Bactericidal/permeability-increasing protein attenuates systemic inflammation and acute lung injury in porcine lower limb ischemia-reperfusion injury.

OBJECTIVE: To investigate the role of recombinant bactericidal/permeability-increasing protein (rBPI21) in the attenuation of the sepsis syndrome and acute lung injury associated with lower limb ischemia-reperfusion (I/R) injury. SUMMARY BACKGROUND DATA: Gut-derived endotoxin has been implicated in the conversion of the sterile inflammatory response to a lethal sepsis syndrome after lower torso I/R injury. rBPI21 is a novel antiendotoxin therapy with proven benefit in sepsis. METHODS: Anesthetized ventilated swine underwent midline laparotomy and bilateral external iliac artery occlusion for 2 hours followed by 2.5 hours of reperfusion. Two groups (n = 6 per group) were randomized to receive, by intravenous infusion over 30 minutes, at the start of reperfusion, either thaumatin, a control-protein preparation, at 2 mg/kg body weight, or rBPI21 at 2 mg/kg body weight. A control group (n = 6) underwent laparotomy without further treatment and was administered thaumatin at 2 mg/kg body weight after 2 hours of anesthesia. Blood from a carotid artery cannula was taken every half-hour for arterial blood gas analysis. Plasma was separated and stored at -70 degrees C for later determination of plasma tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 by bioassay, and IL-8 by enzyme-linked immunosorbent assay (ELISA), as a markers of systemic inflammation. Plasma endotoxin concentration was measured using ELISA. Lung tissue wet-to-dry weight ratio and myeloperoxidase concentration were used as markers of edema and neutrophil sequestration, respectively. Bronchoalveolar lavage protein concentration was measured by the bicinclinoic acid method as a measure of capillary-alveolar protein leak. The alveolar-arterial gradient was measured; a large gradient indicated impaired oxygen transport and hence lung injury. RESULTS: Bilateral hind limb I/R injury increased significantly intestinal mucosal acidosis, intestinal permeability, portal endotoxemia, plasma IL-6 concentrations, circulating phagocytic cell priming and pulmonary leukosequestration, edema, capillary-alveolar protein leak, and impaired gas exchange. Conversely, pigs treated with rBPI21 2 mg/kg at the onset of reperfusion had significantly reduced intestinal mucosal acidosis, portal endotoxin concentrations, and circulating phagocytic cell priming and had significantly less pulmonary edema, leukosequestration, and respiratory failure. CONCLUSIONS: Endotoxin transmigration across a hyperpermeable gut barrier, phagocytic cell priming, and cytokinemia are key events of I/R injury, sepsis, and pulmonary dysfunction. This study shows that rBPI21 ameliorates these adverse effects and may provide a novel therapeutic approach for prevention of I/R-associated sepsis syndrome.

Anti-inflammatory effects of DX-890, a human neutrophil elastase inhibitor

Background
Neutrophil elastase (NE)-mediated inflammation contributes to lung damage in cystic fibrosis (CF). We investigated if DX-890, a small-protein NE inhibitor, could reduce neutrophil trans-epithelial migration and reduce activity released from neutrophils and NE-induced cytokine expression in airway epithelial cells.

Methods
Activated blood neutrophils (CF and healthy) treated ± DX-890 were assayed for NE activity. Transmigration of calcein-labeled neutrophils was studied using a 16HBE14o- epithelial monolayer. IL-8 release from primary nasal epithelial monolayers (CF and healthy) was measured after treatment ± DX-890 and NE or CF sputum.

Results
DX-890 reduced NE activity from neutrophils (CF and healthy) and reduced neutrophil transmigration. DX-890 pre-treatment reduced IL-8 release from epithelial cells of healthy or CF subjects after stimulation with NE and CF sputum sol. All improvements with DX-890 were statistically significant (p < 0.05).

Conclusions
DX-890 reduces NE-mediated transmigration and inflammation. NE inhibition could be useful in managing neutrophilic airway inflammation in CF.

Post-Translational Inhibition of IP-10 Secretion in IEC by Probiotic Bacteria: Impact on Chronic Inflammation

Background: Clinical and experimental studies suggest that the probiotic mixture VSL#3 has protective activities in the context of inflammatory bowel disease (IBD). The aim of the study was to reveal bacterial strain-specific molecular mechanisms underlying the anti-inflammatory potential of VSL#3 in intestinal epithelial cells (IEC).

Methodology/Principal Findings: VSL#3 inhibited TNF-induced secretion of the T-cell chemokine interferon-inducible protein (IP-10) in Mode-K cells. Lactobacillus casei (L. casei) cell surface proteins were identified as active anti-inflammatory components of VSL#3. Interestingly, L. casei failed to block TNF-induced IP-10 promoter activity or IP-10 gene transcription at the mRNA expression level but completely inhibited IP-10 protein secretion as well as IP-10-mediated T-cell transmigration. Kinetic studies, pulse-chase experiments and the use of a pharmacological inhibitor for the export machinery (brefeldin A) showed that L. casei did not impair initial IP-10 production but decreased intracellular IP-10 protein stability as a result of blocked IP-10 secretion. Although L. casei induced IP-10 ubiquitination, the inhibition of proteasomal or lysosomal degradation did not prevent the loss of intracellular IP-10. Most important for the mechanistic understanding, the inhibition of vesicular trafficking by 3-methyladenine (3-MA) inhibited IP-10 but not IL-6 expression, mimicking the inhibitory effects of L. casei. These findings suggest that L. casei impairs vesicular pathways important for the secretion of IP-10, followed by subsequent degradation of the proinflammatory chemokine. Feeding studies in TNF Delta ARE and IL-10(-/-) mice revealed a compartimentalized protection of VSL#3 on the development of cecal but not on ileal or colonic inflammation. Consistent with reduced tissue pathology in IL-10(-/-) mice, IP-10 protein expression was reduced in primary epithelial cells.

Conclusions/Significance: We demonstrate segment specific effects of probiotic intervention that correlate with reduced IP-10 protein expression in the native epithelium. Furthermore, we revealed post-translational degradation of IP-10 protein in IEC to be the molecular mechanism underlying the anti-inflammatory effect.

'That Puissant City': John Milton's Roman Sojourns 1638-1639

John Milton’s sojourns in Rome (1638-9) are attested by his comments in Defensio Secunda, by the minutes of the English College, by Latin encomia which he received from Roman academicians, and, not least, by his Latin letter to Lucas Holstenius (19/29 March 1639), and several Latin poems which he composed in the course of his residency in the capital city: Ad Salsillum, and three Latin epigrams extolling the praises of the virtuosa soprano, Leonora Baroni. Read together, these texts serve to reveal much about Milton’s participation in, and reaction to, the ‘Puissant City’, (History of Britain, Bk 2).

The present monograph presents fresh evidence of Milton's integration into the academic and cultural life of seventeenth-century Rome. It argues that his links with two Roman academies: the Accademia dei Fantastici and Accademia degli Umoristi constitute a sustained participation in an academic community paralleling that of his independently attested performance in Florentine academies (on which I have published extensively). It also investigates his links with Alessandro Cherubini, David Codner, Giovanni Batista Doni, and the Baroni circle hymned in three published anthologies.

Chapter 1: Milton and the Accademia dei Fantastici investigates the cultural climate surrounding Milton's Ad Salsillum by examining two of that academy's publications: the Poesie dei Signori Accademici Fantastici di Roma (Rome, 1637) and the Academia Tenuta da Fantastici a. 12 di Maggio 1655 (Rome, 1655), the latter celebrating the creation of Fabio Chigi as Pope Alexander VII on 5 April 1655. Read in a new light, Milton’s self-fashioning, it is argued, takes its place not only alongside Salzilli’s encomium in Milton's honour, and his Italian sonnets in the 1637 Poesie, but also in relation to other poems in that collection, and the academy's essentially Catholic eulogistic trend. The chapter also provides fresh evidence of Salzilli’s survival of the illness described in Milton’s poem by his epistolary correspondence with Tomaso Stigliani.

Chapter 2: Milton and the Vatican argues for links between Milton’s Latin letter to Holstenius and a range of Holstenius’ published works: his edition of the axioms of the later Pythagoreans gifted by him to Milton, and his published neo-Platonic works. This is achieved by mutual appropriation of Similitudes in a series of Miltonic similes, the anabasis/katabasis motifs in a reworking of the Platonic theory of the transmigration of souls, and allusion to etymological details highlighted in Holstenius’ published editions. The chapter also reveals Milton’s alertness to typographical procedures and, by association, to Holstenius’ recent role (1638) as Director of the press of the Biblioteca Vaticana.

Chapter 3: Milton and the Accademia degli Umoristi argues for Milton’s likely participation in this Roman academy, as suggested by his links with its members. His three Latin epigrams in praise of Leonora Baroni, the only female member of the Umoristi, have hitherto been studied in relation to the 1639 Applausi in her honour. In a new reading, Milton, it is suggested, invokes and interrogates Catholic doctrine before a Catholic audience only to view the whole through the lens of a neo-Platonic Hermeticism (by echoing the phraseology of the sixteenth-century Franciscan Hannibal Rosselli) that refreshingly transcends religious difference. Crucially, the hitherto neglected L’Idea della Veglia (Rome, 1640) includes further encomiastic verse, sonnets to, and by Leonora, and details of the conversazioni hosted by her family at the precise time of Milton’s Roman sojourns. Milton may well have been a participant. The chapter concludes in an assessment of his links with the youthful prodigy Alessandro Cherubini, and of his audience with Francesco Barberini.

Chapter 4: Milton at a Roman Opera analyses the potential impact of ‘Chi Soffre, Speri’, which he attended on 18/28 February 1639, mounted by Francesco Barberini to inaugurate the recently completed theatre of the Palazzo Barberini. A detailed analysis of the opera's libretto, music, and theatricality casts a backward glance to Milton's Comus, and a forward glance to Paradise Lost. It also assesses Milton’s musical interests at this time, as attested by his links with Doni, and his purchase of works by Monteverdi and others.

Chapter 5: Milton’s English Connections in Rome develops the work of Miller and Chaney by investigating Milton’s co-diners at the English College in Rome on 30 October 1638, and by analysing his links between David Codner (alias Matteo Selvaggio), and the family of Jane Savage, Marchioness of Winchester, lamented by Milton in 1631. It also assesses his potential relations with the Englishman Thomas Gawen, who ‘accidentally sometimes fell into the company of John Milton’ (Antony Wood).