Hector R. Wong

Serum neutrophil gelatinase-associated lipocalin (ngal) as a marker of acute kidney injury in critically ill children with septic shock

Objective:To validate serum neutrophil gelatinase-associated lipocalin (NGAL) as an early biomarker for acute kidney injury in critically ill children with septic shock. Design:Observational cohort study. Setting:Fifteen North American pediatric intensive care units (PICUs). Patients:A total of 143 critically ill children with systemic inflammatory response syndrome (SIRS) or septic shock and 25 healthy controls. Interventions:None. Measurements and Main Results:Serum NGAL was measured during the first 24 hrs of admission to the PICU. Acute kidney injury was defined as a blood urea nitrogen concentration >100 mg/dL, serum creatinine >2 mg/dL in the absence of preexisting renal disease, or the need for dialysis. There was a significant difference in serum NGAL between healthy children (median 80 ng/mL, interquartile ratio [IQR] 55.5–85.5 ng/mL), critically ill children with SIRS (median 107.5 ng/mL, IQR 89–178.5 ng/mL), and critically ill children with septic shock (median 302 ng/mL, IQR 151–570 ng/mL; p < .001). Acute kidney injury developed in 22 of 143 (15.4%) critically ill children. Serum NGAL was significantly increased in critically ill children with acute kidney injury (median 355 ng/mL, IQR 166–1322 ng/mL) compared with those without acute kidney injury (median 186 ng/mL, IQR 98–365 ng/mL; p = .009). Conclusions:Serum NGAL is a highly sensitive but nonspecific predictor of acute kidney injury in critically ill children with septic shock. Further validation of serum NGAL as a biomarker of acute kidney injury in this population is warranted.

Salmonella flagellin-dependent proinflammatory responses are localized to the conserved amino and carboxyl regions of the protein.

Flagellin, the monomeric subunit of flagella, is an inducer of proinflammatory mediators. Bacterial flagellin genes have conserved domains (D1 and D2) at the N terminus and C terminus and a middle hypervariable domain (D3). To identify which domains induced proinflammatory activity, r6-histidine (6HIS)-tagged fusion constructs were generated from the Salmonella dublin (SD) fliC flagellin gene. A full-length r6HIS SD flagellin (6HIS flag) induced IκBα loss poststimulation and NF-κB activation in Caco-2BBe cells and was as potent as native-purified SD flagellin. IFN-γ-primed DLD-1 cells stimulated with 1 μg/ml of 6HIS flag induced high levels of NO (60 ± 0.95 μM) comparable to the combination of IL-1β and IFN-γ (77 ± 1.2) or purified native SD flag (66.3 ± 0.98). Selected rSD flagellin proteins representing the D1, D2, or D3 domains alone or in combination were tested for proinflammatory properties. Fusion proteins representing the D3, amino, or carboxyl regions alone did not induce proinflammatory mediators. The results with a recombinant protein containing the amino D1 and D2 and carboxyl D1 and D2 separated by an Escherichia coli hinge (ND1-2/ECH/CD2) indicated that D1 and D2 were bioactive when coupled to an ECH element to allow protein folding. This chimera, but not the hinge alone, induced IκBα degradation, NF-κB activation, and NO and IL-8 production in two intestinal epithelial cell lines. ND1–2/ECH/CD2–1 also induced high levels of TNF-α (900 pg/ml) in human monocytes comparable to native SD flagellin (991.5 pg/ml) and 6HIS flag (987 pg/ml). The potent proinflammatory activity of flagellin, therefore, resides in the highly conserved N and C D1 and D2 regions.

Interactions between the heat shock response and the nuclear factor-κB signaling pathway

The heat shock response (HSR) and the nuclear factor (NF)-kappaB signaling pathway are two fundamental cellular responses. Various laboratories have documented in vitro and in vivo interactions between the HSR and NF-kappaB activation when they are activated sequentially. For example, induction of the HSR before a proinflammatory signal inhibits NF-kappaB activation and NF-kappaB-dependent proinflammatory gene expression. The central point of control appears to be at the level of IkappaBalpha phosphorylation as demonstrated by HSR-mediated inhibition of IkappaB kinase activation and HSR-mediated induction of intracellular phosphatase activity. In addition, induction of the HSR can independently increase de novo expression of the IkappaBalpha gene, thereby providing another potential mechanism through which the HSR can modulate cellular proinflammatory signaling. Another level of interaction is illustrated by the observation that various pharmacologic inhibitors of the NF-kappaB pathway are capable of simultaneously inducing the HSR. In direct contrast, induction of the HSR after a proinflammatory signal can lead to programmed cell death. Further understanding of how these two fundamental cellular responses interact at the molecular level holds the potential to elucidate some of the molecular interactions that occur during disease states common to critical care medicine.

Genomic expression profiling across the pediatric systemic inflammatory response syndrome, sepsis, and septic shock spectrum

Objectives:To advance our biological understanding of pediatric septic shock, we measured the genome-level expression profiles of critically ill children representing the systemic inflammatory response syndrome (SIRS), sepsis, and septic shock spectrum. Design:Prospective observational study involving microarray-based bioinformatics. Setting:Multiple pediatric intensive care units in the United States. Patients:Children ≤10 years of age: 18 normal controls, 22 meeting criteria for SIRS, 32 meeting criteria for sepsis, and 67 meeting criteria for septic shock on day 1. The available day 3 samples included 20 patients still meeting sepsis criteria, 39 patients still meeting septic shock criteria, and 24 patients meeting the exclusive day 3 category, SIRS resolved. Interventions:None other than standard care. Measurements and Main Results:Longitudinal analyses were focused on gene expression relative to control samples and patients having paired day 1 and day 3 samples. The longitudinal analysis focused on up-regulated genes revealed common patterns of up-regulated gene expression, primarily corresponding to inflammation and innate immunity, across all patient groups on day 1. These patterns of up-regulated gene expression persisted on day 3 in patients with septic shock, but not to the same degree in the other patient classes. The longitudinal analysis focused on down-regulated genes demonstrated gene repression corresponding to adaptive immunity-specific signaling pathways and was most prominent in patients with septic shock on days 1 and 3. Gene network analyses based on direct comparisons across the SIRS, sepsis, and septic shock spectrum, and all available patients in the database, demonstrated unique repression of gene networks in patients with septic shock corresponding to major histocompatibility complex antigen presentation. Finally, analyses focused on repression of genes corresponding to zinc-related biology demonstrated that this pattern of gene repression is unique to patients with septic shock. Conclusions:Although some common patterns of gene expression exist across the pediatric SIRS, sepsis, and septic shock spectrum, septic shock is particularly characterized by repression of genes corresponding to adaptive immunity and zinc-related biology.

Increased serum nitrite and nitrate concentrations in children with the sepsis syndrome

OBJECTIVES To measure total serum nitrite and nitrate concentrations in children with the sepsis syndrome as an indicator of endogenous nitric oxide production. To determine if there is an association between total serum nitrite and nitrate concentrations and vascular responsiveness to norepinephrine. DESIGN A prospective, clinical study. SETTING Tertiary, multidisciplinary, pediatric intensive care unit. PATIENTS Thirty-one children with the sepsis syndrome, 18 of whom were also hypotensive. Sixteen critically ill children without signs of the sepsis syndrome served as controls. INTERVENTIONS Blood samples were obtained from indwelling catheters. The norepinephrine dose to reach the age appropriate, 50th percentile mean arterial blood pressure was determined in patients receiving norepinephrine. MEASUREMENTS AND MAIN RESULTS Total serum nitrite and nitrate concentrations were measured on the first three days after the recognition of the sepsis syndrome. Patients with the sepsis syndrome had increased mean total serum nitrite and nitrate concentrations (day 1, 118 +/- 93 microM; day 2, 112 +/- 94 microM; day 3, 112 +/- 93 microM) vs. controls (43 +/- 24 microM, p < .05) on all 3 days. When sepsis syndrome patients were separated into nonhypotensive and hypotensive groups, only the patients with hypotension had increased concentrations vs. controls on all three days (p < .05). Sepsis syndrome patients with hypotension also had higher total serum nitrite and nitrate concentrations (145 +/- 97 microM) than sepsis syndrome patients without hypotension (82 +/- 76 microM, p < .05) on day 1. In five patients receiving norepinephrine infusions, increased total serum nitrite and nitrate concentrations were associated with higher norepinephrine requirements to maintain an age-appropriate, 50th percentile mean arterial blood pressure on each of the three study days (day 1, rs = 0.821, p < .05; day 2, rs = 0.900, p < .05; day 3, rs = 0.872, p < .05). CONCLUSIONS Children with the sepsis syndrome, particularly those patients with hypotension, have increased total serum nitrite and nitrate concentrations that likely reflect increased endogenous production of nitric oxide. Vascular hyporesponsiveness to norepinephrine during the sepsis syndrome may be, in part, a nitric oxide-mediated process.

Absence of inducible nitric oxide synthase modulates early reperfusion-induced NF-κB and AP-1 activation and enhances myocardial damage

The role of nitric oxide (NO) generated by the inducible NO synthase (iNOS) during myocardial ischemia and reperfusion is not understood. We investigated the role of iNOS during early reperfusion damage induced in genetically deficient iNOS (iNOS‐/‐) mice and wild‐type littermates. In wild‐type mice, ischemia (60 min) and reperfusion (60 min) induced an elevation in serum levels of creatine phosphokinase and myocardial injury characterized by the presence of scattered apoptotic myocytes and mild neutrophil infiltration. Northern blot analysis showed increased expression of iNOS, whose activity was markedly elevated after reperfusion. Immunohistochemistry showed staining for nitrotyrosine; Western blot analysis showed elevated expression of heat shock protein 70 (HSP70), a putative cardioprotective mediator. Plasma levels of nitrite and nitrate, tumor necrosis factor α (TNF‐α), interleukin 6 (IL‐6), and IL‐10 were also increased. These events were preceded by degradation of inhibitor κBα (IκBα), activation of IκB kinase complex (IKK) and c‐Jun‐NH2‐terminal kinase (JNK), and subsequently activation of nuclear factor‐κB (NF‐κB) and activator protein 1 (AP‐1) as early as 15 min after reperfusion. In contrast, iNOS‐/‐ mice experienced 35% mortality after reperfusion. The extensive myocardial injury was associated with marked apoptosis and infiltration of neutrophils whereas expression of HSP70 was less pronounced. Nitrotyrosine formation and plasma levels of nitrite and nitrate were undetectable. TNF‐α and IL‐6 were increased and IL‐10 was reduced in earlier stages of reperfusion. Activation of IKK and JNK and binding activity of NF‐κB and AP‐1 were significantly reduced. Thus, we conclude that iNOS plays a beneficial role in modulating the early defensive inflammatory response against reperfusion injury through regulation of signal transduction.—Zingarelli, B., Hake, P. W., Yang, Z., O’Connor, M., Denenberg, A., Wong, H. R. Absence of inducible nitric oxide synthase modulates early reperfusion‐induced NF‐κB and AP‐1 activation and enhances myocardial damage. FASEB J. 16, 327–342 (2002)

The Serine/Threonine Phosphatase, PP2A: Endogenous Regulator of Inflammatory Cell Signaling

We have investigated the regulation of kinases and phosphatases in early gene activation in monocytes because these cells are implicated in the pathogenesis of acute inflammatory states, such as sepsis and acute lung injury. One early gene up-regulated by endotoxin is c-Jun, a member of the activating protein (AP) family. C-Jun is phosphorylated by c-Jun N-terminal kinase (JNK) and associates with c-Fos to form the AP-1 transcriptional activation complex that can drive cytokine expression. Inhibition of the serine/threonine phosphatase, PP2-A, with okadaic acid resulted in a significant increase in JNK activity. This finding was associated with increased phosphorylation of c-Jun, AP-1 transcriptional activity, and IL-1β expression. Activation of PP2A inhibited JNK activity and JNK coprecipitated with the regulatory subunit, PP2A-Aα, supporting the conclusion that PP2A is a key regulator of JNK in the context of an inflammatory stimulus.

Usefulness of corticosteroid therapy in decreasing epinephrine requirements in critically ill infants with congenital heart disease

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The host response to sepsis and developmental impact.

Invasion of the human by a pathogen necessitates an immune response to control and eradicate the microorganism. When this response is inadequately regulated, systemic manifestations can result in physiologic changes described as “sepsis.” Recognition, diagnosis, and management of sepsis remain among the greatest challenges shared by the fields of neonatology and pediatric critical care medicine. Sepsis remains among the leading causes of death in both developed and underdeveloped countries and has an incidence that is predicted to increase each year. Despite these sobering statistics, promising therapies derived from preclinical models have universally failed to obviate the substantial mortality and morbidity associated with sepsis. Thus, there remains a need for well-designed epidemiologic and mechanistic studies of neonatal and pediatric sepsis to improve our understanding of the causes (both early and late) of deaths attributed to the syndrome. In reviewing the definitions and epidemiology, developmental influences, and regulation of the host response to sepsis, it is anticipated that an improved understanding of this host response will assist clinician-investigators in identifying improved therapeutic strategies.

Cerebrospinal fluid and plasma nitrite and nitrate concentrations after head injury in humans.

OBJECTIVES To measure cerebrospinal fluid and plasma nitrite and nitrate concentrations as indicators of nitric oxide production in adults after severe closed-head injury. To determine if there is an association between cerebrospinal fluid and plasma nitrite and nitrate concentrations, and cerebral blood flow, arterio-jugular oxygen content difference, injury severity, and outcome after severe closed-head injury. DESIGN A prospective, clinical study. SETTING Multidisciplinary intensive care unit. PATIENTS Fifteen comatose (Glasgow Coma Scale score of < or = 7) adult patients with severe closed-head injury were studied during the prospective, randomized evaluation of the effect of moderate hypothermia (32 degrees C for 24 hrs) on neurologic outcome after closed-head injury. Seven patients were in the hypothermic group and eight patients were in the normothermic treatment group. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Patients were examined sequentially, every 12 hrs for 2 days. Intraventricular cerebrospinal fluid was assayed for nitrite and nitrate concentrations. Cerebral blood flow was measured by the 133xenon intravenous method. Simultaneous blood samples were obtained for measurements of arterio-jugular oxygen content difference and plasma nitrite and nitrate concentrations. Cerebral metabolic rate for oxygen was calculated. Cerebrospinal fluid nitrite and nitrate concentrations were highest at 30 to 42 hrs vs. 6 to 18, 18 to 30, and 42 to 54 hrs (26.4 +/- 3.3 vs. 17.3 +/- 2.1, 20.0 +/- 2.2, and 18.8 +/- 2.4 microM, respectively, p < .05). There was no difference over time in plasma nitrite and nitrate concentrations. Cerebral blood flow was increased and arterio-jugular oxygen content difference was reduced at 18 to 30, 30 to 42, and 42 to 54 hrs vs. 6 to 18 hrs (p < .05). At 30 to 42 hrs, cerebrospinal fluid nitrite and nitrate concentrations were 80% higher in patients who died vs. survivors (36.4 +/- 3.2 vs. 20.2 +/- 3.6, p < .05). Using a generalized, multivariate, linear regression model, both plasma nitrite and nitrate concentrations and injury Severity Score independently predicted cerebrospinal fluid nitrite and nitrate concentrations (p < .00001 and p = .0053, respectively). Cerebral blood flow and arterio-jugular oxygen content difference were not associated with cerebrospinal fluid or plasma nitrite and nitrate concentrations using this model. Cerebrospinal fluid nitrite and nitrate concentrations were increased over time in hypothermic vs. normothermic patients. But, where this difference occurred could not be determined by multiple comparisons (p = .03). The hypothermic patients had lower admission Glasgow Coma Scale scores than normothermic patients (p = .04) and tended to have higher injury Severity Scores (p = .09). CONCLUSIONS Increases in cerebrospinal fluid nitrite and nitrate concentrations peaked at 30 to 42 hrs after severe closed-head injury. This increase in cerebrospinal fluid nitrite and nitrate concentrations was greater in nonsurvivors. Also, cerebrospinal fluid and plasma nitrite and nitrate concentrations were associated with injury Severity Score, suggesting that increased nitric oxide production in the brain is associated with injury severity and death. Hypothermia did not prevent the increase in cerebrospinal fluid nitrite and nitrate concentrations. Further study is required to determine the source of this increase in cerebrospinal fluid nitrite and nitrate concentrations and to further define the relationship to outcome and the effect of hypothermia on this process.