Immunomodulatory effects of LL-37 in the epithelia

The cationic host defence peptide LL-37 is an immunomodulatory agent that plays an important role in epithelial innate immunity. Previously, concentrations of LL-37 thought to represent levels present during inflammation have been shown to elicit the production of cytokines and chemokines by epithelial cells. To investigate the potential of lower concentrations of LL-37 to alter epithelial cell responses, normal primary keratinocytes and bronchial epithelial cells were treated with pro-inflammatory stimuli in the presence or absence of 1 – 3 μg/ml LL-37. Low, physiologically relevant concentrations of LL-37 synergistically increased IL-8 production by both proliferating and differentiated keratinocytes in response to IL-1β and the TLR5 agonist flagellin, and synergistically increased IL-8 production by bronchial epithelial cells in response to IL-1β, flagellin, and the TLR2/1 agonist PAM3CSK4. Treatment of bronchial epithelial cells with LL-37 and the TLR3 agonist poly(I:C) resulted in synergistic increases in IL-8 release and cytotoxicity. The synergistic increase in IL-8 production observed when keratinocytes were co-stimulated with flagellin and LL-37 was suppressed by pretreatment with inhibitors of Src-family kinase signalling and NF-κB translocation. These data suggest that low concentrations of LL-37 may alter epithelial responses to microbes in vivo. Microarray analysis of keratinocyte transcriptional responses after LL-37 treatment suggest that LL-37 may alter the expression of growth factors and a number of genes important to innate immune responses. LL-37 may thus play a more important role than previously suspected in the regulation of epithelial inflammation; an improved understanding of the mechanisms by which LL-37 alters chemokine responses could lead to the development of novel anti-infective and anti-inflammatory therapeutics.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat

Signaling Pathways Mediating Chemokine Induction in Keratinocytes by Cathelicidin LL-37 and Flagellin

Cathelicidin LL-37 is a multifunctional immunomodulatory and antimicrobial host defense peptide that has an important role in the immune defenses of the skin and other epithelial barriers. We have previously demonstrated that at physiological concentrations LL-37 synergistically augments the production of immune mediators in response to microbial compounds in human primary keratinocytes. Here we define the signaling mechanisms responsible for this activity. We demonstrate that inhibition of Src family kinases (SFKs) strongly inhibited the synergistic chemokine production in response to LL-37 and flagellin in keratinocytes. SFK activation was induced by LL-37 stimulation and was required for the downstream activation of Akt (protein kinase B) and the transcription factors CREB and ATF1. In cells stimulated with LL-37 and flagellin together, Akt activation was primarily induced by LL-37, while both flagellin and LL-37 contributed to the activation of CREB and ATF1 and consequently chemokine induction. The purinergic receptor P2X&lt;sub&gt;7&lt;/sub&gt; was identified as the receptor upstream of SFK activation in LL-37-stimulated keratinocytes. Overall, these findings established the P2X&lt;sub&gt;7&lt;/sub&gt;–SFK–Akt–CREB/ATF1 signaling pathway activated by LL-37 in primary keratinocytes. These signaling mechanisms mediated the synergistic effects of LL-37 on chemokine production in flagellin-stimulated keratinocytes, and thus might have a role in the immune defenses of the skin and possibly other epithelial barriers.</jats:p

Practice Patterns in the Treatment of Patients With Severe Alcohol Withdrawal: A Multidisciplinary, Cross-Sectional Survey

Purpose: To characterize physicians’ stated practices in the treatment of patients with severe acute alcohol withdrawal syndrome (sAAWS) and to use intravenous (IV) phenobarbital as an adjuvant treatment for sAAWS. Methods: A multidisciplinary, cross-sectional, self-administered survey at 2 large academic centers specializing in inner-city healthcare. Results: We analyzed 105 of 195 questionnaires (53.8% response rate). On average, clinicians managed 32 cases of AAWS over a 6-month period, of which 7 (21.9%) were severe. Haloperidol (Haldol; 40 [39%]), clonidine (Catapres; 31 [30%]), phenobarbital (Luminal, Tedral; 29 [27%]) and propofol (Diprivan; 29 [28%]) were the most commonly used adjuvant medications for sAAWS. Sixty-three (60%) of respondents did not use phenobarbital in practice. Of phenobarbital users, 23 (55%) respondents used it early in patients who were refractory to symptom-triggered benzodiazepine treatment. Others waited until patients experienced seizures (5 [10%]) or required intensive care unit admission (8 [18%]). Respondents who used phenobarbital preferred to use the IV versus oral form (66% vs 29%, P &lt; .001). Most respondents, however, were unfamiliar with the pharmacokinetics, side effects, contraindications, and evidence supporting phenobarbital use for sAAWS. Although many respondents (64 [61%]) expressed discomfort using phenobarbital, 87 (83%) expressed comfort or neutrality with enrolling patients in a trial to evaluate IV phenobarbital in sAAWS. Conclusions: Considerable stated practice variation exists in how clinicians treat patients with sAAWS. Our findings support conduct of a pilot trial to evaluate IV phenobarbital as an adjuvant treatment to symptom-triggered benzodiazepines for sAAWS and have informed trial design. </jats:sec

Supplemental Material, Practice_Patterns_in_Phenobarbital_Questionnaire_(1) - Practice Patterns in the Treatment of Patients With Severe Alcohol Withdrawal: A Multidisciplinary, Cross-Sectional Survey

Supplemental Material, Practice_Patterns_in_Phenobarbital_Questionnaire_(1) for Practice Patterns in the Treatment of Patients With Severe Alcohol Withdrawal: A Multidisciplinary, Cross-Sectional Survey by Danielle Buell, Niall Filewod, Jonathan Ailon and Karen E. A. Burns in Journal of Intensive Care Medicine</p