Innate defense regulator peptide 1018 protects against perinatal brain injury.

OBJECTIVE: There is currently no pharmacological treatment that provides protection against brain injury in neonates. It is known that activation of an innate immune response is a key, contributing factor in perinatal brain injury; therefore, the neuroprotective therapeutic potential of innate defense regulator peptides (IDRs) was investigated. METHODS: The anti-inflammatory effects of 3 IDRs was measured in lipopolysaccharide (LPS)-activated murine microglia. IDRs were then assessed for their ability to confer neuroprotection in vivo when given 3 hours after neonatal brain injury in a clinically relevant model that combines an inflammatory challenge (LPS) with hypoxia-ischemia (HI). To gain insight into peptide-mediated effects on LPS-induced inflammation and neuroprotective mechanisms, global cerebral gene expression patterns were analyzed in pups that were treated with IDR-1018 either 4 hours before LPS or 3 hours after LPS+HI. RESULTS: IDR-1018 reduced inflammatory mediators produced by LPS-stimulated microglia cells in vitro and modulated LPS-induced neuroinflammation in vivo. When administered 3 hours after LPS+HI, IDR-1018 exerted effects on regulatory molecules of apoptotic (for, eg, Fadd and Tnfsf9) and inflammatory (for, eg, interleukin 1, tumor necrosis factor α, chemokines, and cell adhesion molecules) pathways and showed marked protection of both white and gray brain matter. INTERPRETATION: IDR-1018 suppresses proinflammatory mediators and cell injurious mechanisms in the developing brain, and postinsult treatment is efficacious in reducing LPS-induced hypoxic-ischemic brain damage. IDR-1018 is effective in the brain when given systemically, confers neuroprotection of both gray and white matter, and lacks significant effects on the brain under normal conditions. Thus, this peptide provides the features of a promising neuroprotective agent in newborns with brain injury.

Functional genetic variation in NFKBIA and susceptibility to childhood asthma, bronchiolitis, and bronchopulmonary dysplasia.

Respiratory diseases are the most frequent chronic illnesses in babies and children. Although a vigorous innate immune system is critical for maintaining lung health, a balanced response is essential to minimize damaging inflammation. We investigated the functional and clinical impact of human genetic variants in the promoter of NFKBIA, which encodes IκBα, the major negative regulator of NF-κB. In this study, we quantified the functional impact of NFKBIA promoter polymorphisms (rs3138053, rs2233406, and rs2233409) on promoter-driven protein expression, allele-specific and total NFKBIA mRNA expression, IκBα protein expression, and TLR responsiveness; mapped innate immune regulatory networks active during respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia; and genotyped and analyzed independent cohorts of children with respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Genetic variants in the promoter of NFKBIA influenced NFKBIA gene expression, IκBα protein expression, and TLR-mediated inflammatory responses. Using a systems biology approach, we demonstrated that NFKBIA/IκBα is a central hub in transcriptional responses of prevalent childhood lung diseases, including respiratory syncytial virus infection, asthma, and bronchopulmonary dysplasia. Finally, by examining independent pediatric lung disease cohorts, we established that this immunologically relevant genetic variation in the promoter of NFKBIA is associated with differential susceptibility to severe bronchiolitis following infection with respiratory syncytial virus, airway hyperresponsiveness, and severe bronchopulmonary dysplasia. These data highlight the importance of negative innate immune regulators, such as NFKBIA, in pediatric lung disease and begin to unravel common aspects in the genetic predisposition to bronchopulmonary dysplasia, bronchiolitis, and childhood asthma.

INMEX--a web-based tool for integrative meta-analysis of expression data.

The widespread applications of various 'omics' technologies in biomedical research together with the emergence of public data repositories have resulted in a plethora of data sets for almost any given physiological state or disease condition. Properly combining or integrating these data sets with similar basic hypotheses can help reduce study bias, increase statistical power and improve overall biological understanding. However, the difficulties in data management and the complexities of analytical approaches have significantly limited data integration to enable meta-analysis. Here, we introduce integrative meta-analysis of expression data (INMEX), a user-friendly web-based tool designed to support meta-analysis of multiple gene-expression data sets, as well as to enable integration of data sets from gene expression and metabolomics experiments. INMEX contains three functional modules. The data preparation module supports flexible data processing, annotation and visualization of individual data sets. The statistical analysis module allows researchers to combine multiple data sets based on P-values, effect sizes, rank orders and other features. The significant genes can be examined in functional analysis module for enriched Gene Ontology terms or Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, or expression profile visualization. INMEX has built-in support for common gene/metabolite identifiers (IDs), as well as 45 popular microarray platforms for human, mouse and rat. Complex operations are performed through a user-friendly web interface in a step-by-step manner. INMEX is freely available at http://www.inmex.ca.

A multibiomarker-based outcome risk stratification model for adult septic shock*.

OBJECTIVES: Clinical trials in septic shock continue to fail due, in part, to inequitable and sometimes unknown distribution of baseline mortality risk between study arms. Investigators advocate that interventional trials in septic shock require effective outcome risk stratification. We derived and tested a multibiomarker-based approach to estimate mortality risk in adults with septic shock. DESIGN: Previous genome-wide expression studies identified 12 plasma proteins as candidates for biomarker-based risk stratification. The current analysis used banked plasma samples and clinical data from existing studies. Biomarkers were assayed in plasma samples obtained from 341 subjects with septic shock within 24 hours of admission to the ICU. Classification and regression tree analysis was used to generate a decision tree predicting 28-day mortality based on a combination of both biomarkers and clinical variables. The derived tree was first tested in an independent cohort of 331 subjects, then calibrated using all subjects (n = 672), and subsequently validated in another independent cohort (n = 209). SETTING: Multiple ICUs in Canada, Finland, and the United States. SUBJECTS: Eight hundred eighty-one adults with septic shock or severe sepsis. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: The derived decision tree included five candidate biomarkers, admission lactate concentration, age, and chronic disease burden. In the derivation cohort, sensitivity for mortality was 94% (95% CI, 87-97), specificity was 56% (50-63), positive predictive value was 50% (43-57), and negative predictive value was 95% (89-98). Performance was comparable in the test cohort. The calibrated decision tree had the following test characteristics in the validation cohort: sensitivity 85% (76-92), specificity 60% (51-69), positive predictive value 61% (52-70), and negative predictive value 85% (75-91). CONCLUSIONS: We have derived, tested, calibrated, and validated a risk stratification tool and found that it reliably estimates the probability of mortality in adults with septic shock.

Knockout of extracytoplasmic function sigma factor ECF-10 affects stress resistance and biofilm formation in Pseudomonas putida KT2440.

Pseudomonas putida is a Gram-negative soil bacterium which is well-known for its versatile lifestyle, controlled by a large repertoire of transcriptional regulators. Besides one- and two-component regulatory systems, the genome of P. putida reveals 19 extracytoplasmic function (ECF) sigma factors involved in the adaptation to changing environmental conditions. In this study, we demonstrate that knockout of extracytoplasmic function sigma factor ECF-10, encoded by open reading frame PP4553, resulted in 2- to 4-fold increased antibiotic resistance to quinolone, ß-lactam, sulfonamide, and chloramphenicol antibiotics. In addition, the ECF-10 mutant exhibited enhanced formation of biofilms after 24 h of incubation. Transcriptome analysis using Illumina sequencing technology resulted in the detection of 12 genes differentially expressed (>2-fold) in the ECF-10 knockout mutant strain compared to their levels of expression in wild-type cells. Among the upregulated genes were ttgA, ttgB, and ttgC, which code for the major multidrug efflux pump TtgABC in P. putida KT2440. Investigation of an ECF-10 and ttgA double-knockout strain and a ttgABC-overexpressing strain demonstrated the involvement of efflux pump TtgABC in the stress resistance and biofilm formation phenotypes of the ECF-10 mutant strain, indicating a new role for this efflux pump beyond simple antibiotic resistance in P. putida KT2440.

Atypical activation of the unfolded protein response in cystic fibrosis airway cells contributes to p38 MAPK-mediated innate immune responses.

Inflammatory lung disease is the major cause of morbidity and mortality in cystic fibrosis (CF); understanding what produces dysregulated innate immune responses in CF cells will be pivotal in guiding the development of novel anti-inflammatory therapies. To elucidate the molecular mechanisms that mediate exaggerated inflammation in CF following TLR signaling, we profiled global gene expression in immortalized human CF and non-CF airway cells at baseline and after microbial stimulation. Using complementary analysis methods, we observed a signature of increased stress levels in CF cells, specifically characterized by endoplasmic reticulum (ER) stress, the unfolded protein response (UPR), and MAPK signaling. Analysis of ER stress responses revealed an atypical induction of the UPR, characterized by the lack of induction of the PERK-eIF2α pathway in three complementary model systems: immortalized CF airway cells, fresh CF blood cells, and CF lung tissue. This atypical pattern of UPR activation was associated with the hyperinflammatory phenotype in CF cells, as deliberate induction of the PERK-eIF2α pathway with salubrinal attenuated the inflammatory response to both flagellin and Pseudomonas aeruginosa. IL-6 production triggered by ER stress and microbial stimulation were both dependent on p38 MAPK activity, suggesting a molecular link between both signaling events. These data indicate that atypical UPR activation fails to resolve the ER stress in CF and sensitizes the innate immune system to respond more vigorously to microbial challenge. Strategies to restore ER homeostasis and normalize the UPR activation profile may represent a novel therapeutic approach to minimize lung-damaging inflammation in CF.

Endotoxin tolerance represents a distinctive state of alternative polarization (M2) in human mononuclear cells.

Classical (M1) and alternative (M2) polarization of mononuclear cells (MNCs) such as monocyte and macrophages is known to occur in response to challenges within a microenvironment, like the encounter of a pathogen. LPS, also known as endotoxin, is a potent inducer of inflammation and M1 polarization. LPS can also generate an effect in MNCs known as endotoxin tolerance, defined as the reduced capacity of a cell to respond to LPS activation after an initial exposure to this stimulus. Using systems biology approaches in PBMCs, monocytes, and monocyte-derived macrophages involving microarrays and advanced bioinformatic analysis, we determined that gene responses during endotoxin tolerance were similar to those found during M2 polarization, featuring gene and protein expression critical for the development of key M2 MNC functions, including reduced production of proinflammatory mediators, expression of genes involved in phagocytosis, as well as tissue remodeling. Moreover, expression of different metallothionein gene isoforms, known for their role in the control of oxidative stress and in immunomodulation, were also found to be consistently upregulated during endotoxin tolerance. These results demonstrate that after an initial inflammatory stimulus, human MNCs undergo an M2 polarization probably to control hyperinflammation and heal the affected tissue.

Identification of novel host defense peptides and the absence of alpha-defensins in the bovine genome.

Host defense peptides (historically called antimicrobial peptides, AMPs) are key components in the mammalian innate immune system, and are responsible for both direct killing and immunomodulatory effects in host defense against pathogenic organisms. In order to identify novel host defense peptides by sequence analysis, we constructed the AMPer resource (http://www.cnbi2.com/cgi-bin/amp.pl) that utilizes hidden Markov models to recognize sequences of antimicrobial peptides. In the current work, we utilized the AMPer resource to search bovine expressed sequence tags from the NCBI dbEST project and the bovine genome sequence for novel host defense peptides. Of the 34 known bovine AMPs, 27 were identified with high confidence in the AMPs predicted from ESTs. A further potential 68 AMPs predicted from the EST data were found that appear to be novel giving a total estimate of 102 AMPs present in the genome. Two of these were cathelicidins and selected for experimental verification in RNA derived from bovine tissue. One predicted AMP, most similar to rabbit '15 kDa protein' AMP, was confirmed to be present in infected bovine intestinal tissue using PCR. These findings demonstrated the practical applicability of the developed bioinformatics approach and laid a foundation for future discoveries of gene-coded AMPs. No members of the alpha-defensin family were found in the bovine sequences. Since we could find no technical reasons these would be missed and no references to bovine alpha-defensins in the literature, this suggests that cattle lack this important family of host defense peptides.

AMPer: a database and an automated discovery tool for antimicrobial peptides.

MOTIVATION: Increasing antibiotics resistance in human pathogens represents a pressing public health issue worldwide for which novel antibiotic therapies based on antimicrobial peptides (AMPs) may offer one possible solution. In the current study, we utilized publicly available data on AMPs to construct hidden Markov models (HMMs) that enable recognition of individual classes of antimicrobials peptides (such as defensins, cathelicidins, cecropins, etc.) with up to 99% accuracy and can be used for discovering novel AMP candidates. RESULTS: HMM models for both mature peptides and propeptides were constructed. A total of 146 models for mature peptides and 40 for propeptides have been developed for individual AMP classes. These were created by clustering and analyzing AMP sequences available in the public sources and by consequent iterative scanning of the Swiss-Prot database for previously unknown gene-coded AMPs. As a result, an additional 229 additional AMPs have been identified from Swiss-Prot, and all but 34 could be associated with known antimicrobial activities according to the literature. The final set of 1045 mature peptides and 253 propeptides have been organized into the open-source AMPer database. AVAILABILITY: The developed HMM-based tools and AMP sequences can be accessed through the AMPer resource at http://www.cnbi2.com/cgi-bin/amp.pl

Short linear cationic antimicrobial peptides: screening, optimizing, and prediction.

The problem of pathogenic antibiotic-resistant bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa is worsening, demonstrating the urgent need for new therapeutics that are effective against multidrug-resistant bacteria. One potential class of substances is cationic antimicrobial peptides. More than 1000 natural occurring peptides have been described so far. These peptides are short (less than 50 amino acids long), cationic, amphiphilic, demonstrate different three-dimensional structures, and appear to have different modes of action. A new screening assay was developed to characterize and optimize short antimicrobial peptides. This assay is based on peptides synthesized on cellulose, combined with a bacterium, where a luminescence gene cassette was introduced. With help of this method tens of thousands of peptides can be screened per year. Information gained by this high-throughput screening can be used in quantitative structure-activity relationships (QSAR) analysis. QSAR analysis attempts to correlate chemical structure to measurement of biological activity using statistical methods. QSAR modeling of antimicrobial peptides to date has been based on predicting differences between peptides that are highly similar. The studies have largely addressed differences in lactoferricin and protegrin derivatives or similar de novo peptides. The mathematical models used to relate the QSAR descriptors to biological activity have been linear models such as principle component analysis or multivariate linear regression. However, with the development of high-throughput peptide synthesis and an antibacterial activity assay, the numbers of peptides and sequence diversity able to be studied have increased dramatically. Also, "inductive" QSAR descriptors have been recently developed to accurately distinguish active from inactive drug-like activity in small compounds. "Inductive" QSAR in combination with more complex mathematical modeling algorithms such as artificial neural networks (ANNs) may yield powerful new methods for in silico identification of novel antimicrobial peptides.

Increased Plasma PCSK9 Levels Are Associated with Reduced Endotoxin Clearance and the Development of Acute Organ Failures during Sepsis.

PURPOSE: We have recently shown that PCSK9 reduces the clearance of endotoxin and is therefore a critical regulator of the innate immune response during infection. However, plasma PCSK9 levels during human sepsis and their relationship to outcomes are not known. Our objective was to determine the relationship between plasma PCSK9 levels and the rate of endotoxin clearance, and then correlate PCSK9 levels with the development of acute organ failures in a cohort of patients with sepsis. METHODS: Using human hepatocyte cells, we determined the threshold at which PCSK9 is able to reduce Escherichia coli endotoxin uptake by cultured human hepatocytes. In a single-centre observational cohort at St. Paul's Hospital in Vancouver, Canada, we recruited 200 patients who activated our Emergency Department's sepsis protocol and measured plasma PCSK9 and lipid levels at triage and throughout the admission. Outcomes were the development of sepsis-induced cardiovascular or respiratory failure. RESULTS: We reviewed the literature and determined that the normal human range of PCSK9 found in plasma is 170-220 ng/ml, while levels of 250 ng/ml and above reduced E. coli endotoxin clearance in cultured human hepatocytes. In septic patients, the median levels associated with new-onset respiratory and cardiovascular failure were 370 (250-500) and 380 (270-530) ng/ml, respectively, versus 270 (220-380) ng/ml in patients who did not go on to develop any organ failure (p = 0.003 and 0.005, respectively). CONCLUSIONS: Plasma PCSK9 levels are greatly increased in sepsis. At normal levels, PCSK9 has no influence upon hepatocyte bacterial endotoxin clearance, but as levels rise, there is a progressive inhibition of clearance. During sepsis, PCSK9 levels are highly correlated with the development of subsequent multiple organ failure. Inhibition of PCSK9 activity is an attractive target for treating the spectrum of sepsis and septic shock.

TNFAIP2 Inhibits Early TNFα-Induced NF-x03BA;B Signaling and Decreases Survival in Septic Shock Patients.

During septic shock, tumor necrosis factor alpha (TNFα) is an early response gene and induces a plethora of genes and signaling pathways. To identify robust signals in genes reliably upregulated by TNFα, we first measured microarray gene expression in vitro and searched methodologically comparable, publicly available data sets to identify concordant signals. Using tag single-nucleotide polymorphisms in the genes common to all data sets, we identified a genetic variant of the TNFAIP2 gene, rs8126, associated with decreased 28-day survival and increased organ dysfunction in an adult cohort in the Vasopressin and Septic Shock Trial. Similar to this cohort, we found that an association with rs8126 and increased organ dysfunction is replicated in a second cohort of septic shock patients in the St. Paul's Hospital Intensive Care Unit. We found that TNFAIP2 inhibits NF-x03BA;B activity, impacting the downstream cytokine interleukin (IL)-8. The minor G allele of TNFAIP2 rs8126 resulted in greater TNFAIP2 expression, decreased IL-8 production and was associated with decreased survival in patients experiencing septic shock. These data suggest that TNFAIP2 is a novel inhibitor of NF-x03BA;B that acts as an autoinhibitor of the TNFα response during septic shock.

In Vivo, In Vitro, and In Silico Characterization of Peptoids as Antimicrobial Agents.

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.

VPS13D Gene Variant Is Associated with Altered IL-6 Production and Mortality in Septic Shock.

BACKGROUND: Genetic variations contribute to septic shock mortality. To discover a novel locus, we performed in vitro genome-wide association studies (GWAS) and further tested the result in a cohort of septic shock patients. METHODS: Two in vitro GWAS using a quantitative trait locus analysis of stimulated IL-6 production in lymphoblastoid cells from 60 individuals of European ancestry were performed. VPS13D rs6685273 was genotyped in European ancestry patients (n = 498). The VPS13D gene was silenced in vitro. RESULTS: Two GWAS using lymphoblastoid cells identified the locus of VPS13D rs6685273 that was significant in the same direction in both GWAS. The VPS13D rs6685273 C allele was associated with increased IL-6 production. Patients with septic shock who had the VPS13D rs6685273 CC genotype had an increased 28-day mortality (p = 0.023) and more organ failure (p < 0.05) compared to the CT/TT genotypes. VPS13D in vitro gene silencing in the HeLa cell line increased IL-6 production. Furthermore, the rs6685273 genotype was associated with differential VPS13D splice variant expression. CONCLUSIONS: The VPS13D rs6685273 C allele was associated with increased IL-6 production in vitro. The patients with the VPS13D rs6685273 CC genotype had increased 28-day mortality and increased organ failure. VPS13D appears to regulate IL-6 production.

Prolonged QTc affects short-term and long-term outcomes in patients with normal left ventricular function undergoing cardiac surgery.

OBJECTIVE: Although it is known that preoperative decreased left ventricular ejection fraction (LVEF) is a risk for morbidity and mortality after cardiac surgery, there are no reliable markers of risk in patients with preserved LVEF. This study examines whether a prolonged QTc interval is associated with adverse outcomes in patients with preoperative LVEF greater than 40% undergoing cardiac surgery. METHODS: A retrospective chart review of patients who had cardiac surgery at St. Paul's Hospital in Vancouver, Canada, between 2004 and 2009, who had a preoperative LVEF greater than 40%, was undertaken. We tested for association of preoperative prolonged QTc interval with mortality and morbidity using unadjusted and adjusted analyses. RESULTS: Five-hundred and fifty-five patients with a preoperative LVEF greater than 40% were included in the study; 496 (89.4%) had cardiopulmonary bypass and the remainder were off pump. Preoperative prolonged QTc was associated with increased mortality at 30 days (P < .01), 90 days (P < .01), and 8 years (P < .01), and these results remained significant after adjusting for the clinical variables significantly associated with mortality (8-year odds ratio, 2.42; 95% confidence interval, 1.34-4.34; P = .003). Similar results were found when the analysis was restricted to the more homogeneous group of patients undergoing on-pump coronary artery bypass (CABG, n = 408). Prolonged QTc was also associated with prolonged intensive care unit stay (P = .02), prolonged hospital stay (P < .01), development of atrial arrhythmias (P = .02), and low cardiac output syndrome (on-pump CABG, P = .02). CONCLUSIONS: In patients undergoing cardiac surgery and a preoperative LVEF greater than 40%, a prolonged QTc interval is associated with increased short-term and long-term mortality and increased perioperative morbidity, and therefore should be considered when assessing risk preoperatively.

An Endotoxin Tolerance Signature Predicts Sepsis and Organ Dysfunction at Initial Clinical Presentation.

BACKGROUND: Sepsis involves aberrant immune responses to infection, but the exact nature of this immune dysfunction remains poorly defined. Bacterial endotoxins like lipopolysaccharide (LPS) are potent inducers of inflammation, which has been associated with the pathophysiology of sepsis, but repeated exposure can also induce a suppressive effect known as endotoxin tolerance or cellular reprogramming. It has been proposed that endotoxin tolerance might be associated with the immunosuppressive state that was primarily observed during late-stage sepsis. However, this relationship remains poorly characterised. Here we clarify the underlying mechanisms and timing of immune dysfunction in sepsis. METHODS: We defined a gene expression signature characteristic of endotoxin tolerance. Gene-set test approaches were used to correlate this signature with early sepsis, both newly and retrospectively analysing microarrays from 593 patients in 11 cohorts. Then we recruited a unique cohort of possible sepsis patients at first clinical presentation in an independent blinded controlled observational study to determine whether this signature was associated with the development of confirmed sepsis and organ dysfunction. FINDINGS: All sepsis patients presented an expression profile strongly associated with the endotoxin tolerance signature (p < 0.01; AUC 96.1%). Importantly, this signature further differentiated between suspected sepsis patients who did, or did not, go on to develop confirmed sepsis, and predicted the development of organ dysfunction. INTERPRETATION: Our data support an updated model of sepsis pathogenesis in which endotoxin tolerance-mediated immune dysfunction (cellular reprogramming) is present throughout the clinical course of disease and related to disease severity. Thus endotoxin tolerance might offer new insights guiding the development of new therapies and diagnostics for early sepsis.

Synthetic cationic peptide IDR-1018 modulates human macrophage differentiation.

Macrophages play a critical role in the innate immune response. To respond in a rapid and efficient manner to challenges in the micro-environment, macrophages are able to differentiate towards classically (M1) or alternatively (M2) activated phenotypes. Synthetic, innate defense regulators (IDR) peptides, designed based on natural host defence peptides, have enhanced immunomodulatory activities and reduced toxicity leading to protection in infection and inflammation models that is dependent on innate immune cells like monocytes/macrophages. Here we tested the effect of IDR-1018 on macrophage differentiation, a process essential to macrophage function and the immune response. Using transcriptional, protein and systems biology analysis, we observed that differentiation in the presence of IDR-1018 induced a unique signature of immune responses including the production of specific pro and anti-inflammatory mediators, expression of wound healing associated genes, and increased phagocytosis of apoptotic cells. Transcription factor IRF4 appeared to play an important role in promoting this IDR-1018-induced phenotype. The data suggests that IDR-1018 drives macrophage differentiation towards an intermediate M1-M2 state, enhancing anti-inflammatory functions while maintaining certain pro-inflammatory activities important to the resolution of infection. Synthetic peptides like IDR-1018, which act by modulating the immune system, could represent a powerful new class of therapeutics capable of treating the rising number of multidrug resistant infections as well as disorders associated with dysregulated immune responses.

Cytokines and signaling molecules predict clinical outcomes in sepsis.

INTRODUCTION: Inflammatory response during sepsis is incompletely understood due to small sample sizes and variable timing of measurements following the onset of symptoms. The vasopressin in septic shock trial (VASST) compared the addition of vasopressin to norepinephrine alone in patients with septic shock. During this study plasma was collected and 39 cytokines measured in a 363 patients at both baseline (before treatment) and 24 hours. Clinical features relating to both underlying health and the acute organ dysfunction induced by the severe infection were collected during the first 28 days of admission. HYPOTHESIS: Cluster analysis of cytokines identifies subgroups of patients at differing risk of death and organ failure. METHODS: Circulating cytokines and other signaling molecules were measured using a Luminex multi-bead analyte detection system. Hierarchical clustering was performed on plasma values to create patient subgroups. Enrichment analysis identified clinical outcomes significantly different according to these chemically defined patient subgroups. Logistic regression was performed to assess the importance of cytokines for predicting patient subgroups. RESULTS: Plasma levels at baseline produced three subgroups of patients, while 24 hour levels produced two subgroups. Using baseline cytokine data, one subgroup of 47 patients showed a high level of enrichment for severe septic shock, coagulopathy, renal failure, and risk of death. Using data at 24 hours, a larger subgroup of 81 patients that largely encompassed the 47 baseline subgroup patients had a similar enrichment profile. Measurement of two cytokines, IL2 and CSF2 and their product were sufficient to classify patients into these subgroups that defined clinical risks. CONCLUSIONS: A distinct pattern of cytokine levels measured early in the course of sepsis predicts disease outcome. Subpopulations of patients have differing clinical outcomes that can be predicted accurately from small numbers of cytokines. Design of clinical trials and interventions may benefit from consideration of cytokine levels.

A systems biology approach to nutritional immunology - focus on innate immunity.

Innate immunity and nutrient metabolism are complex biological systems that must work in concert to sustain and preserve life. The effector cells of the innate immune system rely on essential nutrients to generate energy, produce metabolic precursors for macromolecule biosynthesis and tune their responses to infectious agents. Thus disruptions to nutritional status have a substantial impact on immune competence and can result in increased susceptibility to infection in the case of nutrient deficiency, or chronic inflammation in the case of over-nutrition. The traditional, reductionist methods used in the study of nutritional immunology are incapable of exploring the extremely complex interactions between nutrient metabolism and innate immunity. Here, we review a relatively new analytical approach, systems biology, and highlight how it can be applied to nutritional immunology to provide a comprehensive view of the mechanisms behind nutritional regulation of the innate immune system.

Designing antimicrobial peptides: form follows function.

Multidrug-resistant bacteria are a severe threat to public health. Conventional antibiotics are becoming increasingly ineffective as a result of resistance, and it is imperative to find new antibacterial strategies. Natural antimicrobials, known as host defence peptides or antimicrobial peptides, defend host organisms against microbes but most have modest direct antibiotic activity. Enhanced variants have been developed using straightforward design and optimization strategies and are being tested clinically. Here, we describe advanced computer-assisted design strategies that address the difficult problem of relating primary sequence to peptide structure, and are delivering more potent, cost-effective, broad-spectrum peptides as potential next-generation antibiotics.