Rapamycin Augments the NMDA-Mediated TNF Suppression of MRSA-Stimulated RAW264.7 Murine Macrophages

Background. Methicillin-resistant Staphylococcus aureus (MRSA) can stimulate massive cytokine release. Ketamine suppresses tumor necrosis factor (TNF) secretion by MRSA-stimulated RAW264.7 macrophages, and the mechanism likely involves N-methyl-D-aspartic acid (NMDA) receptor antagonism. The downstream effects of NMDA-mediated TNF suppression, specifically the PI3K/Akt and mTOR modulation, have not been described. Methods. RAW264.7 cells were stimulated for 18 hrs with 10(5) to 10(7) CFU/mL inocula of either of two prototypical community-acquired- (CA-) MRSA isolates, USA300 strain LAC and USA400 strain MW2. Then we added the NMDA inhibitors ketamine or 2R-amino-5-phosphonopentanoate (AP5), NMDA substrate, LY294002, and rapamycin in various combinations. Results. NMDA inhibition suppressed TNF secretion by almost a third compared to the no-ketamine control. When NMDA substrate was added, the TNF secretion increased by 10%. Addition of LY294002 suppressed TNF production by macrophages by 20%. Rapamycin exhibited a concentration-dependent TNF induction-suppression response: induction at doses of 0.1 and 1 ng/mL and suppression at 10 and 100 ng/mL. Induction of TNF was abolished when LY294002 was added and the suppression became uniform. Ketamine-induced suppression of TNF secretion was intensified 10–15% when rapamycin was added, but not when LY294002 was added. Conclusion. These findings suggest that NMDA-induced TNF suppression can be augmented by concurrent mTOR inhibition

Rapamycin Augments the NMDA-Mediated TNF Suppression of MRSA-Stimulated RAW264.7 Murine Macrophages

Background. Methicillin-resistant Staphylococcus aureus (MRSA) can stimulate massive cytokine release. Ketamine suppresses tumor necrosis factor (TNF) secretion by MRSA-stimulated RAW264.7 macrophages, and the mechanism likely involves Nmethyl-D-aspartic acid (NMDA) receptor antagonism. The downstream effects of NMDA-mediated TNF suppression, specifically the PI3K/Akt and mTOR modulation, have not been described. Methods. RAW264.7 cells were stimulated for 18 hrs with 10 5 to 10 7 CFU/mL inocula of either of two prototypical community-acquired-(CA-) MRSA isolates, USA300 strain LAC and USA400 strain MW2. Then we added the NMDA inhibitors ketamine or 2R-amino-5-phosphonopentanoate (AP5), NMDA substrate, LY294002, and rapamycin in various combinations. Results. NMDA inhibition suppressed TNF secretion by almost a third compared to the no-ketamine control. When NMDA substrate was added, the TNF secretion increased by 10%. Addition of LY294002 suppressed TNF production by macrophages by 20%. Rapamycin exhibited a concentration-dependent TNF inductionsuppression response: induction at doses of 0.1 and 1 ng/mL and suppression at 10 and 100 ng/mL. Induction of TNF was abolished when LY294002 was added and the suppression became uniform. Ketamine-induced suppression of TNF secretion was intensified 10-15% when rapamycin was added, but not when LY294002 was added. Conclusion. These findings suggest that NMDA-induced TNF suppression can be augmented by concurrent mTOR inhibition

Bedside sonographic measurement of optic nerve sheath diameter as a predictor of increased intracranial pressure in children

Study objective: We investigate the test performance of emergency physician-performed sonographic measurement of optic nerve sheath diameter for diagnosis of increased intracranial pressure. Methods: Children between the ages of 0 and 18 years with suspected increased intracranial pressure were prospectively recruited from the emergency department and ICU of an urban, tertiary-level, freestanding pediatric facility. Pediatric emergency physicians with goal-directed training in ophthalmic sonography measured optic nerve sheath diameter. Images were recorded and subsequently reviewed by a pediatric ophthalmologist and an ophthalmic sonographer, both of whom were blind to the patient's clinical condition. Measurements obtained by the ophthalmic sonographer were considered the criterion standard. An optic nerve sheath diameter greater than 4.0 mm in subjects younger than 1 year and greater than 4.5 mm in older children was considered abnormal. The diagnosis of increased intracranial pressure was based on results of cranial imaging or direct measurement of intracranial pressure. Results: Sixty-four patients were recruited, of whom 24 (37%) had a confirmed diagnosis of increased intracranial pressure. The sensitivity of optic nerve sheath diameter as a screening test for increased intracranial pressure was 83% (95% confidence interval [CI] 0.60 to 0.94); specificity was 38% (95% CI 0.23 to 0.54); positive likelihood ratio was 1.32 (95% CI 0.97 to 1.79) and negative likelihood ratio was 0.46 (95% CI 0.18 to 1.23). There was fair to good interobserver agreement between the pediatric emergency physician and ophthalmic sonographer ( 0.52) and pediatric ophthalmologist ( 0.64). Conclusion: The sensitivity and specificity of bedside sonographic measurement of optic nerve sheath diameter is inadequate to aid medical decisionmaking in children with suspected increased intracranial pressure. Pediatric emergency physicians with focused training by a pediatric ophthalmologist familiar with ophthalmic sonography can measure optic nerve sheath diameter accurately. [Ann Emerg Med. 2009;53:785-791.] Provide feedback on this article at the journal's Web site, www.annemergmed.com

Differential Role of Rapamycin and Torin/KU63794 in Inflammatory Response of 264.7 RAW Macrophages Stimulated by CA-MRSA

Background. Rapamycin suppresses the RAW264.7 macrophage mediated inflammatory response but in lower doses induces it. In the present study, we tested the suppression of the inflammatory response in the presence of mTOR 1 and 2 inhibitors, Torin and KU63794. Methods. RAW264.7 cells were stimulated for 18 hrs with 106 to 107 CFU/mL inocula of community-acquired- (CA-) MRSA isolate, USA400 strain MW2, in the presence of Vancomycin. Then, in sequential experiments, we added Torin, KU63794, and Rapamycin alone and in various combinations. Supernatants were collected and assayed for TNF, IL-1, IL-6, INF, and NO. Results. Rapamycin induces 10–20% of the inflammatory cascade at dose of 0.1 ng/mL and suppresses it by 60% at dose of 10 ng/mL. The induction is abolished in the presence of Torin KU63794. Torin and KU63794 are consistently suppressing cytokine production 50–60%. Conclusions. There is a differential response between Rapamycin (mTOR-1 inhibitor) and Torin KU63794 (mTOR 1 and 2 inhibitors). Torin and KU63794 exhibit a dose related suppression. Rapamycin exhibits a significant induction-suppression biphasic response. Knowledge of such response may allow manipulation of the septic inflammatory cascade for clinical advantages

Differential Role of Rapamycin and Torin/KU63794 in Inflammatory Response of 264.7 RAW Macrophages Stimulated by CA-MRSA

Background. Rapamycin suppresses the RAW264.7 macrophage mediated inflammatory response but in lower doses induces it. In the present study, we tested the suppression of the inflammatory response in the presence of mTOR 1 and 2 inhibitors, Torin and KU63794. Methods. RAW264.7 cells were stimulated for 18 hrs with 106 to 107 CFU/mL inocula of community-acquired- (CA-) MRSA isolate, USA400 strain MW2, in the presence of Vancomycin. Then, in sequential experiments, we added Torin, KU63794, and Rapamycin alone and in various combinations. Supernatants were collected and assayed for TNF, IL-1, IL-6, INF, and NO. Results. Rapamycin induces 10–20% of the inflammatory cascade at dose of 0.1 ng/mL and suppresses it by 60% at dose of 10 ng/mL. The induction is abolished in the presence of Torin KU63794. Torin and KU63794 are consistently suppressing cytokine production 50–60%. Conclusions. There is a differential response between Rapamycin (mTOR-1 inhibitor) and Torin KU63794 (mTOR 1 and 2 inhibitors). Torin and KU63794 exhibit a dose related suppression. Rapamycin exhibits a significant induction-suppression biphasic response. Knowledge of such response may allow manipulation of the septic inflammatory cascade for clinical advantages

Pulmonary function in infants with swallowing dysfunction.

Swallowing dysfunction can lead to recurring aspiration and is frequently associated with chronic symptoms such as cough and wheezing in infants. Our objective was to describe the characteristics of infants with swallowing dysfunction, determine if pulmonary function abnormalities are detectable, and if they improve after therapy.We studied 38 infants with a history of coughing and wheezing who had pulmonary function tests performed within two weeks of their diagnosis of swallowing dysfunction. The raised lung volume rapid thoracoabdominal compression technique was used. After 6 months of therapy, 17 of the infants repeated the tests.Initially, 25 had abnormal spirometry, 18 had abnormal plethysmography, and 15 demonstrated bronchodilator responsiveness. Six months later test were repeated for seventeen patients. Ten patients had continued abnormal spirometry, two patients remained normal, three patients' abnormal spirometry had normalized, and two patients' previously normal studies became abnormal. Eight of the 17 patients had continued abnormal plethysmography, six had continued normal plethysmography, and three patients' normal plethysmography became abnormal. After 6 months of treatment, eight patients demonstrated bronchodilator responsiveness, of which five continued to demonstrate bronchodilator responsiveness and three developed responsiveness. The remainder either continued to be non- bronchodilator responsive (two) or lost responsiveness (three.) The findings of the abnormal tests in most infants tested is complicated by frequent occurrence of other co-morbidities in this population, including gastroesophageal reflux in 23 and passive smoke exposure in 13 of the infants.The interpretation of lung function changes is complicated by the frequent association of swallowing dysfunction with gastroesophageal reflux and passive smoke exposure in this population. Six months of medical therapy for swallowing dysfunction/gastroesophageal reflux did not significantly improve pulmonary function in these infants. Long-term studies will be necessary to determine which of these changes persists into adulthood

Initial pulmonary function test results for subjects who underwent repeat testing 6 months after therapy.

<p>BD, bronchodilator; FEF_25–75%, forced expiratory flow between 25% and 75% of expiration; FEV_0.5, forced expiratory volume during the first half-second of expiration; ND, not done; RV, residual volume; TLC, total lung capacity; % pre, percent predicted.</p><p>Initial pulmonary function test results for subjects who underwent repeat testing 6 months after therapy.</p

Demographics of patients who had both first and second pulmonary function tests.

<p>M, male; F, female; AA, African American; C, Caucasian; Y, yes; N, no; NE, not evaluated; PA, penetration-aspiration, PBT, peribronchial thickening.</p><p>Demographics of patients who had both first and second pulmonary function tests.</p

Clinical characteristics of study subjects (n = 38).

<p>Clinical characteristics of study subjects (n = 38).</p