Validity of self-reported migraine in adolescents and children.

OBJECTIVE: To assess agreement for migraine day between self-report and diagnostic guidelines for children and adolescents using a headache diary. BACKGROUND: Trial guidelines recommend prospective collection of headache features and adoption of migraine day as an outcome measure, but there is no clear consensus on the definition of migraine day. METHODS: This is a secondary analysis of data from two projects-a prospective cohort study validating a pediatric scale of treatment expectancy and a clinical trial of occipital nerve blocks to treat status migrainosus. Participants completed a text message-based diary for 4 or 12 weeks (depending on treatment), and a detailed headache assessment on a random 20% of headache days. Using this assessment, we determined whether a headache day qualified for migraine or probable migraine, based on the International Classification of Headache Disorders, 3rd edition (ICHD-3). RESULTS: Of 122 enrolled children and adolescents, 106 (86.9%) completed ≥1 detailed headache assessment (438 entries). We found moderate agreement between self-reported and ICHD-derived migraine day, with a Cohen's Kappa of 0.50 (positive predictive value [PPV]: 0.66; negative predictive value [NPV]: 0.85; correlation: 0.51). Allowing for ICHD-derived probable migraine significantly increased PPV (0.66 vs. 0.94; 95% CI: 0.57-0.74 vs. 0.90-0.97), but decreased NPV (0.85 vs. 0.293; CI: 0.77-0.90 vs. 0.199-0.40), Cohen's Kappa (0.50 vs. 0.237; CI: 0. 389-0.60 vs. 0.139-0.352), and correlation (r = 0.51 vs. 0.302; CI: 0.41-0.61 vs. 0.192-0.41). Pain severity (OR: 5.7; CI: 2.39-13.8), photophobia (OR: 4.1; CI: 1.02-16.6), and phonophobia (OR: 7.5; CI: 1.95-29.3) were significantly associated with participants' perception of migraine. CONCLUSION: We found only moderate agreement between self-reported and ICHD-derived migraine day, suggesting both measures are not equal but may represent overlapping aspects of migraine as a disease. This highlights the difficulty of applying ICHD criteria to individual attacks. We recommend greater methodological transparency in future research to avoid readers conflating both measures.

Development of a text message-based headache diary in adolescents and children.

BACKGROUND: International guidelines recommend diaries in migraine trials for prospective collection of headache symptoms. Studies in other patient populations suggest higher adherence with electronic diaries instead of pen-and-paper. This study examines the feasibility of a text message-based (texting) diary for children and adolescents with headache. METHODS: This is a secondary analysis of data from a study validating a pediatric scale of treatment expectancy. We developed a Health Insurance Portability and Accountability Act-compliant texting diary collecting headache characteristics, medication use, and disability with 3-5 core daily questions for 4 or 12 weeks depending on headache treatment. Adherence was incentivized. RESULTS: 93 participants consented to the expectancy study. Five participants opted for a paper diary for follow-up. 88 participants chose the texting diary with 28 4-week and 60 12-week participants. Five participants did not complete the enrollment visit. Of those remaining 83, 89% of 4-week and 93% of 12-week participants responded on at least 80% of days. On average, participants fully completed 88% (4-week cohort) and 90% (12-week) of diary entries. CONCLUSIONS: Text messages are a promising method for collecting patient-reported data. Adherence was similar to that reported for paper diaries in other pediatric migraine trials, but time-stamped entries ensure real-time data collection.

The spectrum of indomethacin-responsive headaches in children and adolescents.

BACKGROUND: Headaches with marked, specific response to indomethacin occur in children, but the phenotypic spectrum of this phenomenon has not been well-studied. METHODS: We reviewed pediatric patients with headache showing ≥80% improvement with indomethacin, from seven academic medical centers. RESULTS: We included 32 pediatric patients (16 females). Mean headache onset age was 10.9 y (range 2-16 y). Headache syndromes included hemicrania continua (n = 13), paroxysmal hemicrania (n = 10), primary stabbing headache (n = 2), short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (n = 1), primary exercise headache (n = 1) and primary cough headache (n = 1). Adverse events were reported in 13, most commonly gastrointestinal symptoms, which often improved with co-administration of gastro-protective agents. CONCLUSION: Indomethacin-responsive headaches occur in children and adolescents, and include headache syndromes, such as primary cough headache, previously thought to present only in adulthood. The incidence of adverse events is high, and patients must be co-treated with a gastroprotective agent.

Orexinergic activity modulates altered vital signs and pituitary hormone secretion in experimental sepsis.

OBJECTIVES: Sepsis is a common, lethal poorly understood disorder affecting nearly a million Americans annually. The syndrome is characterized by altered cardiodynamics, respiration, metabolism, pituitary function, arousal, and impaired interaction among organ systems. The immunologic and endocrine systems, which are in part responsible for organ-organ communication, have been studied extensively in sepsis. However, little is known about sepsis-induced changes in central nervous system activity. HYPOTHESIS: A defect in hypothalamic neurons secreting the neurotransmitter orexin modulates physiologic derangements in sepsis. DESIGN: Animal study. SETTING: University Research Laboratory. INTERVENTIONS: Male C57Bl6 mice were made septic using cecal ligation and puncture. Data were collected 24 or 48 hours later, blood was collected, animals were killed, and brain tissue was harvested, fixed, and sectioned. Hypothalamic sections were subjected to immunohistochemistry using antibodies to orexin and c-Fos, a marker of neuronal activity. In a separate cohort of mice, cannulas were placed in the right lateral cerebral ventricle. Cecal ligation and puncture was performed 1 week later. At 24 or 48 hours post-cecal ligation and puncture, vital signs were measured, and1 µL of saline with or without 3 nmol orexin-A was infused. Vital signs were repeated at 25 or 49 hours post-cecal ligation and puncture, blood was collected, animals were killed, and brains were removed, fixed, sectioned, and stained. MEASUREMENTS AND MAIN RESULTS: Orexinergic activity decreased six-fold following cecal ligation and puncture. This change was associated with decreases in arousal, temperature, and heart and respiratory rates. Levels of selected pituitary hormones increased 24 hours post-cecal ligation and puncture but were significantly lower than baseline at 48 hours. Injection of orexin-A increased vital signs to baseline levels. Hormone levels were unaffected at 25 hours but increased to supranormal levels at 49 hours. CONCLUSIONS: Sepsis-induced changes in activity, vital signs, and pituitary hormones are modulated by the orexinergic system. This finding implicates central nervous system dysfunction in the pathogenesis of sepsis, suggesting further study of neurological dysfunction to identify novel approaches to management.

Failed interleukin-6 signal transduction in murine sepsis: attenuation of hepatic glycoprotein 130 phosphorylation.

OBJECTIVE: Sepsis impairs the activation of the interleukin (IL)-6 dependent transcription factor signal transducer and activator of transcription (STAT)-3. However, the molecular basis for depressed functionality has not been characterized. In this study, we test the hypothesis that altered signal transduction results from a change in the activation state of one or more of the components of the intracellular IL-6-linked pathway. DESIGN: Randomized prospective experimental study. SETTING: University medical laboratory. SUBJECTS: Male, 6-8-week-old C57/Bl6 mice. INTERVENTIONS: Cecal ligation and single puncture (CLP) or cecal ligation and double puncture (2CLP) was used to model mild and fulminant sepsis, respectively. Sham-operated and unoperated animals served as controls. All animals were fluid resuscitated at the time of surgery and every 24 hours thereafter. Surviving animals were euthanized at 3, 6, 16, 24, 48, and 72 hours; blood samples were obtained and liver tissue was harvested. MEASUREMENTS AND MAIN RESULTS: Serum IL-6 levels were elevated in both CLP and 2CLP relative to controls. STAT-3 DNA binding activity and nuclear phosphorylated-STAT-3 levels were elevated in CLP but decreased abruptly 24 hours after 2CLP. This 2CLP-induced alteration was associated with attenuated phosphorylation of the key transcellular glycoprotein (gp) 130. Abundance and phosphorylation of the other key component of IL-6 signal transduction pathway, janus kinase-1, was unchanged following either CLP or 2CLP. 2CLP also did not cause disassociation of the gp130-janus kinase-1 complex. CONCLUSIONS: Impaired gp130 phosphorylation may be responsible for IL-6 hyporesponsiveness during sepsis.

Trigeminal Pain Responses in Obese ob/ob Mice Are Modality-Specific.

How obesity exacerbates migraine and other pain disorders remains unknown. Trigeminal nociceptive processing, crucial in migraine pathophysiology, is abnormal in mice with diet induced obesity. However, it is not known if this is also true in genetic models of obesity. We hypothesized that obese mice, regardless of the model, have trigeminal hyperalgesia. To test this, we first evaluated trigeminal thermal nociception in leptin deficient (ob/ob) and control mice using an operant thermal assay. Unexpectedly, we found significant hypoalgesia in ob/ob mice. Because thermal hypoalgesia also occurs in mice lacking the transient receptor potential vanilloid 1 channel (TRPV1), we tested capsaicin-evoked trigeminal nociception. Ob/ob and control mice had similar capsaicin-evoked nocifensive behaviors, but ob/ob mice were significantly less active after a facial injection of capsaicin than were diet-induced obese mice or lean controls. Conditioned place aversion in response to trigeminal stimulation with capsaicin was similar in both genotypes, indicating normal negative affect and pain avoidance. Supporting this, we found no difference in TRPV1 expression in the trigeminal ganglia of ob/ob and control mice. Finally, we assessed the possible contribution of hyperphagia, a hallmark of leptin deficiency, to the behavior observed in the operant assay. Ob/ob and lean control mice had similar reduction of intake when quinine or capsaicin was added to the sweetened milk, excluding a significant contribution of hyperphagia. In summary, ob/ob mice, unlike mice with diet-induced obesity, have trigeminal thermal hypoalgesia but normal responses to capsaicin, suggesting specificity in the mechanisms by which leptin acts in pain processing.

Enhanced expression of 70-kilodalton heat shock protein limits cell division in a sepsis-induced model of acute respiratory distress syndrome.

OBJECTIVE: Fibrotic changes are initiated early in acute respiratory distress syndrome. This may involve overproliferation of alveolar type II cells. In an animal model of acute respiratory distress syndrome, we have shown that the administration of an adenoviral vector overexpressing the 70-kd heat shock protein (AdHSP) limited pathophysiological changes. We hypothesized that this improvement may be modulated, in part, by an early AdHSP-induced attenuation of alveolar type II cell proliferation. DESIGN: Laboratory investigation. SETTING: Hadassah-Hebrew University and University of Pennsylvania animal laboratories. SUBJECTS: Sprague-Dawley Rats (250 g). INTERVENTIONS: Lung injury was induced in male Sprague-Dawley rats via cecal ligation and double puncture. At the time of cecal ligation and double puncture, we injected phosphate-buffered saline, AdHSP, or AdGFP (an adenoviral vector expressing the marker green fluorescent protein) into the trachea. Rats then received subcutaneous bromodeoxyuridine. In separate experiments, A549 cells were incubated with medium, AdHSP, or AdGFP. Some cells were also stimulated with tumor necrosis factor-alpha. After 48 hrs, cytosolic and nuclear proteins from rat lungs or cell cultures were isolated. These were subjected to immunoblotting, immunoprecipitation, electrophoretic mobility shift assay, fluorescent immunohistochemistry, and Northern blot analysis. MEASUREMENTS AND MAIN RESULTS: Alveolar type I cells were lost within 48 hrs of inducing acute respiratory distress syndrome. This was accompanied by alveolar type II cell proliferation. Treatment with AdHSP preserved alveolar type I cells and limited alveolar type II cell proliferation. Heat shock protein 70 prevented overexuberant cell division, in part, by inhibiting hyperphosphorylation of the regulatory retinoblastoma protein. This prevented retinoblastoma protein ubiquitination and degradation and, thus, stabilized the interaction of retinoblastoma protein with E2F1, a key cell division transcription factor. CONCLUSIONS: : Heat shock protein 70-induced attenuation of cell proliferation may be a useful strategy for limiting lung injury when treating acute respiratory distress syndrome if consistent in later time points.

IL-6 modulates sepsis-induced decreases in transcription of hepatic organic anion and bile acid transporters.

Sepsis, a lethal inflammatory syndrome, is characterized by organ system dysfunction. In the liver, we have observed decreased expression of genes encoding proteins modulating key processes. These include organic anion and bile acid transport. We hypothesized that the inflammatory mediator IL-6 modulates altered expression of several key hepatic genes in sepsis via induction of the intracellular transcription factor signal transducer and activator of transcription (Stat) 3. Sepsis was induced in IL-6 +/+ and IL-6 -/- mice, and expression of the liver-restricted genes encoding the sodium-taurocholate cotransporter (Ntcp), the multidrug resistant protein (MRP) 2 and the organic anion transporter protein (OATP), was determined. As demonstrated previously, cecal ligation and puncture decreases expression of Ntcp, MRP-2, and OATP in IL-6 +/+ mice. Transcription elongation analysis demonstrated that altered expression resulted from decreased transcription. These changes were not observed in IL-6 -/- animals. Cecal ligation and puncture increased the DNA binding activity of Stat-3 in IL-6 +/+ but not IL-6 -/- mice. Because the promoters of Ntcp, MRP-2, and OATP do not contain Stat-3 binding sites, we postulated that altered Ntcp, MRP-2, and OATP expression resulted from activation of hepatocyte nuclear factor (HNF) 1alpha, which is IL-6 dependent. Cecal ligation and puncture decreased HNF-1alpha expression and DNA binding activity in IL-6 +/+ but not IL-6 -/- mice. Recombinant human IL-6 restored the sepsis-induced decrease in Ntcp, MRP-2, OATP, and HNF-1alpha expression in IL-6 -/- mice. We conclude that sepsis decreases the expression of three key hepatic genes via a transcriptional mechanism that is IL-6, Stat-3, and HNF-1alpha dependent.

Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.

TAT-HSP70 Attenuates Experimental Lung Injury.

Sepsis, a poorly understood syndrome of disordered inflammation, is the leading cause of death in critically ill patients. Lung injury, in the form of acute respiratory distress syndrome (ARDS), is the most common form of organ injury in sepsis. The heat shock response, during which heat shock proteins (HSPs) are expressed, is an endogenous mechanism to protect cells from injury. We have found that the abundance of pulmonary HSP70 is not increased after cecal ligation and double puncture (CLP) in a rat model of sepsis-induced ARDS. Using the HIV-1 trans-activator of transcription (TAT) cell-penetrating protein, we enhanced HSP70 protein abundance in the lung. We found that intratracheal administration of HSP70 using the TAT methodology, just after CLP (CLP-TAT-HSP70), when compared with treatment with phosphate buffered saline (CLP-phosphate buffered saline), significantly increased HSP70 abundance in the lung 24 and 48 h after surgery. Treatment of septic rats with TAT-HSP70 increased HSP70 abundance in histologically normal and abnormal lung regions. In addition, TAT-HSP70 treatment significantly decreased the levels of macrophage inflammatory protein 2 and cytokine-induced neutrophil chemoattractant 1 24 h after CLP. The TAT-HSP70 treatment reduced myeloperoxidase abundance 48 h after CLP and attenuated histological evidence of inflammation at both 24 and 48 h. Administration of TAT-HSP70 also improved 48-h survival in this rat model of sepsis. Thus, intratracheal administration of TAT-HSP70 increased HSP70 abundance in the lung and attenuated the lung injury. Enhancing pulmonary HSP70 using TAT is a novel potential therapeutic strategy for the treatment of ARDS that will be explored further.

Competitive and noncompetitive inhibition of myocardial cytochrome C oxidase in sepsis.

Sepsis is the most common cause of death in intensive care units worldwide. The basic pathophysiologic defect in sepsis, causing functional abnormalities in many organ systems, remains elusive. One potential cause is disruption of oxidative phosphorylation in mitochondria. Here, we report that oxidation of cytochrome c by myocardial cytochrome c oxidase, the terminal oxidase in the electron transport chain, is competitively inhibited early in experimental sepsis (cecal ligation with single or double 23-gauge puncture) in mice. In severe sepsis (cecal ligation and double puncture, 75% mortality at 48 h), inhibition becomes noncompetitive by 48 h. The development of noncompetitive inhibition is associated with a decrease in heme a,a3 content, which is the key active site in the functional subunit (I) and catalyzes the reduction of molecular oxygen. In addition, there are persistently decreased steady-state levels of subunit I mRNA and protein after cecal ligation and double puncture. Both loss of heme and loss of subunit I could explain the observed irreversible inhibition of cytochrome c oxidase. Noncompetitive inhibition of cytochrome c oxidase may interrupt oxidative phosphorylation, leading to sepsis-associated cardiac depression. Importantly, this abnormality may underlie sepsis-associated dysfunction in other organ systems.

Transcription factors C/EBP-alpha and HNF-1alpha are associated with decreased expression of liver-specific genes in sepsis.

Previous studies have demonstrated sepsis-specific changes in the transcription of key hepatic genes. However, the role of hepatic transcription factors in sepsis-associated organ dysfunction has not been well established. We hypothesize that the binding activities of C/EBPalpha and beta, HNF-1alpha, and HNF-3 transiently decrease during mild sepsis but persistently decrease after fulminant sepsis, and that the decrease in this binding activity correlates in time and severity with previously described decreases in the transcription of key hepatic genes. Male C57/BL6 mice had nonlethal sepsis induced by cecal ligation and single puncture (CLP) and fulminant sepsis via cecal ligation and double puncture (2CLP). Sham-operated and unoperated animals served as controls. Transcription factor binding activity was assessed with electrophoretic mobility shift assays. C/EBP-a and HNF-1alpha binding activity decreased transiently after CLP and persistently after 2CLP. Binding activity of both C/EBP-beta and HNF-3 were unchanged. The decrease in C/EBP-a and HNF-1alpha binding activities correlated in time and magnitude with the decreased hepatic gene transcription previously observed in sepsis. Furthermore, the loss of activity after 2CLP correlated in time with outcome. Sepsis decreases DNA binding activities of C/EBPalpha and HNF-1alpha, two key hepatocyte transcription factors, in a time course consistent with down-regulation of their target hepatic genes. Therefore, alterations in transcription factor binding are likely important in the transcriptional modulation that is characteristic of hepatic dysfunction in sepsis.

Enhanced Hsp70 expression protects against acute lung injury by modulating apoptotic pathways.

The Acute respiratory distress syndrome (ARDS) is a highly lethal inflammatory lung disorder. Apoptosis plays a key role in its pathogenesis. We showed that an adenovirus expressing the 70 kDa heat shock protein Hsp70 (AdHSP) protected against sepsis-induced lung injury. In this study we tested the hypothesis that AdHSP attenuates apoptosis in sepsis-induced lung injury. Sepsis was induced in rats via cecal ligation and double puncture (2CLP). At the time of 2CLP PBS, AdHSP or AdGFP (an adenoviral vector expressing green fluorescent protein) were injected into the tracheas of septic rats. 48 hours later, lungs were isolated. One lung was fixed for TUNEL staining and immunohistochemistry. The other was homogenized to isolate cytosolic and nuclear protein. Immunoblotting, gel filtration and co-immunoprecipitation were performed in these extracts. In separate experiments MLE-12 cells were incubated with medium, AdHSP or AdGFP. Cells were stimulated with TNFα. Cytosolic and nuclear proteins were isolated. These were subjected to immunoblotting, co-immunoprecipitation and a caspase-3 activity assay. TUNEL assay demonstrated that AdHSP reduced alveolar cell apoptosis. This was confirmed by immunohistochemical detection of caspase 3 abundance. In lung isolated from septic animals, immunoblotting, co-immunoprecipitation and gel filtration studies revealed an increase in cytoplasmic complexes containing caspases 3, 8 and 9. AdHSP disrupted these complexes. We propose that Hsp70 impairs apoptotic cellular pathways via interactions with caspases. Disruption of large complexes resulted in stabilization of lower molecular weight complexes, thereby, reducing nuclear caspase-3. Prevention of apoptosis in lung injury may preserve alveolar cells and aid in recovery.

Protection against sepsis-induced lung injury by selective inhibition of protein kinase C-δ (δ-PKC).

Inflammation and proinflammatory mediators are activators of δ-PKC. In vitro, δ-PKC regulates proinflammatory signaling in neutrophils and endothelial and epithelial cells, cells that can contribute to lung tissue damage associated with inflammation. In this study, a specific δ-PKC TAT peptide inhibitor was used to test the hypothesis that inhibition of δ-PKC would attenuate lung injury in an animal model of ARDS. Experimental ARDS was induced in rats via 2CLP, a model of polymicrobial sepsis. Following 2CLP surgery, the δ-PKC TAT inhibitory peptide (2CLP+δ-PKC TAT in PBS) or PBS (2CLP+PBS) was administered intratracheally. Controls consisted of SO, where animals underwent a laparotomy without 2CLP. Twenty-four hours after SO or 2CLP, blood, BALF, and lung tissue were collected. 2CLP induced δ-PKC phosphorylation in the lung within 24 h. Treatment with the δ-PKC TAT inhibitory peptide significantly decreased pulmonary δ-PKC phosphorylation, indicating effective inhibition of δ-PKC activation. Plasma and BALF levels of the chemokines CINC-1 and MIP-2 were elevated in 2CLP + PBS rats as compared with SO rats. Treatment with δ-PKC TAT reduced 2CLP-induced elevations in chemokine levels in BALF and plasma, suggesting that δ-PKC modulated chemokine expression. Most importantly, intratracheal administration of δ-PKC TAT peptide significantly attenuated inflammatory cell infiltration, disruption of lung architecture, and pulmonary edema associated with 2CLP. Thus, δ-PKC is an important regulator of proinflammatory events in the lung. Targeted inhibition of δ-PKC exerted a lung-protective effect 24 h after 2CLP.

Impaired hepatocellular regeneration in murine sepsis is dependent on regulatory protein levels.

Sepsis is a poorly understood syndrome. Therefore, we examined the mechanisms underlying failed regeneration in sham-operated (SO), mildly septic (cecal ligation and single puncture [CLP]), and severely septic (cecal ligation with two punctures [2CLP]) C57Bl6 mice. Relative to no operation (T0) or SO, CLP, but not 2CLP, increased the number of cells staining for proliferating cell nuclear antigen, a marker for cell division. Levels of the retinoblastoma protein (pRb) were detected at T0 and after SO. CLP increased pRb abundance, whereas 2CLP decreased it. Changes in phosphorylated pRb were similar but more profound. The abundance of the transcription factor E2F was unaltered by SO, CLP, or 2CLP. However, E2F DNA binding activity, although unchanged after SO, increased after CLP and decreased after 2CLP. The abundance of cyclin D1 in nuclear fractions increased following CLP but decreased after 2CLP. Neither SO nor 2CLP altered the abundance of the cyclin-dependent kinase (cdk) 4. However, cdk-4 abundance increased after CLP. Finally, CLP increased the steady-state abundance of the mRNAs encoding thymidine kinase, DNA polymerase α, and dihydrofolate reductase, all required for DNA replication. No changes were noted after 2CLP. We conclude that 2CLP impaired hepatocyte proliferation following 2CLP in part via impaired cyclin D1/cdk-4-induced phosphorylation of pRb, maintaining the association between pRb and E2F and inhibited E2F transcriptional activity.

Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome.

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.

Sepsis-induced cholestasis, steatosis, hepatocellular injury, and impaired hepatocellular regeneration are enhanced in interleukin-6 -/- mice.

OBJECTIVE: Hepatic dysfunction is an important but poorly understood component of sepsis. In severe sepsis, liver dysfunction is characterized by cholestasis, steatosis, hepatocellular injury, impaired regeneration, a decreased response to the cytokine interleukin-6, and high mortality. To determine whether loss of interleukin-6 activity caused hepatic dysfunction and mortality, we induced sepsis in wild-type (interleukin-6 +/+) and interleukin-6 knockout (interleukin-6 -/-) mice. We hypothesized that sepsis in interleukin-6 -/- mice would increase cholestasis, steatosis, hepatocellular injury, and mortality and impair hepatocyte regeneration. DESIGN: Randomized prospective experimental study. SETTING: University medical laboratory. SUBJECTS: Male adolescent C57Bl6 interleukin-6 +/+ and interleukin-6 -/- mice. INTERVENTIONS: Mild sepsis was induced using cecal ligation and single puncture (CLP). Severe, lethal sepsis was induced using cecal ligation and double puncture (2CLP). Some mice received recombinant human interleukin-6 at the time of CLP/2CLP. All animals were fluid resuscitated at the time of surgery and every 24 hrs thereafter. In survival cohorts, mortality at 16, 24, 48, and 72 hrs was recorded. In separate cohorts, surviving animals were killed at 24 and 48 hrs, and liver tissue was harvested. A separate cohort of mice received bromodeoxyuridine for detection of regeneration. MEASUREMENTS AND MAIN RESULTS: 2CLP was 100% fatal within the first 12 hrs in interleukin-6 -/- mice. Mortality from 2CLP in interleukin-6 +/+ mice before 24 hrs was nil but was 90% by 72 hrs. At 72 hrs, CLP was 40% fatal in interleukin-6 +/+ mice but 90% in interleukin-6 -/- mice. CLP induced cholestasis, steatosis, and hepatocellular injury in interleukin-6 -/-, but not interleukin-6 +/+, mice. Regeneration was absent following CLP in interleukin-6 -/- animals but occurred in interleukin-6 +/+ mice. Early administration of recombinant human interleukin-6 did not reverse abnormalities in interleukin-6 -/- mice. CONCLUSIONS: The absence of interleukin-6 is an important determinant of hepatic dysfunction and mortality in sepsis.