Retinoid receptors are expressed in mouse and human lungs.

Retinoid receptors are members of nuclear receptor superfamily consisting of two distinct families: RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each family contains three receptor subtypes α, ß, and γ. Retinoids transduce their effects through binding to retinoid receptors and inhibit transcription factors such as activator protein-1 and nuclear factor-κB (NF-κB) both of which regulate the transcription of several inflammatory genes. Considering the role of retinoid receptors in lung physiology, we need a precise understanding of their expression in normal and inflamed lungs. We used light and electron microscopic immunohistochemistry and Western blot to determine the expression of retinoid receptors in a murine model of endotoxin-induced (E. coli; 055:B5, 80 µg intranasal) acute lung inflammation and normal human lungs. Western blot showed expression of all six retinoid receptor subtypes in normal and inflamed mouse lungs. Immunohistology localized differential expression of retinoid receptors in airway epithelium, alveolar/septal macrophages, vascular endothelium, and alveolar septum in mouse lungs. Intranasal LPS challenge in mice resulted in increased expression of RXRα in airway epithelium compared to control animals. All six retinoid receptor subtypes were expressed in normal human lungs. Immunoelectron microscopy further confirmed the localization of all the receptors in various lung cells including the nucleus of these cells. The basal and altered expression of retinoid receptors in normal and inflamed lungs, respectively, may suggest their roles in lung pathophysiology.

Efficacy of chimeric ectolysin P128 in drug-resistant Staphylococcus aureus bacteraemia in mice.

Objectives: P128 is a recombinant chimeric ectolysin with potent antistaphylococcal activity. P128 was evaluated as monotherapy and in combination with two standard-of-care (SoC) antibiotics, vancomycin and daptomycin, in mouse models of Staphylococcus aureus bacteraemia. Methods: Healthy BALB/c mice were challenged (intraperitoneally) with 109 cfu of MRSA strain COL or USA300 and treated with a single dose of P128 (0.2-10 mg/kg). Drug synergy was tested using a single dose of P128 (0.2 or 2.5 mg/kg) along with sub-therapeutic dose levels of vancomycin (27.5 or 55 mg/kg) or daptomycin (12.5 mg/kg). Bacterial load was checked in peritoneal fluid and in blood, at different time intervals. Synergy against drug-resistant strains was tested using the P128/vancomycin combination against vancomycin-resistant S. aureus (VRSA). Results: In MRSA bacteraemia, P128, vancomycin and daptomycin monotherapy resulted in 31%, 46% and 46% survival, respectively. The P128/vancomycin and P128/daptomycin combinations afforded increased survival of 85% and 88%, respectively. P128 showed a rapid bactericidal effect with a reduction of cfu in both the peritoneal fluid and the blood within 1 h. In VRSA bacteraemia, a mouse-equivalent therapeutic dose of vancomycin (110 mg/kg) failed to rescue animals. P128 (1-20 mg/kg) as monotherapy resulted in dose-dependent efficacy. Survival (37%) with 2.5 mg/kg P128 increased to 63% with the P128/vancomycin combination. Conclusions: P128 exerted a rapid bactericidal effect in vivo and rescued animals from fatal invasive MRSA and VRSA infections. P128/SoC antibiotic combinations exerted a synergistic effect. P128 restored the susceptibility of VRSA to vancomycin. P128 is a novel, potent therapeutic agent for antibiotic-resistant, systemic S. aureus infections.

Restoration of sensitivity of a diverse set of drug-resistant Staphylococcus clinical strains by bactericidal protein P128.

PURPOSE: P128, a phage-derived lysin, exerts antibacterial activity on staphylococci by cleaving the pentaglycine-bridge of peptidoglycan. We sought to determine whether the presence of P128 could re-sensitize drug-resistant bacteria to antibiotics by virtue of its cell wall degrading property. METHODOLOGY: P128 was tested in combination with standard-of-care (SoC) drugs by chequerboard assays on planktonic cells and biofilms of strains individually resistant to these drugs. The bactericidal effect of P128 and drug combinations on planktonic cells and biofilms was measured by c.f.u. reduction assays. A mouse model of MRSA bacteraemia was used to test the efficacy of P128 and oxacillin in combination. RESULTS: A combination of sub-MIC P128 (0.025-0.20 µg ml-1) and 0.5 µg ml-1 of oxacillin resulted in inhibition of bacterial growth in four MRSA strains. Similar results were seen with all the other drugs tested, wherein sub-MIC of P128 re-sensitized S. aureus and CoNS strains to SoC drugs. The chequerboard assays on strains of S. aureus and CoNS showed that combinations of P128 and antibiotics consistently inhibited bacterial growth on biofilms. Data from scanning electron microscopy and c.f.u. reduction assays on drug-resistant S. aureus and CoNS demonstrated that sub-MICs of P128 and SoC antibiotics could kill biofilm-embedded bacteria. In vivo, a combination of sub-therapeutic doses of P128 and oxacillin could help protect animals from fatal bacteraemia. CONCLUSION: The ability of P128 to re-sensitize bacteria to SoC drugs suggests that combinations of P128 and SoC antibiotics can potentially be developed to treat infections caused by drug-resistant strains of staphylococci.

Phage-derived lysins as potential agents for eradicating biofilms and persisters.

Bacterial biofilms are highly resistant to the action of antibiotics. Presence of persisters, phenotypically resistant populations of bacterial cells, is thought to contribute toward recalcitrance of biofilms. The phage-derived lysins, by virtue of their ability to cleave the peptidoglycan of bacterial cells in an enzymatic manner, have the unique ability to kill dormant cells. Several lysins have shown potent antibiofilm activity in vitro. The fact that lysins have shown better efficacy than conventional drugs in animal models of endocarditis and other infections involving biofilms suggests that the lysins can potentially be developed against difficult-to-treat bacterial infections.

Efficacy of Novel Antistaphylococcal Ectolysin P128 in a Rat Model of Methicillin-Resistant Staphylococcus aureus Bacteremia.

Staphylococcus aureus causes systemic infections with high morbidity and mortality, and the emergence of drug-resistant strains is a rapidly growing clinical concern. Novel therapeutic agents are required to tackle S. aureus infections. P128 is a bacteriophage-derived chimeric ectolysin with potent and rapid bactericidal activity against S. aureus In the present study, the efficacy of P128 was evaluated in a newly developed rat model of S. aureus bacteremia. Prior to in vivo testing, P128 was shown to be stable in whole blood by incubation in rat blood for up to 6 h and testing its bactericidal activity against the methicillin-resistant S. aureus isolate USA300. Rats succumbed to intravenous challenge with 109 CFU of S. aureus USA300, resulting in 80 to 100% mortality by day 14. Evaluation of the bacterial load in various organs at 96 h postinfection revealed high bacterial counts in the kidney, and this correlated with the presence of renal abscesses. Treatment of infected animals with P128 either by intravenous bolus administration via tail vein or by 1-h infusion via the jugular vein at 2 h postinfection resulted in the dose-dependent survival of rats. P128 treatment also resulted in very few or no abscesses in the kidneys. These data show that P128 is stable in the physiological milieu and that intravenous treatment with P128 is highly effective in rescuing rats from S. aureus bacteremia. P128 can be a novel therapeutic option for treatment of S. aureus systemic infections.

Toll-like receptor 9 partially regulates lung inflammation induced following exposure to chicken barn air.

BACKGROUND: Exposure to animal barn air is an occupational hazard that causes lung dysfunction in barn workers. Respiratory symptoms experienced by workers are typically associated with endotoxin and TLR4 signalling, but within these environments gram negative bacteria constitute only a portion of the total microbial population. In contrast, unmethylated DNA can be found in all bacteria, some viruses, and mold. We hypothesized that in such environments TLR9, which binds unmethylated DNA, contributes to the overall immune responses in the lung. METHODS: Using a mouse model, wild-type and TLR9(-/-) mice were exposed to chicken barn air for 1, 5, or 20 days. Blood serum and bronchiolar lavage fluid was tested against a panel of six TLR9-induced cytokines (IL-1ß, IL-6, IL-10, IL-12, TNFα, and IFNγ) for changes in expression. Bronchiolar lavage fluid (BAL) was also tested for macrophage as well as monocyte migration. RESULTS: There were significant decreases in serum TNFα after a single day exposure in TLR9(-/-) mice. BAL concentrations of TNFα and IFNγ, as well as TNFα in serum in TLR9(-/-) mice were also reduced after barn exposure for 5 days. After 20 days of exposure IFNγ was significantly reduced in lavage of TLR9(-/-) mice. Myeloperoxidase (MPO) accumulation in the lung was reduced at 20 days of exposure in TLR9(-/-) mice, as was total lavage cell counts. However, Masson's staining revealed no apparent lung histological differences between any of the treatment groups. CONCLUSIONS: Taken together our data show TLR9 plays a partial role in lung inflammation induced following exposure to chicken barn air potentially through binding of unmethylated DNA.

Leukocyte-specific protein 1 regulates neutrophil recruitment in acute lung inflammation.

The mechanisms of excessive migration of activated neutrophils into inflamed lungs, credited with tissue damage, are not fully understood. We explored the hitherto unknown expression of leukocyte-specific protein 1 (LSP1) in human and mouse lungs and neutrophils and examined its role in neutrophil migration in acute lung inflammation. Autopsied septic human lungs showed increased LSP1 labeling in epithelium, endothelium, and leukocytes, including in their nuclei compared with normal lungs. We induced acute lung inflammation through intranasal administration of E. coli lipopolysaccharide (LPS) (80 µg) in LSP1-deficient (Lsp1(-/-)) and wild-type (WT) 129/SvJ mice. Immunocytochemistry and Western blots showed increased expression of LSP1 and phosphorylated LSP1 in lungs of LPS-treated WT mice. Histology showed more congestion, inflammation, and Gr-1(+) neutrophils in lung of WT mice than Lsp1(-/-) mice. LPS-treated WT mice had significantly more neutrophils in bronchoalveolar lavage (BAL) and myeloperoxidase levels in lungs compared with Lsp1(-/-) mice. However, there were no differences in lung tissue and BAL concentrations of keratinocyte-derived chemokine, monocyte chemoattractant protein-1, macrophage inflammatory protein-1α and -1ß, vascular permeability, and phosphorylated p38 MAPK between LPS-treated WT and Lsp1(-/-) mice, whereas TNF-α concentration was higher in BAL fluid from LPS-treated WT. Immunoelectron microscopy showed increased LSP1 in the nuclei of LPS-treated neutrophils. We also found increased levels of phosphorylated LSP1 associated with plasma membrane, nucleus, and cytosol at various times after LPS treatment of murine bone marrow-derived neutrophils, suggesting its role in modulation of neutrophil cytoskeleton and the membrane. These data collectively show increased expression of LSP1 in inflamed mouse and human lungs and its role in neutrophil recruitment and lung inflammation.

Expression of retinoid receptors in lungs of cattle, dogs, and pigs.

Retinoids play an important role in lung development and immune response. The effects of retinoids are mediated through 2 families of retinoid receptors: retinoic acid receptors (RARs) and retinoid X receptors (RXRs), with alpha (α), beta (ß), and gamma (γ) subtypes in each family. To date, no data exist on the expression pattern of retinoid receptors in lungs of cattle, dogs, and pigs. Because of the biomedical importance of retinoid receptors in inflammation and immune responses, Western blot, immunohistology, and immunoelectron microscopy were used to determine the expression of retinoid receptors in normal lungs of cattle, dogs, and pigs (n = 2 for each species). Western blot showed expression of all 6 retinoid receptor subtypes in pig lungs. Immunohistology data indicated differential expression of retinoid receptors in airway epithelium, vascular endothelium, alveolar/septal macrophages, and alveolar septum in all 3 species. Electron microscopy showed nuclear localization of retinoid receptors in neutrophils and pulmonary intravascular macrophages. Retinoic acid receptors (RAR) α subtype were localized in cytoplasmic vacuoles of pig monocytes. These data indicate constitutive expression of retinoid receptors in the lungs of cattle, dogs, and pigs.

Les rétinoïdes jouent un rôle important dans le développement des poumons et la réponse immunitaire. Les effets des rétinoïdes sont médiés via deux familles de récepteurs des rétinoïdes : les récepteurs de l'acide rétinoïque (RAR) et les récepteurs du rétinoïde X (RXR), avec des sous-types alpha (α), beta (ß) et gamma (γ) dans chaque famille. Aucune information n'existe à ce jour sur le patron d'expression des récepteurs des rétinoïdes dans les poumons des bovins, chiens et porcs. Étant donné l'importance biomédicale des récepteurs des rétinoïdes lors de l'inflammation et des réponses immunitaires, l'immunobuvardage, l'immunohistologie et l'immunomicroscopie électronique ont été utilisés afin de déterminer l'expression des récepteurs des rétinoïdes dans des poumons normaux de bovins, chiens et porcs (n = 2 pour chaque espèce). L'immunobuvardage a permis de démontrer l'expression des six sous-types de récepteurs des rétinoïdes dans les poumons de porc. Les données de l'immunohistologie indiquaient une expression différente des récepteurs des rétinoïdes dans l'épithélium des voies respiratoires, l'endothélium vasculaire, les macrophages alvéolaires/septaux, et le septum alvéolaire chez les trois espèces. La microscopie électronique permettait de visualiser la localisation nucléaire des récepteurs des rétinoïdes dans les neutrophiles et les macrophages pulmonaires intravasculaires. Les RAR de sous-type α étaient localisés dans des vacuoles cytoplasmiques des monocytes porcins. Ces données indiquent l'expression constitutive des récepteurs des rétinoïdes dans les poumons des bovins, chiens et porcs.(Traduit par Docteur Serge Messier).

Retinoic acid induces apoptosis in activated canine neutrophils.

Activated neutrophils live longer, produce toxic metabolites and cause considerable tissue injury, which is central to the pathogenesis of many inflammatory conditions. Retinoids are a class of lipophilic compounds with anti-inflammatory effects. We examined the effect of retinoic acid on apoptosis in normal and activated neutrophils. Our results showed that treatment with 1 µg/ml Escherichia coli lipopolysaccharide (LPS) for 12 and 36 h delayed the spontaneous neutrophil apoptosis compared to untreated cells. But exposure of LPS-treated cells to retinoic acid (1 and 5 µM) abolished the inhibitory effects of LPS on neutrophil apoptosis in a concentration-dependent manner based on annexin V staining, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, light and electron microscopy. These results show that retinoic acid increases apoptosis in activated canine neutrophils and this effect could enhance the resolution of inflammation in vivo.