However, procalcitonin kinetics peaked on postoperative day 2 and fell more sharply than C-reactive protein kinetics, which peaked at postoperative day 3. The monitoring of procalcitonin kinetics following cardiac surgery may help guide rational antimicrobial use.

Loss of endothelial protein C receptors links coagulation and inflammation to parasite sequestration in cerebral malaria in African children.

Cerebral malaria (CM) is a major cause of mortality in African children and the mechanisms underlying its development, namely how malaria-infected erythrocytes (IEs) cause disease and why the brain is preferentially affected, remain unclear. Brain microhemorrhages in CM suggest a clotting disorder, but whether this phenomenon is important in pathogenesis is debated. We hypothesized that localized cerebral microvascular thrombosis in CM is caused by a decreased expression of the anticoagulant and protective receptors thrombomodulin (TM) and endothelial protein C receptor (EPCR) and that low constitutive expression of these regulatory molecules in the brain make it particularly vulnerable. Autopsies from Malawian children with CM showed cerebral fibrin clots and loss of EPCR, colocalized with sequestered IEs. Using a novel assay to examine endothelial phenotype ex vivo using subcutaneous microvessels, we demonstrated that loss of EPCR and TM at sites of IE cytoadherence is detectible in nonfatal CM. In contrast, although clotting factor activation was seen in the blood of CM patients, this was compensated and did not disseminate. Because of the pleiotropic nature of EPCR and TM, these data implicate disruption of the endothelial protective properties at vulnerable sites and particularly in the brain, linking coagulation and inflammation with IE sequestration.

Assessment and clinical course of hypocalcemia in critical illness.

INTRODUCTION: Hypocalcemia is common in critically ill patients. However, its clinical course during the early days of admission and the role of calcium supplementation remain uncertain, and the assessment of calcium status is inconsistent. We aimed to establish the course of hypocalcemia during the early days of critical illness in relation to mortality and to assess the impact of calcium supplementation on calcium normalization and mortality. METHODS: Data were collected on 1,038 admissions to the critical care units of a tertiary care hospital. One gram of calcium gluconate was administered intravenously once daily to patients with adjusted calcium (AdjCa)<2.2 mmol/L. Demographic and outcome data were compared in normocalcemic (ionized calcium, iCa, 1.1-1.3 mmol/L) and mildly and severely hypocalcemic patients (iCa 0.9-1.1 mmol/L and <0.9 mmol/L, respectively). The change in iCa concentrations was monitored during the first four days of admission and comparisons between groups were made using Repeated Measures ANOVA. Comparisons of normalization and outcome were made between hypocalcemic patients who did and did not receive calcium replacement according to the local protocol. The suitability of AdjCa to predict low iCa was determined by analyzing sensitivity, specificity and receiver operating characteristic (ROC) curves. Multivariate logistic regression was performed to determine associations of other electrolyte derangements with hypocalcemia. RESULTS: 55.2% of patients were hypocalcemic on admission; 6.2% severely so. Severely hypocalcemic patients required critical care for longer (P=0.001) compared to normocalcemic or mildly hypocalcemic patients, but there was no difference in mortality between groups (P=0.48). iCa levels normalized within four days in most, with no difference in normalization between those who died and survived (P=0.35). Severely hypocalcemic patients who failed to normalize their iCa by day 4 had double the mortality (38% vs. 19%, P=0.15). Neither iCa normalization nor survival were superior in hypocalcemic patients receiving supplementation on admission. AdjCa<2.2 mmol/L had a sensitivity of 78.2% and specificity of 63.3% for predicting iCa<1.1 mmol/L. Low magnesium, sodium and albumin were independently associated with hypocalcemia on admission. CONCLUSIONS: Hypocalcemia usually normalizes within the first four days after admission to ICU and failure to normalize in severely hypocalcemic patients may be associated with increased mortality. Calcium replacement appears not to improve normalization or mortality. AdjCa is not a good surrogate of iCa in an ICU setting.

The clinical and functional relevance of microparticles induced by activated protein C treatment in sepsis.

INTRODUCTION: Activated protein C (APC) induces release of microparticles (MP) from primary physiological cells, which are found in patients undergoing treatment with recombinant human APC (rhAPC) for severe sepsis. We hypothesised that APC on these circulating MPs activate endothelial protease-activated receptor 1 (PAR1) to induce anti-apoptotic and anti-inflammatory properties that can improve patient outcome. METHODS: This was an experimental study on clinical samples in an intensive care setting, and included patients with severe sepsis who fulfilled criteria for treatment with rhAPC. The number of CD13+ MPs from the patients were analysed to determine their origin. They were also quantified for endothelial protein C receptor (EPCR) and APC expression. Clinical relevance of these MPs were ascertained by comparing survival between the group receiving rhAPC (n = 25) and a control group of untreated patients (n = 25). MPs were also incubated with endothelial cells to analyse apoptotic gene expression, cytoprotection and anti-inflammatory effects. RESULTS: rhAPC treatment induced a significant increase in circulating MP-associated EPCR by flow cytometry (P < 0.05) and by quantitative ELISA (P < 0.005). APC expression also showed significant increases (P < 0.05). Numerically, CD13+ MPs were higher in rhAPC-treated survivors versus non-survivors. However, the number of non-survivors was low and this was not significantly different. APC on MPs was demonstrated to induce anti-apoptotic and endothelial barrier effects through the activation of endothelial PAR1. CONCLUSIONS: rhAPC treatment in patients with sepsis significantly increases circulating EPCR + MPs. These MPs were noted to express APC, which has specific anti-apoptotic and anti-inflammatory effects, with a non-significant correlative trend towards survival. This suggests that MPs could disseminate APC function and activate endothelial PAR1 at distal vascular sites.

Diagnostic efficacy of activated partial thromboplastin time waveform and procalcitonin analysis in pediatric meningococcal sepsis.

OBJECTIVE: A biphasic activated partial thromboplastin time waveform predicts sepsis and disseminated intravascular coagulation in adults. This has not been previously investigated in children. Our aim is to ascertain whether there are changes in the activated partial thromboplastin time waveform in children with meningococcal disease and to compare its diagnostic use with procalcitonin. SETTING: Alder Hey Children's National Health Service Foundation Trust, Liverpool, UK. PATIENTS: Thirty-six children admitted to the hospital for the treatment of suspected meningococcal disease had activated partial thromboplastin time waveform and procalcitonin analysis performed at admission. The light transmittance level at 18 secs was used to quantitate the waveform. Severity of disease was assessed using the Glasgow Meningococcal Septicaemia Prognostic Score, Pediatric Risk of Mortality III score, and the Pediatric Logistic Organ Dysfunction score. MEASUREMENTS AND MAIN RESULTS: Twenty-four children had proven meningococcal disease, 12 had a presumed viral illness, and 20 control subjects were recruited. Transmittance level at 18 secs was lower in children with meningococcal disease and those with a viral illness (p < .0001) and control subjects (p < .0005). Sensitivity and specificity was 0.91 and 0.96 for transmittance level at 18 secs and 0.92 and 1 for procalcitonin in identifying meningococcal disease. There was a significant difference in procalcitonin between children with meningococcal disease and those with a viral illness and control subjects (p < .0005). A negative correlation was found between transmittance level at 18 secs and length of hospital stay (p < .0001), C-reactive protein (p < .0001), procalcitonin (p < .0001), Glasgow Meningococcal Septicaemia Prognostic Score (p < .01), Pediatric Risk of Mortality III score (p < .0001), and Pediatric Logistic Organ Dysfunction score score (p < .0001). CONCLUSION: The activated partial thromboplastin time waveform is abnormal in children with meningococcal disease and may be a useful adjunct in the diagnosis and management of sepsis in children.

Procalcitonin, C-reactive protein, neutrophil gelatinase-associated lipocalin, resistin and the APTT waveform for the early diagnosis of serious bacterial infection and prediction of outcome in critically ill children.

OBJECTIVE: Bacterial Infections remains a leading cause of death in the Paediatric Intensive Care Unit (PICU). In this era of rising antimicrobial resistance, new tools are needed to guide antimicrobial use. The aim of this study was to investigate the accuracy of procalcitonin (PCT), neutrophil gelatinase-associated lipocalin (NGAL), resistin, activated partial thromboplastin time (aPTT) waveform and C-reactive protein (CRP) for the diagnosis of serious bacterial infection (SBI) in children on admission to PICU and their use as prognostic indicators. SETTING: A regional PICU in the United Kingdom. PATIENTS: Consecutive PICU admissions between October 2010 and June 2012. MEASUREMENTS: Blood samples were collected daily for biomarker measurement. The primary outcome measure was performance of study biomarkers for diagnosis of SBI on admission to PICU based on clinical, radiological and microbiological criteria. Secondary outcomes included durations of PICU stay and invasive ventilation and 28-day mortality. Patients were followed up to day 28 post-admission. MAIN RESULTS: A total of 657 patients were included in the study. 92 patients (14%) fulfilled criteria for SBI. 28-day mortality was 2.6% (17/657), but 8.7% (8/92) for patients with SBI. The combination of PCT, resistin, plasma NGAL and CRP resulted in the greatest net reclassification improvement compared to CRP alone (0.69, p<0.005) with 10.5% reduction in correct classification of patients with SBI (p 0.52) but a 78% improvement in correct classification of patients without events (p <0.005). A statistical model of prolonged duration of PICU stay found log-transformed maximum values of biomarkers performed better than first recorded biomarkers. The final model included maximum values of CRP, plasma NGAL, lymphocyte and platelet count (AUC 79%, 95% CI 73.7% to 84.2%). Longitudinal profiles of biomarkers showed PCT levels to decrease most rapidly following admission SBI. CONCLUSION: Combinations of biomarkers, including PCT, may improve accurate and timely identification of SBI on admission to PICU.

Microparticle-associated endothelial protein C receptor and the induction of cytoprotective and anti-inflammatory effects.

BACKGROUND: The endothelial protein C receptor plays an important role within the protein C pathway in regulating coagulation and inflammation. Recently, we described that endothelial protein C receptor can be released in vitro in microparticulate form from primary endothelial cells by exogenous activated protein C. Activated protein C bound to this endothelial protein C receptor retains anticoagulant activity and we hypothesize that this microparticulate endothelial protein C receptor-activated protein C complex can also cleave endothelial protease-activated receptor 1 to modulate inflammation and increase cell survival. Our main objective was, therefore, to study the effect that microparticle-associated endothelial protein C receptor-activated protein C has on endothelial function. DESIGN AND METHODS: Mini-arrays were used and probed with cDNA obtained from endothelial cells after treatment with microparticle-associated endothelial protein C receptor-activated protein C and results were confirmed by real time polymerase chain reaction. The functional relevance of changes at gene level were further analyzed by endothelial apoptosis and permeability assays, in the presence and absence of specific blockade of endothelial protein C receptor, protein C and protease-activated receptor 1. RESULTS: Gene profiling of endothelial cells stimulated by 40 nmol/L activated protein C on microparticles showed significant changes in anti-apoptotic and inflammatory pathways. This was accompanied by protease-activated receptor 1-dependent anti-apoptotic and barrier protective effects, the latter of which also involved sphingosine 1-phosphate receptor and vascular endothelial growth factor receptor-2/ kinase insert domain receptor. Protein C blockade reversed these effects showing specificity for activated protein C on microparticles. Furthermore, confocal microscopy and enzyme-linked immunosorbent assay of plasma obtained from septic patients during recombinant activated protein C treatment showed evidence of their presence in vivo. CONCLUSIONS: Activated protein C on microparticle-associated endothelial protein C receptor release can induce protease-activated receptor 1-dependent endothelial effects. The mechanisms underlying barrier protection involve sphingosine 1-phosphate receptor and kinase insert domain receptor.

Influence of stimulus size on revealing non-cardinal color mechanisms.

Multiple studies have shown that performance of subjects on a number of visual tasks is worse for non-cardinal than cardinal colors, especially in the red-green/luminance (RG/LUM) and tritan/luminance (TRIT/LUM) color planes. Inspired by neurophysiological evidence that suppressive surround input to receptive fields is particularly sensitive to luminance, we hypothesized that non-cardinal mechanisms in the RG/LUM and TRIT/LUM planes would be more sensitive to stimulus size than are isoluminant non-cardinal mechanisms. In Experiment 1 we tested 9-10 color-normal subjects in each of the three color planes (RG/TRIT, RG/LUM, and TRIT/LUM) on visual search at four bull's-eye dot sizes (0.5°/1°, 1°/2°, 2°/4°, and 3°/6° center/annulus dot diameter). This study yielded a significant main effect of dot size in each of the three color planes. In Experiment 2 we tested the same hypothesis using noise masking, at three stimulus sizes (3°, 6° and 9° diameter Gabors), again in all three color planes (5 subjects per color plane). This experiment yielded, in the RG/TRIT plane, a significant main effect of stimulus size; in the RG/LUM plane, significant evidence for non-cardinal mechanisms only for the 9° stimulus; but in the TRIT/LUM plane no evidence for non-cardinal mechanisms at any stimulus size. These results suggest that non-cardinal mechanisms, particularly in the RG/LUM color plane, are more sensitive to stimulus size than are non-cardinals in the RG/TRIT plane, supporting our hypothesis.

Arterial ischemic stroke in HIV: Defining and classifying etiology for research studies.

HIV infection, and potentially its treatment, increases the risk of an arterial ischemic stroke. Multiple etiologies and lack of clear case definitions inhibit progress in this field. Several etiologies, many treatable, are relevant to HIV-related stroke. To fully understand the mechanisms and the terminology used, a robust classification algorithm to help ascribe the various etiologies is needed. This consensus paper considers the strengths and limitations of current case definitions in the context of HIV infection. The case definitions for the major etiologies in HIV-related strokes were refined (e.g., varicella zoster vasculopathy and antiphospholipid syndrome) and in some instances new case definitions were described (e.g., HIV-associated vasculopathy). These case definitions provided a framework for an algorithm to help assign a final diagnosis, and help classify the subtypes of HIV etiology in ischemic stroke.

Back to the future: testing in disseminated intravascular coagulation.

Following on from the first clinical observations on disseminated intravascular coagulation (DIC) in the nineteenth century, the dawn of laboratory testing for DIC began with the availability of assays that characterized the extrinsic and intrinsic pathways of coagulation. Markedly increased clotting times were the hallmark of DIC. As the understanding of the biochemistry of haemostasis and thrombosis improved, new tests were developed based on the molecular players that participate in the process. However, many are non-specific for DIC and/or are unwieldy in performance to keep apace with the demands of the acute clinical setting. The renewed emphasis in DIC for the modern laboratory of the twenty-first century has seen a return to the simple, rapid and practical global tests of coagulation within scoring systems that also capture the pathophysiological continuum by trend analysis. Additionally, new technologies based on these simple tests of coagulation hold promise in also indicating the in vivo interplay between coagulation and inflammation during DIC.

Performance and prognostic importance of a new clinical and laboratory scoring system for identifying non-overt disseminated intravascular coagulation.

A template for diagnosing the non-overt phase of disseminated intravascular coagulation (DIC) has recently been proposed. However, validation of its performance and the proposal of a defining score are required. The aim was to assess feasibility of the non-overt DIC scoring template and its potential prognostic significance. Consecutive patients admitted to a university hospital intensive care unit were initially assessed over 2 months. Following this, a 12-month study examined the prognostic performance of the derived scores prospectively. Outcome parameters were overt DIC and 28-day mortality. The 2-month study, involving 66 patients and 919 time points, demonstrated practical feasibility and prognostic associations for mortality with scores of 5 and greater. The 12-month study involving 450 patients showed that the mortality rate was 29% (105 of 360) and 78% (70 of 90) for scores below 5 and scores of 5 or above, respectively. The mortality rate for overt DIC was also 78% (38 of 49). The non-overt DIC scoring template is workable and has prognostic relevance. A score of 5 and greater is recommended as diagnostic of non-overt DIC.

Lipoprotein-complexed C-reactive protein and the biphasic transmittance waveform in critically ill patients.

The 'biphasic transmittance waveform' (BTW) refers to a decrease in light transmittance that often occurs prior to clotting in coagulation assays of critically ill patient plasmas. It correlates with disseminated intravascular coagulation and mortality. The present work shows that the BTW is due to the rapid formation of a precipitate and a coincident change in turbidity in re-calcified plasma. The precipitate was isolated from patient plasma and contained lipids typical of very low density lipoprotein (VLDL), plus the proteins apolipoprotein B-100 and C-reactive protein (CRP). Precipitation also occurred in normal plasma supplemented with CRP. In addition, CRP precipitated with VLDL and intermediate density lipoprotein, but not low density lipoprotein or high density lipoprotein. The Kd value for the CRP/VLDL interaction is 340 nM. The IC50 value of Ca2+ for complex formation is 5.0 mM, and epsilon-aminocaproic acid inhibits the process. In 15 plasmas with the BTW from critically ill patients, CRP was highly elevated (77-398 microg/mL) and VLDL cholesterol ranged from 0.082 to 1.32 mM. The magnitude of the turbidity change on re-calcification correlated well with the calculated level of the CRP/VLDL complex. Thus, the Ca2+-dependent formation of a complex between CRP and VLDL accounts for the BTW.

Thrombin activatable fibrinolysis inhibitor and its fibrinolytic effect in normal pregnancy.

We investigated changes in both thrombin activatable fibrinolysis inhibitor (TAFI) antigen levels and its functional effect on in vitro fibrinolysis in normal pregnancy. 152 pregnant women and 31 women in the immediate postpartum period were studied, with pregnancy divided into 6 windows at 4 weekly intervals. As TAFI influences and is in turn influenced by components of the protein C (PC) pathway, its measurements were correlated with levels of soluble thrombomodulin, PC, protein S (PS) and the overall phenotype of activated PC resistance (APCR). Compared with mean TAFI levels at booking gestation (6.6 +/- 1.2 microg/ml), levels peaked at 35-39 weeks gestation (9.6 +/- 2 microg/ml, p = 0.001), followed by a significant drop within 24 hours of delivery (7.2 +/- 1.1 microg/ml). In functional terms, the mean clot lysis time (CLT) (101 +/- 13 min at booking) also peaked at 35-39 weeks gestation (141 +/- 42 min, p = 0.007) and dropped after delivery (99 +/- 33 min), and was significantly correlated with gestational age (r = 0.410, p = 0.001) and could be abrogated in the presence of an inhibitor to TAFI activation. A significant negative correlation was found between TAFI levels and APCR (r = -0.478, p <0.001), APCRV (r = -0.598; p <0.001), PS (r = -0.490, P <0.001) and PC (r = -0.198, p = 0.02). In summary, there is a significant increase in TAFI levels, which translates into increased CLT during pregnancy. Furthermore, changes in TAFI contribute to the increasing APCR of pregnancy.

Prothrombinase enhancement through quantitative and qualitative changes affecting very low density lipoprotein in complex with C-reactive protein.

The biphasic waveform that can predict for disseminated intravascular coagulation (DIC) is due to the formation of a calcium-dependent complex between C reactive protein (CRP) and very low density lipoprotein (VLDL). As thrombin generation is pivotal to DIC, this aspect has been specifically investigated and the VLDL component has been found to increase prothrombinase activity via both quantitative and qualitative changes. The specific prothrombinase activity of VLDL from patients manifesting the biphasic waveform was 2.5 times that of normal individuals or critically ill patients without the biphasic waveform. This activity was due to an increase in anionic phospholipid surfaces that could be inhibited with excess annexin V and which was dependent on structurally intact apolipoprotein B. The qualitative change appeared to be due to a deficiency of phosphatidylethanolamine in VLDL from patients with the biphasic waveform. The functional consequence of this enhanced prothrombinase activity was an increased procoagulant effect in plasma. Using a modified activated partial thromboplastin time assay, the mean normal clot time decreased significantly when VLDL from patients with biphasic waveforms was substituted. These results indicate that VLDL derived from patients with the biphasic waveform can enhance thrombin procoagulant activity. As the CRP-VLDL complex exists in vivo, it could have a pathogenic role in disseminating the process of intravascular coagulation.

Early identification of sepsis and mortality risks through simple, rapid clot-waveform analysis. Implications of lipoprotein-complexed C reactive protein formation.

OBJECTIVE: To determine if the rapid waveform profile of the activated partial thromboplastin time (aPTT) assay, which detects lipoprotein-complexed C reactive protein (LCCRP) formation, predicts sepsis and mortality in critically ill patients. DESIGN: Observational, cohort study. SETTING: General intensive therapy unit (ITU) of a tertiary care hospital. PATIENTS AND PARTICIPANTS: A total of 1187 consecutive patients admitted to the ITU. INTERVENTION: Activated partial thromboplastin time transmittance waveform analysis was performed within the first hour of admission to the ITU. The degree of change causing a biphasic waveform was quantified through the drop in light transmittance level. MEASUREMENTS AND RESULTS: Three hundred forty-six patients had a biphasic waveform on admission to the ITU with a mortality rate of 44% compared with 26% for those with normal waveforms. Logistic regression models showed direct correlation between the likelihood for sepsis and in-patient mortality with increasing waveform abnormalities. The mortality fraction was 0.3 with normal waveforms versus 0.6 when the light transmittance decreased by 30%. The odds ratio (OR) for mortality and sepsis were 4.5 and 11, respectively, from the most abnormal to normal aPTT waveforms. These were comparable with APACHE II scores and superior to those estimated by CRP for mortality (OR 2.3) / sepsis (OR 6.4) prediction. CONCLUSION: Waveform analysis within the first hour of ITU admission is a single, simple and rapid method of identifying the risks of mortality and sepsis. Its measure of LCCRP formation shows superior prediction over CRP alone and it warrants further assessment as a tool to triage and target prompt, appropriate treatment in the ITU.