Frank Radella

Randomized, Prospective Trial of Antioxidant Supplementation in Critically Ill Surgical Patients

ObjectiveTo determine the effectiveness of early, routine antioxidant supplementation using α-tocopherol and ascorbic acid in reducing the rate of pulmonary morbidity and organ dysfunction in critically ill surgical patients.Summary Background DataOxidative stress has been associated with the develo

Toll-like Receptor 1 Polymorphisms Affect Innate Immune Responses and Outcomes in Sepsis

RATIONALE Polymorphisms affecting Toll-like receptor (TLR)-mediated responses could predispose to excessive inflammation during an infection and contribute to an increased risk for poor outcomes in patients with sepsis. OBJECTIVES To identify hypermorphic polymorphisms causing elevated TLR-mediated innate immune cytokine and chemokine responses and to test whether these polymorphisms are associated with increased susceptibility to death, organ dysfunction, and infections in patients with sepsis. METHODS We screened single-nucleotide polymorphisms (SNPs) in 43 TLR-related genes to identify variants affecting TLR-mediated inflammatory responses in blood from healthy volunteers ex vivo. The SNP associated most strongly with hypermorphic responses was tested for associations with death, organ dysfunction, and type of infection in two studies: a nested case-control study in a cohort of intensive care unit patients with sepsis, and a case-control study using patients with sepsis, patients with sepsis-related acute lung injury, and healthy control subjects. MEASUREMENTS AND MAIN RESULTS The SNP demonstrating the most hypermorphic effect was the G allele of TLR1(-7202A/G) (rs5743551), which associated with elevated TLR1-mediated cytokine production (P < 2 x 10(-20)). TLR1(-7202G) marked a coding SNP that causes higher TLR1-induced NF-kappaB activation and higher cell surface TLR1 expression. In the cohort of patients with sepsis TLR1(-7202G) predicted worse organ dysfunction and death (odds ratio, 1.82; 95% confidence interval, 1.07-3.09). In the case-control study TLR1(-7202G) was associated with sepsis-related acute lung injury (odds ratio, 3.40; 95% confidence interval, 1.59-7.27). TLR1(-7202G) also associated with a higher prevalence of gram-positive cultures in both clinical studies. CONCLUSIONS Hypermorphic genetic variation in TLR1 is associated with increased susceptibility to organ dysfunction, death, and gram-positive infection in sepsis.

Modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome

Objective: To determine whether bronchoalveolar lavage fluid (BALF) from patients either at risk for the acute respiratory distress syndrome (ARDS) or with sustained ARDS modulates neutrophil apoptosis; to measure the BALF concentrations of the apoptosis inhibitors granulocyte colony‐stimulating factor (G‐CSF) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF) before and after the onset of ARDS; and to determine whether the BALF concentrations of G‐CSF and/or GM‐CSF are associated with clinical outcome. Design: Prospective cohort study. Setting: Tertiary university hospital. Patients: Twenty patients at risk for ARDS and 45 patients with established ARDS. Interventions: Patients at risk for ARDS underwent bronchoalveolar lavage within 24 hrs of being identified, then again 72 hrs later. Patients with ARDS underwent bronchoalveolar lavage within 24 hrs of meeting ARDS criteria, then again on days 3, 7, and 14 of the disease. Measurements and Main Results: Normal peripheral blood neutrophil were incubated overnight in BALF from normal volunteers, from patients at risk for ARDS, or from patients with ARDS. neutrophil apoptosis was determined by flow cytometric analysis of annexin V binding. G‐CSF and GM‐CSF were measured in BALF by immunoassays. Compared with normal BALF, BALF from patients on days 1 and 3 of ARDS inhibited neutrophil apoptosis, but BALF from patients at later stages of ARDS, or from patients at risk for ARDS, did not. The BALF concentrations of both G‐CSF and GM‐CSF were elevated early in ARDS and decreased toward later stages. Patients who lived had significantly higher concentrations of GM‐CSF in the BALF than those who died. Conclusions: We conclude that the antiapoptotic effect of ARDS BALF on normal neutrophil is highest during early ARDS, and decreases during late ARDS. G‐CSF and GM‐CSF are present in BALF from patients with ARDS, and their concentrations parallel the antiapoptotic effect of ARDS BALF. These data support the concept that the life‐span of neutrophil in the air spaces is modulated during acute inflammation. GM‐CSF in the air spaces is associated with improved survival in patients with ARDS.

Identification of high and low responders to lipopolysaccharide in normal subjects: an unbiased approach to identify modulators of innate immunity.

LPS stimulates a vigorous inflammatory response from circulating leukocytes that varies greatly from individual to individual. The goal of this study was to use an unbiased approach to identify differences in gene expression that may account for the high degree of interindividual variability in inflammatory responses to LPS in the normal human population. We measured LPS-induced cytokine production ex vivo in whole blood from 102 healthy human subjects and identified individuals who consistently showed either very high or very low responses to LPS (denoted lpshigh and lpslow, respectively). Comparison of gene expression profiles between the lpshigh and lpslow individuals revealed 80 genes that were differentially expressed in the presence of LPS and 21 genes that were differentially expressed in the absence of LPS (p < 0.005, ANOVA). Expression of a subset of these genes was confirmed using real-time RT-PCR. Functional relevance for one gene confirmed to be expressed at a higher level in lpshigh, adipophilin, was inferred when reduction in adipophilin mRNA by small interfering RNA in the human monocyte-like cell line THP-1 resulted in a modest but significant reduction in LPS-induced MCP-1 mRNA expression. These data illustrate a novel approach to the identification of factors that determine interindividual variability in innate immune inflammatory responses and identify adipophilin as a novel potential regulator of LPS-induced MCP-1 production in human monocytes.

Interleukin-17A Is Associated with Alveolar Inflammation and Poor Outcomes in Acute Respiratory Distress Syndrome

Objective:Interleukin-17A is a proinflammatory cytokine known to play a role in host defense and pathologic inflammation in murine models of lung injury. The relationship between interleukin-17A and inflammation in human lung injury is unknown. Our primary objective was to determine whether interleukin-17A levels are associated with alveolar measures of inflammation and injury in patients with acute respiratory distress syndrome. Our secondary objective was to test whether interleukin-17A levels are associated with acute respiratory distress syndrome–related outcomes. Design:Observational study. Setting:Six North American medical centers. Patients:We studied two groups of patients with acute respiratory distress syndrome: 1) patients previously enrolled in a placebo-controlled clinical trial of omega-3 fatty acids performed at five North American medical centers (n = 86, acute respiratory distress syndrome 1), and 2) patients with systemic inflammatory response syndrome admitted to an ICU who developed acute respiratory distress syndrome (n = 140, acute respiratory distress syndrome 2). In acute respiratory distress syndrome 1, we used paired serum and bronchoalveolar lavage fluid samples obtained within 48 hours of acute respiratory distress syndrome onset, whereas in acute respiratory distress syndrome 2, we used plasma obtained within the first 24 hours of ICU admission. Interventions:None. Measurements and Main Results:We measured circulating interleukin-17A in acute respiratory distress syndrome 1 and acute respiratory distress syndrome 2. We also measured interleukin-17A, neutrophil counts, and total protein in bronchoalveolar lavage fluid from acute respiratory distress syndrome 1. We found that bronchoalveolar lavage interleukin-17A was strongly associated with higher bronchoalveolar lavage percent neutrophils (p < 0.001) and bronchoalveolar lavage total protein (p < 0.01) in acute respiratory distress syndrome1. In both acute respiratory distress syndrome 1 and acute respiratory distress syndrome 2, elevated interleukin-17A was associated with higher Sequential Organ Failure Assessment scores (p < 0.05). Conclusions:Elevated circulating and alveolar levels of interleukin-17A are associated with increased percentage of alveolar neutrophils, alveolar permeability, and organ dysfunction in acute respiratory distress syndrome.

Cutting Edge: Genetic Variation in TLR1 Is Associated with Pam3CSK4-Induced Effector T Cell Resistance to Regulatory T Cell Suppression

TLR play essential roles in the initiation and modulation of immune responses. TLR1/TLR2 heterodimers recognize triacylated bacterial lipopeptides, including the synthetic TLR1/2 lipopeptide Pam3CSK4. Genetic variation in TLR1 is associated with outcomes in diseases in which regulatory T cells (Treg) play a role, including asthma and allergy. To determine whether genetic polymorphisms in TLR1 are associated with alterations in Treg suppression of effector T cells (Teff), we performed in vitro suppression assays in healthy individuals with various haplotypes in TLR1. We show that functional genetic polymorphisms in TLR1 modify surface expression of TLR1 on T lymphocytes and confer enhanced Teff resistance to Treg suppression in the presence of Pam3CSK4. These effects are mediated, in part, by IL-6 and inhibited by blocking IL-6 signaling through STAT3. These findings suggest that TLR1 polymorphisms could influence immune-related disease through Teff resistance to Treg suppression.

Synthetic Macromolecular Antibiotic Platform for Inhalable Therapy against Aerosolized Intracellular Alveolar Infections

Lung-based intracellular bacterial infections remain one of the most challenging infectious disease settings. For example, the current standard for treating Franciscella tularensis pneumonia (tularemia) relies on administration of oral or intravenous antibiotics that poorly achieve and sustain pulmonary drug bioavailability. Inhalable antibiotic formulations are approved and in clinical development for upper respiratory infections, but sustained drug dosing from inhaled antibiotics against alveolar intracellular infections remains a current unmet need. To provide an extended therapy against alveolar intracellular infections, we have developed a macromolecular therapeutic platform that provides sustained local delivery of ciprofloxacin with controlled dosing profiles. Synthesized using RAFT polymerization, these macromolecular prodrugs characteristically have high drug loading (16-17 wt % drug), tunable hydrolysis kinetics mediated by drug linkage chemistry (slow-releasing alkyllic vs fast-releasing phenolic esters), and, in general, represent new fully synthetic nanotherapeutics with streamlined manufacturing profiles. In aerosolized and completely lethal F.t. novicida mouse challenge models, the fast-releasing ciprofloxacin macromolecular prodrug provided high cure efficiencies (75% survival rate under therapeutic treatment), and the importance of release kinetics was demonstrated by the inactivity of the similar but slow-releasing prodrug system. Pharmacokinetics and biodistribution studies further demonstrated that the efficacious fast-releasing prodrug retained drug dosing in the lung above the MIC over a 48 h period with corresponding Cmax/MIC and AUC0-24h/MIC ratios being greater than 10 and 125, respectively; the thresholds for optimal bactericidal efficacy. These findings identify the macromolecular prodrug platform as a potential therapeutic system to better treat alveolar intracellular infections such as F. tularensis, where positive patient outcomes require tailored antibiotic pharmacokinetic and treatment profiles.

A Two-Biomarker Model Predicts Mortality in the Critically Ill with Sepsis.

Rationale: Improving the prospective identification of patients with systemic inflammatory response syndrome (SIRS) and sepsis at low risk for organ dysfunction and death is a major clinical challenge. Objectives: To develop and validate a multibiomarker‐based prediction model for 28‐day mortality in critically ill patients with SIRS and sepsis. Methods: A derivation cohort (n = 888) and internal test cohort (n = 278) were taken from a prospective study of critically ill intensive care unit (ICU) patients meeting two of four SIRS criteria at an academic medical center for whom plasma was obtained within 24 hours. The validation cohort (n = 759) was taken from a prospective cohort enrolled at another academic medical center ICU for whom plasma was obtained within 48 hours. We measured concentrations of angiopoietin‐1, angiopoietin‐2, IL‐6, IL‐8, soluble tumor necrosis factor receptor‐1, soluble vascular cell adhesion molecule‐1, granulocyte colony‐stimulating factor, and soluble Fas. Measurements and Main Results: We identified a two‐biomarker model in the derivation cohort that predicted mortality (area under the receiver operator characteristic curve [AUC], 0.79; 95% confidence interval [CI], 0.74‐0.83). It performed well in the internal test cohort (AUC, 0.75; 95% CI, 0.65‐0.85) and the external validation cohort (AUC, 0.77; 95% CI, 0.72‐0.83). We determined a model score threshold demonstrating high negative predictive value (0.95) for death. In addition to a low risk of death, patients below this threshold had shorter ICU length of stay, lower incidence of acute kidney injury, acute respiratory distress syndrome, and need for vasopressors. Conclusions: We have developed a simple, robust biomarker‐based model that identifies patients with SIRS/sepsis at low risk for death and organ dysfunction.

Peripheral and Alveolar Cell Transcriptional Programs Are Distinct in Acute Respiratory Distress Syndrome

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Glycan targeted polymeric antibiotic prodrugs for alveolar macrophage infections

Alveolar macrophages resident in the lung are prominent phagocytic effector cells of the pulmonary innate immune response, and paradoxically, are attractive harbors for pathogens. Consequently, facultative intracellular bacteria, such as Francisella tularensis, can cause severe systemic disease and sepsis, with high morbidity and mortality associated with pulmonary infection. Current clinical treatment, which involves exhaustive oral or intravenous antibiotic therapy, has limitations such as systemic toxicity and off-target effects. Pulmonary administration represents a promising alternative to systemic dosing for delivering antibiotics directly to the lung. Here, we present synthesized mannosylated ciprofloxacin polymeric prodrugs for efficient pulmonary delivery, targeting, and subsequent internalization by alveolar macrophages. We demonstrate significant improvement in efficacy against intracellular infections in an otherwise uniformly lethal airborne Francisella murine model (F. novicida). When administered to the lungs of mice in a prophylactic regimen, the mannosylated ciprofloxacin polymeric prodrugs led to 50% survival. In a treatment regimen that was concurrent with infection, the survival of mice increased to 87.5%. Free ciprofloxacin antibiotic was ineffective in both cases. This significant difference in antibacterial efficacy demonstrates the impact of this delivery platform based on improved physiochemical, pharmacokinetic, and pharmacodynamic properties of ciprofloxacin administered via our glycan polymeric prodrug. This modular platform provides a route for overcoming the limitations of free drug and increasing efficacy in treatment of intracellular infection.