Michael W. Peterson

Lipopolysaccharide Activates Akt in Human Alveolar Macrophages Resulting in Nuclear Accumulation and Transcriptional Activity of β-Catenin

Exposure of human alveolar macrophages to bacterial LPS results in activation of a number of signal transduction pathways. An early event after the alveolar macrophage comes in contact with LPS is activation of the phosphatidylinositol 3 kinase (PI 3-kinase). This study evaluates the downstream effects of that activation. We observed that LPS exposure results in phosphorylation of Akt (serine 473). We found this using both phosphorylation-specific Abs and also by in vivo phosphorylation with 32P-loaded cells. AKT activation resulted in the phosphorylation-dependent inactivation of glycogen synthase kinase (GSK-3) (serine 21/9). We found that both of these events were linked to PI 3-kinase because the PI 3-kinase inhibitors, wortmannin and LY294002, inhibited LPS-induced phosphorylation of both AKT and GSK-3. Inactivation of GSK-3 has been shown to reduce the ubiquitination of β-catenin, resulting in nuclear accumulation and transcriptional activity of β-catenin. Consistent with this, we found that LPS caused an increase in the amounts of PI 3-kinase-dependent nuclear β-catenin in human alveolar macrophages and expression of genes that require nuclear β-catenin for their activation. This is the first demonstration that LPS exposure activates AKT, inactivates GSK-3, and causes accumulation and transcriptional activity of β-catenin in the nucleus of any cell, including alveolar macrophages.

Acute Kidney Injury in Patients with Acute Lung Injury: Impact of Fluid Accumulation on Classification of Acute Kidney Injury and Associated Outcomes

Objective:It has been suggested that fluid accumulation may delay recognition of acute kidney injury. We sought to determine the impact of fluid balance on the incidence of nondialysis requiring acute kidney injury in patients with acute lung injury and to describe associated outcomes, including mortality. Design:Analysis of the Fluid and Catheter Treatment Trial, a factorial randomized clinical trial of conservative vs. liberal fluid management and of management guided by a central venous vs. pulmonary artery catheter. Setting:Acute Respiratory Distress Syndrome Network hospitals. Patients:One thousand patients. Interventions:None. Measurements and Main Results:The incidence of acute kidney injury, defined as an absolute rise in creatinine of ≥0.3 mg/dL or a relative change of >50% over 48 hrs, was examined before and after adjustment of serum creatinine for fluid balance. The incidence of acute kidney injury before adjustment for fluid balance was greater in those managed with the conservative fluid protocol (57% vs. 51%, p = .04). After adjustment for fluid balance, the incidence of acute kidney injury was greater in those managed with the liberal fluid protocol (66% vs. 58%, p = .007). Patients who met acute kidney injury criteria after adjustment of creatinine for fluid balance (but not before) had a mortality rate that was significantly greater than those who did not meet acute kidney injury criteria both before and after adjustment for fluid balance (31% vs. 12%, p < .001) and those who had acute kidney injury before but not after adjustment for fluid balance (31% vs. 11%, p = .005). The mortality of those patients meeting acute kidney injury criteria after but not before adjustment for fluid balance was similar to patients with acute kidney injury both before and after adjustment for fluid balance (31% vs. 38%, p = .18). Conclusions:Fluid management influences serum creatinine and therefore the diagnosis of acute kidney injury using creatinine-based definitions. Patients with “unrecognized” acute kidney injury that is identified after adjusting for positive fluid balance have higher mortality rates, and patients who have acute kidney injury before but not after adjusting for fluid balance have lower mortality rates. Future studies of acute kidney injury should consider potential differences in serum creatinine caused by changes in fluid balance and the impact of these differences on diagnosis and prognosis.

Randomized Clinical Trial of Activated Protein C for the Treatment of Acute Lung Injury

RATIONALE Microvascular injury, inflammation, and coagulation play critical roles in the pathogenesis of acute lung injury (ALI). Plasma protein C levels are decreased in patients with acute lung injury and are associated with higher mortality and fewer ventilator-free days. OBJECTIVES To test the efficacy of activated protein C (APC) as a therapy for patients with ALI. METHODS Eligible subjects were critically ill patients who met the American/European consensus criteria for ALI. Patients with severe sepsis and an APACHE II score of 25 or more were excluded. Participants were randomized to receive APC (24 microg/kg/h for 96 h) or placebo in a double-blind fashion within 72 hours of the onset of ALI. The primary endpoint was ventilator-free days. MEASUREMENTS AND MAIN RESULTS APC increased plasma protein C levels (P = 0.002) and decreased pulmonary dead space fraction (P = 0.02). However, there was no statistically significant difference between patients receiving placebo (n = 38) or APC (n = 37) in the number of ventilator-free days (median [25-75% interquartile range]: 19 [0-24] vs. 19 [14-22], respectively; P = 0.78) or in 60-day mortality (5/38 vs. 5/37 patients, respectively; P = 1.0). There were no differences in the number of bleeding events between the two groups. CONCLUSIONS APC did not improve outcomes from ALI. The results of this trial do not support a large clinical trial of APC for ALI in the absence of severe sepsis and high disease severity.

Protein Kinase C ζ Plays a Central Role in Activation of the p42/44 Mitogen-Activated Protein Kinase by Endotoxin in Alveolar Macrophages

Human alveolar macrophages respond to endotoxin (LPS) by activation of a number of mitogen-activated protein kinase pathways, including the p42/44 (extracellular signal-related kinase (ERK)) kinase pathway. In this study, we evaluated the role of the atypical protein kinase C (PKC) isoform, PKC ζ, in LPS-induced activation of the ERK kinase pathway. Kinase activity assays showed that LPS activates PKC ζ, mitogen-activated protein/ERK kinase (MEK, the upstream activator of ERK), and ERK. LPS did not activate Raf-1, the classic activator of MEK. Pseudosubstrate-specific peptides with attached myristic acid are cell permeable and can be used to block the activity of specific PKC isoforms in vivo. We found that a peptide specific for PKC ζ partially blocked activation of both MEK and ERK by LPS. We also found that this peptide blocked in vivo phosphorylation of MEK after LPS treatment. In addition, we found that LPS caused PKC ζ to bind to MEK in vivo. These observations suggest that MEK is an LPS-directed target of PKC ζ. PKC ζ has been shown in other systems to be phosphorylated by phosphatidylinositol (PI) 3-kinase-dependent kinase. We found that LPS activates PI 3-kinase and causes the formation of a PKC ζ/PI 3-kinase-dependent kinase complex. These data implicate the PI 3-kinase pathway as an integral part of the LPS-induced PKC ζ activation. Taken as a whole, these studies suggest that LPS activates ERK kinase, in part, through activation of an atypical PKC isoform, PKC ζ.

Tanning bed exposure increases the risk of malignant melanoma.

Background  Epidemiologic studies have associated tanning bed exposure and cutaneous melanoma. The relationship between the extent of tanning bed exposure and the risk of melanoma has not been elucidated in detail.

Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase 2 mRNA.

Human alveolar macrophages have both lipopolysaccharide (LPS)-induced and constitutive phosphatidylinositol 3-kinase (PI3K) activity. We observed that blocking PI3K activity increased release of prostaglandin E2 after LPS exposure, and increasing PI3K activity (interleukin-13) decreased release of prostaglandin E2 after LPS exposure. This was not because of an effect of PI3K on phospholipase 2 activity. PI3K inhibition resulted in an increase in cyclooxygenase 2 (COX2) protein, mRNA, and mRNA stability. PI3K negatively regulated activation of the p38 pathway (p38, MKK3/6, and MAPKAP2), and an active p38 was necessary for COX2 production. The data suggest that PI3K inhibition of p38 modulates COX2 expression via destabilization of LPS-induced COX2 mRNA.

FDG-PET imaging and the diagnosis of non-small cell lung cancer in a region of high histoplasmosis prevalence

STUDY OBJECTIVE Determine the sensitivity and specificity of [F-18]-fluorine-2-deoxy-D-glucose positron emission tomography (FDG-PET) in differentiating non-small cell lung cancer (NSCLC) from benign solitary pulmonary nodules (SPNs) in a region with a high endemic rate of histoplamosis. DESIGN Prospective, clinical study. SETTING University, tertiary referral hospital in the upper Mississippi River valley. PATIENTS Ninety patients with SPNs. INTERVENTIONS Independent interpretation of FDG-PET imaging, computed tomography and pathologic evaluation of the SPNs. MEASUREMENTS AND RESULTS To detect malignant SPNs, FDG-PET imaging had a sensitivity of 93%, a specificity of 40%, a positive predictive value (PPV) of 88% and a negative predictive value (NPV) of 55%. CONCLUSIONS In a region with a high prevalence of pulmonary fungal infection, FDG-PET is sensitive but has a low specificity and NPV for identifying NSCLC. In our study cohort, FDG-PET does not appear to reduce the need for SPN biopsies.

Medical students' use of information resources: is the digital age dawning?

PurposeOne of the many challenges clinicians face is applying growing medical knowledge to specific patients; however, there is an information gap between information needs and delivery. Digital information resources could potentially bridge this gap. Because most medical students are exposed to per

Ceramide Regulates Lipopolysaccharide-Induced Phosphatidylinositol 3-Kinase and Akt Activity in Human Alveolar Macrophages

The phosphatidylinositol (PI) 3-kinase pathway is an important regulator of cell survival. In human alveolar macrophages, we found that LPS activates PI 3-kinase and its downstream effector, Akt. LPS exposure of alveolar macrophages also results in the generation of ceramide. Because ceramide exposure induces apoptosis in other cell types and the PI 3-kinase pathway is known to inhibit apoptosis, we determined the relationship between LPS-induced ceramide and PI 3-kinase activation in alveolar macrophages. We found that ceramide exposure activated PI 3-kinase and Akt. When we blocked LPS-induced ceramide with the inhibitor D609, we blocked LPS-induced PI 3-kinase and Akt activation. Evaluating cell survival after ceramide or LPS exposure, we found that blocking PI 3-kinase induced a significant increase in cell death. Because these effects of PI 3-kinase inhibition were more pronounced in ceramide- vs LPS-treated alveolar macrophages, we also evaluated NF-κB, which has also been linked to cell survival. We found that LPS, to a greater degree than ceramide, induced NF-κB translocation to the nucleus. As a composite, these studies suggest that the effects of ceramide exposure in alveolar macrophages may be very different from the effects described for other cell types. We believe that LPS induction of ceramide results in PI 3-kinase activation and represents a novel effector mechanism that promotes survival of human alveolar macrophages in the setting of pulmonary sepsis.

Human neutrophil elastase abolishes interleukin-8 chemotactic activity

A large body of literature supports the role of interleukin‐8 (IL‐8) in inflammatory lung disease. Numerous factors induce the local synthesis and secretion of this potent chemokine leading to the recruitment and activation of polymorphonuclear leukocytes. However, little is currently known about the fate of IL‐8 secreted at sites of inflammatory injury. We have found that incubation of recombinant human IL‐8 with purified human neutrophil elastase (HNE) results in the loss of IL‐8 chemotactic activity in a dose‐ and time‐dependent fashion. This loss in bioactivity is accompanied by a similar loss of IL‐8 immunoreactivity. Western blot analysis revealed that IL‐8 chemotactic activity is lost by proteolysis of the parent molecule into undetectable small fragments. The terminal digestion of IL‐8 was specific to HNE as no loss of bioactivity was observed with equimolar concentrations of the serine proteases urokinase, plasmin, thrombin, or cathepsin G. This effect on chemotactic activity is not limited to recombinant IL‐8 because HNE also digested IL‐8 secreted by human monocytes. HNE‐mediated proteolysis offers a novel mechanism for down‐regulating the inflammatory cascade initiated by IL‐8. J. Leukoc. Biol. 61: 361–366; 1997.