Host immunoglobulin G selectively identifies pathobionts in pediatric inflammatory bowel diseases.

BACKGROUND: Inflammatory bowel diseases (IBD) are a group of complex and multifactorial disorders with unknown etiology. Chronic intestinal inflammation develops against resident intestinal bacteria in genetically susceptible hosts. We hypothesized that host intestinal immunoglobulin (Ig) G can be used to identify bacteria involved in IBD pathogenesis. RESULTS: IgG-bound and -unbound microorganisms were collected from 32 pediatric terminal ileum aspirate washes during colonoscopy [non-IBD (n = 10), Crohn disease (n = 15), and ulcerative colitis (n = 7)], and composition was assessed using the Illumina MiSeq platform. In vitro analysis of invasive capacity was evaluated by fluorescence in situ hybridization and gentamicin invasion assay; immune activation was measured by qPCR. Despite considerable inter-individual variations, IgG binding favored specific and unique mucosa-associated species in pediatric IBD patients. Burkholderia cepacia, Flavonifractor plautii, and Rumminococcus sp. demonstrated increased IgG binding, while Pseudomonas ST29 demonstrated reduced IgG binding, in IBD. In vitro validation confirmed that B. cepacia, F. plautii, and Rumminococcus display invasive potential while Pseudomonas protogens did not. CONCLUSION: Using IgG as a marker of pathobionts in larger patient cohorts to identify microbes and elucidate their role in IBD pathogenesis will potentially underpin new strategies to facilitate development of novel, targeted diagnostic, and therapeutic approaches. Interestingly, this method can be used beyond the scope of this manuscript to evaluate altered gut pathobionts in a number of diseases associated with altered microbiota including arthritis, obesity, diabetes mellitus, alcoholic liver disease, cirrhosis, metabolic syndrome, and carcinomas.

Inflammasome Activation by ATP Enhances Citrobacter rodentium Clearance through ROS Generation.

BACKGROUND: Nod-like receptor family, pyrin domain containing 3 (NLRP3) is an important cytosolic sensor of cellular stress and infection. Once activated, NLRP3 forms a multiprotein complex (inflammasome) that triggers the maturation and secretion of interleukin (IL)-1ß and IL-18. We aimed to define the consequences of NLRP3 induction, utilizing exogenous adenosine triphosphate (ATP) as an inflammasome activator, to determine if inflammasome activation increases macrophage killing of Citrobacter rodentium and define mechanisms. METHODS: Bacterial survival was measured using a gentamicin protection assay. Inflammasome activation or inhibition in mouse J774A.1 macrophages were assessed by measuring IL-1ß; cytokines and reactive oxygen species (ROS) were measured by ELISA and DCFDA, respectively. RESULTS: Activation of the inflammasome increased bacterial killing by macrophages and its inhibition attenuated this effect with no impact on phagocytosis or cell death. Furthermore, inflammasome activation suppressed pro-inflammatory cytokines during infection, possibly due to more effective bacterial killing. While the infection increased ROS production, this effect was reduced by inflammasome inhibitors, indicating that ROS is inflammasome-dependent. ROS inhibitors increased bacterial survival in the presence of ATP, suggesting that inflammasome-induced bacterial killing is mediated, at least in part, by ROS activity. CONCLUSION: Improving inflammasome activity during infection may increase bacterial clearance by macrophages and reduce subsequent microbe-induced inflammation.

Mucosal Barrier Depletion and Loss of Bacterial Diversity are Primary Abnormalities in Paediatric Ulcerative Colitis.

BACKGROUND AND AIMS: Ulcerative colitis [UC] is associated with colonic mucosa barrier defects and bacterial dysbiosis, but these features may simply be the result of inflammation. Therefore, we sought to assess whether these features are inherently abrogated in the terminal ileum [TI] of UC patients, where inflammation is absent. METHODS: TI biopsies from paediatric inflammatory bowel disease [IBD] subsets [Crohn's disease [CD; n = 13] and UC [n = 10]], and non-IBD disease controls [n = 12] were histologically graded, and alcian blue/periodic acid-Schiff stained biopsies were quantified. The mucosal barrier was assessed for mucin [MUC2], immunoglobulin [Ig]A, IgG, and total bacteria (fluorescence in-situ hybridisation [FISH probe EUB338]) by immunofluorescence. The regulation of mucin secretion was investigated by NLRP6 gene expression and immunofluorescence. The composition of the active mucosa-associated microbiota was explored by sequencing the 16S rRNA amplicon generated from total RNA. RESULTS: Despite the absence of ileitis, UC patients displayed ileal barrier depletion illustrated by reductions in mucin-containing goblet cells and mucin production and altered epithelial NLRP6 expression. In both CD patients with ileitis and UC patients with normal histology, bacteria coated with IgA and IgG penetrated the TI mucin layer. Biopsy 16S rRNA sequencing revealed a reduction in α-diversity by three methods [Shannon, Simpson, and Equitability indices] between UC and non-IBD paediatric patients. CONCLUSIONS: These findings suggest an underlying defect in the UC-afflicted intestinal tract even in the absence of inflammation, implicating barrier and microbial changes as primary abnormalities in UC that may play a causative role in disease development.

The bioactivity and toxicological actions of carvacrol.

Carvacrol is a monoterpenic phenol produced by an abundant number of aromatic plants, including thyme and oregano. Presently, carvacrol is used in low concentrations as a food flavoring ingredient and preservative, as well as a fragrance ingredient in cosmetic formulations. In recent years, considerable research has been undertaken in an effort to establish the biological actions of carvacrol for its potential use in clinical applications. Results from in vitro and in vivo studies show that carvacrol possess a variety of biological and pharmacological properties including antioxidant, antibacterial, antifungal, anticancer, anti-inflammatory, hepatoprotective, spasmolytic, and vasorelaxant. The focus of this review is to evaluate the existing knowledge regarding the biological, pharmacological, and toxicological effects of carvacrol.

Liposomal antibiotic formulations for targeting the lungs in the treatment of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a Gram-negative bacterium that causes serious lung infections in cystic fibrosis, non-cystic fibrosis bronchiectasis, immunocompromised, and mechanically ventilated patients. The arsenal of conventional antipseudomonal antibiotic drugs include the extended-spectrum penicillins, cephalosporins, carbapenems, monobactams, polymyxins, fluoroquinolones, and aminoglycosides but their toxicity and/or increasing antibiotic resistance are of particular concern. Improvement of existing therapies against Pseudomonas aeruginosa infections involves the use of liposomes - artificial phospholipid vesicles that are biocompatible, biodegradable, and nontoxic and able to entrap and carry hydrophilic, hydrophobic, and amphiphilic molecules to the site of action. The goal of developing liposomal antibiotic formulations is to improve their therapeutic efficacy by reducing drug toxicity and/or by enhancing the delivery and retention of antibiotics at the site of infection. The focus of this review is to appraise the current progress of the development and application of liposomal antibiotic delivery systems for the treatment pulmonary infections caused by P. aeruginosa.

A balanced IL-1ß activity is required for host response to Citrobacter rodentium infection.

Microbial sensing plays essential roles in the innate immune response to pathogens. In particular, NLRP3 forms a multiprotein inflammasome complex responsible for the maturation of interleukin (IL)-1ß. Our aim was to delineate the role of the NLRP3 inflammasome in macrophages, and the contribution of IL-1ß to the host defense against Citrobacter rodentium acute infection in mice. Nlrp3(-/-) and background C57BL/6 (WT) mice were infected by orogastric gavage, received IL-1ß (0.5 µg/mouse; ip) on 0, 2, and 4 days post-infection (DPI), and assessed on 6 and 10 DPI. Infected Nlrp3(-/-) mice developed severe colitis; IL-1ß treatments reduced colonization, abrogated dissemination of bacteria to mesenteric lymph nodes, and protected epithelial integrity of infected Nlrp3(-/-) mice. In contrast, IL-1ß treatments of WT mice had an opposite effect with increased penetration of bacteria and barrier disruption. Microscopy showed reduced damage in Nlrp3(-/-) mice, and increased severity of disease in WT mice with IL-1ß treatments, in particular on 10 DPI. Secretion of some pro-inflammatory plasma cytokines was dissipated in Nlrp3(-/-) compared to WT mice. IL-1ß treatments elevated macrophage infiltration into infected crypts in Nlrp3(-/-) mice, suggesting that IL-1ß may improve macrophage function, as exogenous administration of IL-1ß increased phagocytosis of C. rodentium by peritoneal Nlrp3(-/-) macrophages in vitro. As well, the exogenous administration of IL-1ß to WT peritoneal macrophages damaged the epithelial barrier of C. rodentium-infected polarized CMT-93 cells. Treatment of Nlrp3(-/-) mice with IL-1ß seems to confer protection against C. rodentium infection by reducing colonization, protecting epithelial integrity, and improving macrophage activity, while extraneous IL-1ß appeared to be detrimental to WT mice. Together, these findings highlight the importance of balanced cytokine responses as IL-1ß improved bacterial clearance in Nlrp3(-/-) mice but increased tissue damage when given to WT mice.

Therapeutic effect of liposomal-N-acetylcysteine against acetaminophen-induced hepatotoxicity.

BACKGROUND: Acetaminophen (APAP) is an antipyretic analgesic drug that when taken in overdose causes depletion of glutathione (GSH) and hepatotoxicity. N-acetylcysteine (NAC) is the antidote of choice for the treatment of APAP toxicity; however, due to its short-half-life repeated dosing of NAC is required. PURPOSE: To determine whether a NAC-loaded liposomal formulation (Lipo-NAC) is more effective than the conventional NAC in protecting against acute APAP-induced hepatotoxicity. METHODS: Male Sprague-Dawley rats were challenged with an intragastric dose of APAP (850 mg/kg b.wt.); 4 h later, animals were administered saline, NAC, Lipo-NAC or empty liposomes and sacrificed 24 h post-APAP treatment. RESULTS: APAP administration resulted in hepatic injury as evidenced by increases in plasma bilirubin, alanine (AST) and aspartate (ALT) aminotransferase levels and tissue levels of lipid peroxidation and myeloperoxidase as well as decreases in hepatic levels of reduced GSH, GSH peroxidase and GSH reductase. Treatment of animals with Lipo-NAC was significantly more effective than free NAC in reducing APAP-induced hepatotoxicity. Histological evaluation showed that APAP caused periacinar hepatocellular apoptosis and/or necrosis of hepatocytes around the terminal hepatic venules which was reduced by NAC treatment, the degree of reduction being greater for Lipo-NAC. CONCLUSION: These data suggest that administration of Lipo-NAC ameliorated the APAP-induced hepatotoxicity.

Epithelial cell extrusion leads to breaches in the intestinal epithelium.

BACKGROUND: Two distinct forms of intestinal epithelial cell (IEC) extrusion are described: 1 with preserved epithelial integrity and 1 that introduced breaches in the epithelial lining. In this study, we sought to determine the mechanism underlying the IEC extrusion that alters the permeability of the gut epithelium. METHODS: IEC extrusions in polarized T84 monolayer were induced with nigericin. Epithelial permeability was assessed with transepithelial electrical resistance and movements of latex microspheres and green fluorescent protein-transfected Escherichia coli across the monolayer. In vivo IEC extrusion was modulated in wild-type and a colitic (interleukin-10 knock-out) mouse model with caspase-1 activation and inhibition. Luminal aspirates and mucosal biopsies from control patients and patients with inflammatory bowel disease were analyzed for caspase-1 and caspase-3&7 activation. RESULTS: Caspase-1-induced IEC extrusion in T84 monolayers resulted in dose-dependent and time-dependent barrier dysfunction, reversible with caspase-1 inhibition. Moreover, the movements of microspheres and microbes across the treated epithelial monolayers were observed. Increased caspase-1-mediated IEC extrusion in interleukin-10 knock-out mice corresponded to enhanced permeation of dextran, microspheres, and translocation of E. coli compared with wild type. Caspase-1 inhibition in interleukin-10 knock-out mice resulted in a time-dependent reduction in cell extrusion and normalization of permeability to microspheres. Increased IEC extrusion in wild-type mice was induced with caspase-1 activation. In human luminal aspirates, the ratio of positively stained caspase-1 to caspase-3&7 cells were 1:1 and 2:1 in control patients and patients with inflammatory bowel disease, respectively; these observations were confirmed by cytochemical analysis of mucosal biopsies. CONCLUSIONS: IEC extrusion mediated by caspase-1 activation contributes to altered intestinal permeability in vitro and in vivo.

Safety and pharmacokinetic studies of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol or γ-tocopherol in beagle dogs.

The safety and pharmacokinetic profile of liposomal formulations containing combinations of the antioxidants α-tocopherol, γ-tocopherol or N-acetylcysteine in beagle dogs was examined. Each group consisted of beagle dogs of both genders with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (330 mg/kg DPPC, EL), and test groups receiving liposomes prepared from DPPC lipids with (i) N-acetylcysteine (NAC) (60 mg/kg NAC [L-NAC]); (ii) NAC and α-tocopherol (αT) (60 mg/kg NAC and 25 mg/kg α-tocopherol [L-αT-NAC]) and (iii) NAC and γ-tocopherol (60 mg/kg NAC and 25 mg/kg γ-tocopherol (γT) [L-γT-NAC]). The dogs in the control group (EL) and three test groups exhibited no signs of toxicity during the dosing period or day 15 post treatment. Weight gain, feed consumption and clinical pathology findings (hematology, coagulation, clinical chemistry, urinalysis) were unremarkable in all dogs and in all groups. Results from the pharmacokinetic study revealed that the inclusion of tocopherols in the liposomal formulation significantly increased the area under the curve (AUC) and ß-half life for NAC; the tocopherols had greater impact on the clearance of NAC, where reductions of central compartment clearance (CL) ranged from 56% to 60% and reductions of tissue clearance (CL2) ranged from 73% to 77%. In conclusion, there was no treatment-related toxicity in dogs at the maximum feasible dose level by a single bolus intravenous administration while the addition of tocopherols to the liposomal formulation prolonged the circulation of NAC in plasma largely due to a decreased clearance of NAC.

Toxicity of ricin toxin A chain in rats.

Ricin toxin A chain (RTA) is the cytotoxic component of the dimeric protein, ricin, one of the most potent and deadly plant toxins extracted from the seeds of Ricinus communis. RTA has been investigated as a potential candidate for cancer chemotherapy, in the form of immunotoxins, and as a method for depleting macrophages in vivo. The toxicity of RTA immunotoxins is mostly characterized by inflammation and necrosis and has been attributed to the RTA moiety of the conjugate. The present study was carried out to investigate the toxicity of intravenously (i.v.) administered RTA alone and to assess whether the observed tissue injuries are associated with increases in oxidative stress (OS) and inflammation. RTA (10 or 90 µg/kg body weight) was administered to animals i.v., and 5 or 24 hours later, liver, lungs, kidneys, and hearts were examined. RTA, at a dose of 90 µg/kg (i.v.), resulted in significant increases (P < 0.05) in an inflammatory response (i.e., increases in hepatic and lung myeloperoxidase activity) and increases in oxidant response (increases in lipid peroxidation and decreases in glutathione levels in hepatic and lung homogenates). These data suggest that i.v. administration of RTA resulted in organ injuries that were associated with inflammation and OS.

Acute toxicity study of liposomal antioxidant formulations containing N-acetylcysteine, α-tocopherol, and γ-tocopherol in rats.

Liposomes have been used for the delivery of antioxidants to different tissues and organs for the treatment of oxidative stress-induced injuries. In this study, the acute toxicity of a single dose of intravenously (i.v.) administered liposomal antioxidant formulation, containing N-acetylcysteine (NAC) with or without α-tocopherol (α-T) or γ-tocopherol (γ-T), in rats was examined. Each group consisted of 5 male and 5 female Sprague-Dawley rats, with a control group receiving empty dipalmitoylphosphatidylcholine (DPPC) liposomes (660 mg/kg) and test groups receiving DPPC liposomes (660 mg/kg) entrapped with 1) NAC (200 mg/kg), 2) NAC (200 mg/kg) and α-T (83.3 mg/kg), and 3) NAC (200 mg/kg) and γ-T (71.4 mg/kg). These dose levels were determined from the dose-range-finding study and were considered to be the maximum feasible dose (MFD) levels, based on the volume of 10 mL/kg and physical properties and viscosity of the test articles that could be safely administered to rats by an i.v. injection. Two weeks after treatment (day 15), rats in the control group and three test groups exhibited no clinical signs of toxicity during the dosing period or during the 14-day post-treatment period. Weight gain and food consumption in all animals was appropriate for the age and sex of animals. Clinical pathology findings (e.g., hematology, coagulation, clinical chemistry, and urinalysis) were unremarkable in all rats and in all groups. In conclusion, the results of this study showed no treatment-related toxicity in rats at the MFD level by a single bolus i.v. administration.

Ginseng aqueous extract attenuates the production of virulence factors, stimulates twitching and adhesion, and eradicates biofilms of Pseudomonas aeruginosa.

This study was carried out to examine the antimicrobial activity of the aqueous extract of Panax quinquefolius from North American ginseng (NAGE) root against Pseudomonas aeruginosa . The minimum inhibitory concentrations of reference and clinical isolates of Pseudomonas aeruginosa were measured by a standard agar-dilution method. At subinhibitory NAGE concentrations, the secretion of virulence factors, motility on agar, and adhesion to 96-well microplates were studied on the nonmucoid Pseudomonas aeruginosa O1 strain. At suprainhibitory concentrations, the activity of NAGE against mature biofilm complexes formed in the Calgary Biofilm Device and the Stovall flow cell were assessed. NAGE possessed an antibacterial activity against all the Pseudomonas aeruginosa strains at 1.25%-2.5% w/v. NAGE also significantly attenuated pyocyanin, pyoverdine, and lipase concentrations, stimulated twitching, and attenuated swarming and swimming motility. At 1.25% w/v, NAGE augmented adhesion, and at 5% w/v detached 1-day-old biofilms in microplates. The extract also eradicated 6-day-old mature biofilms (5% w/v), and fluorescence microscopy displayed a reduction of live cells and biofilm complexes compared with nontreated biofilms. These data suggest that the aqueous extract from North American ginseng possesses antimicrobial activities in vitro.

Treatment of ricin A-chain-induced hepatotoxicity with liposome-encapsulated N-acetylcysteine.

BACKGROUND: The toxicity of ricin resides in the ricin A-chain (RTA) and is attributed to the inhibition of protein synthesis but inflammation and oxidative stress have also been implicated. RTA can independently enter cells producing comparable tissue injury and inflammation, although at much higher concentrations than intact ricin. Treatment for exposure to ricin or RTA is supportive. PURPOSE: To examine the effectiveness of conventional or liposome-encapsulated N-acetylcysteine (Lipo-NAC) in ameliorating RTA-induced hepatotoxicity. METHODS: Four hours after RTA administration (90 µg/kg b.wt, iv), rats were treated with conventional NAC or Lipo-NAC (25 mg/kg NAC). The hepatoprotective effects of the antioxidant formulations were assessed by measuring indexes for liver injury (alanine [ALT] and aspartate [AST] aminotransferase activities), inflammation (myeloperoxidase, tumor necrosis factor-α, chloramine levels), and oxidant response (lipid peroxidation, nitrotyrosine, glutathione levels) 24-h post-RTA exposure. RESULTS: Administration of RTA to animals resulted in hepatotoxicity as demonstrated by elevated plasma ALT and AST levels, increases in an inflammatory response, and increases in oxidant response. Treatment of animals with the antioxidant formulations reversed the RTA-induced hepatotoxicity, being most evident following the administration of Lipo-NAC. CONCLUSION: NAC, administered in a liposomal form, may serve as a potentially effective pharmacological agent in the treatment of RTA-induced liver injuries.

Effectiveness of liposomal paclitaxel against MCF-7 breast cancer cells.

Paclitaxel is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including breast, ovarian, and non-small-cell lung cancer. Due to its high lipophilicity, paclitaxel is difficult to administer and requires solubilization with Cremophor EL (polyethoxylated castor oil) and ethanol, which often lead to adverse side effects, including life-threatening anaphylaxis. Incorporation of paclitaxel in dimyristoylphosphatidylcholine:dimyristoylphosphatidylglycerol (DPPC:DMPG) liposomes can facilitate its delivery to cancer cells and eliminate the adverse reactions associated with the Cremophor EL vehicle. Accordingly, the effectiveness of liposomal paclitaxel on MCF-7 breast cancer cells was examined. The results from this study showed that (i) the lipid components of the liposomal formulation were nontoxic, (ii) the cytotoxic effects of liposomal paclitaxel were improved when compared with those seen with conventional paclitaxel, and (iii) the intracellular paclitaxel levels were higher in MCF-7 cells treated with the liposomal paclitaxel formulation. The results of these studies showed that delivery of paclitaxel as a liposomal formulation could be a promising strategy for enhancing its chemotherapeutic effects.

Importance of DNase and alginate lyase for enhancing free and liposome encapsulated aminoglycoside activity against Pseudomonas aeruginosa.

OBJECTIVES: This study evaluated the potential of DNase, alginate lyase (AlgL) and N-acetylcysteine (NAC) in enhancing the in vitro bactericidal activity of conventional (free) and vesicle-entrapped (liposomal) gentamicin, amikacin and tobramycin. METHODS: The MICs and biofilm eradication for two clinical isolates of Pseudomonas aeruginosa (a mucoid strain and a non-mucoid strain) were determined in the presence and absence of AlgL. The co-activity of aminoglycosides with DNase and/or AlgL against endogenous P. aeruginosa in cystic fibrosis (CF) sputum was also measured. The inhibitory effects of mucin in the presence and absence of the mucolytic agent NAC on aminoglycosidic activity were also examined. RESULTS: The MIC values of the liposomal aminoglycosides were similar to or lower than those of free aminoglycosides. Biofilm formation increased the bactericidal concentrations of these drugs by 8- to 256-fold and treatment with AlgL improved killing of the mucoid strain. The activity of some aminoglycosides against the sputum was increased by the addition of DNase or AlgL (P < 0.05), and was increasingly evident with concurrent DNase and AlgL administration. Addition of mucin inhibited liposomal aminoglycosidic activity (up to 32-fold) evidently more than the free aminoglycosides (up to 8-fold). The addition of NAC did not improve activity significantly (P > 0.05). Tobramycin was the most effective aminoglycoside to reduce biofilms and sputum. CONCLUSIONS: Liposomal aminoglycosides do not fare better than conventional forms. The co-administration of DNase and AlgL is essential for enhanced activity in reducing biofilm growth and sputum bacterial counts. While mucin retards bactericidal activity, NAC does not improve aminoglycosidic activity.

Activity and interactions of liposomal antibiotics in presence of polyanions and sputum of patients with cystic fibrosis.

BACKGROUND: To compare the effectiveness of liposomal tobramycin or polymyxin B against Pseudomonas aeruginosa in the Cystic Fibrosis (CF) sputum and its inhibition by common polyanionic components such as DNA, F-actin, lipopolysaccharides (LPS), and lipoteichoic acid (LTA). METHODOLOGY: Liposomal formulations were prepared from a mixture of 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) or 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC) and Cholesterol (Chol), respectively. Stability of the formulations in different biological milieus and antibacterial activities compared to conventional forms in the presence of the aforementioned inhibitory factors or CF sputum were evaluated. RESULTS: The formulations were stable in all conditions tested with no significant differences compared to the controls. Inhibition of antibiotic formulations by DNA/F-actin and LPS/LTA was concentration dependent. DNA/F-actin (125 to 1000 mg/L) and LPS/LTA (1 to 1000 mg/L) inhibited conventional tobramycin bioactivity, whereas, liposome-entrapped tobramycin was inhibited at higher concentrations--DNA/F-actin (500 to 1000 mg/L) and LPS/LTA (100 to 1000 mg/L). Neither polymyxin B formulation was inactivated by DNA/F-actin, but LPS/LTA (1 to 1000 mg/L) inhibited the drug in conventional form completely and higher concentrations of the inhibitors (100 to 1000 mg/L) was required to inhibit the liposome-entrapped polymyxin B. Co-incubation with inhibitory factors (1000 mg/L) increased conventional (16-fold) and liposomal (4-fold) tobramycin minimum bactericidal concentrations (MBCs), while both polymyxin B formulations were inhibited 64-fold. CONCLUSIONS: Liposome-entrapment reduced antibiotic inhibition up to 100-fold and the CFU of endogenous P. aeruginosa in sputum by 4-fold compared to the conventional antibiotic, suggesting their potential applications in CF lung infections.

Effectiveness of liposomal-N-acetylcysteine against LPS-induced lung injuries in rodents.

Acute lung injury (ALI) and its most severe form, the acute respiratory distress syndrome (ARDS) are frequent complications in critically ill patients and are responsible for significant morbidity and mortality. So far, experimental evidence supports the role of oxidants and oxidative injury in the pathogenesis of ALI/ARDS. In this study, the antioxidant effects of conventional N-acetylcysteine (NAC) and liposomally entrapped N-acetylcysteine (L-NAC) were evaluated in experimental animals challenged with lipopolysaccharide (LPS). Rats were pretreated with empty liposomes, NAC, or L-NAC (25mg/kg body weight, iv); 4h later were challenged with LPS (E. coli, LPS 0111:B4) and sacrificed 20h later. Challenge of saline (SAL)-pretreated animals with LPS resulted in lung injury as evidenced by increases in wet lung weight (edema), increases in lipid peroxidation (marker of oxidative stress), decreases of lung angiotensin-converting enzyme (ACE) (injury marker for pulmonary endothelial cells) and increases in the pro-inflammatory eicosanoids, thromboxane B(2) and leukotriene B(4). The LPS challenge also increased pulmonary myeloperoxidase activity and chloramine concentrations indicative of neutrophil infiltration and activation of the inflammatory response. Pretreatment of animals with L-NAC resulted in significant increases in the levels of non-protein thiols and NAC levels in lung homogenates (p<0.05) and bronchoalveolar lavage fluids (p<0.001), respectively. L-NAC was significantly (p<0.05) more effective than NAC or empty liposomes in attenuating the LPS-induced lung injuries as indicated by the aforementioned injury markers. Our results suggested that the delivery of NAC as a liposomal formulation improved its prophylactic effectiveness against LPS-induced lung injuries.

Liposomal bismuth-ethanedithiol formulation enhances antimicrobial activity of tobramycin.

Pseudomonas aeruginosa and Burkholderia cenocepacia (formally, genomovar III genotype of Burkholderia cepacia complex) have emerged as serious opportunistic resistant pathogens in patients with cystic fibrosis (CF). We have developed a liposomal formulation containing bismuth-ethanedithiol (BiEDT) and tobramycin to overcome bacterial resistance. The stability of liposomal BiEDT-tobramycin (LipoBiEDT-TOB) was studied in phosphate buffered saline (PBS) and human pooled plasma at 4 and 37 degrees C. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) for free tobramycin and LipoBiEDT-TOB against clinical isolates of P. aeruginosa and B. cenocepacia were determined by the broth dilution method. The toxicity profile and the influence on bacterial adhesion of LipoBiEDT-TOB formulation were determined using a human lung carcinoma cell line (A549). LipoBiEDT-TOB exhibited lower MICs than the conventional antibiotic (0.25mg/L vs. 1024 mg/L) and eradicated this highly resistant bacterial strain of P. aeruginosa (PA-48913) at very low concentrations (4 mg/L vs. 4096 mg/L). LipoBiEDT-TOB was significantly less toxic when compared to the free BiEDT, as evaluated by the MTT and LDH assay. The LipoBiEDT-TOB formulation suppressed bacterial adhesion (B. cenocepacia M13642R) to A549 cells. These data suggest that the novel LipoBiEDT-TOB drug delivery system could be utilized as a new strategy to enhance the efficacy of existing antibiotics against resistant organisms that commonly affect individuals with chronic lung infections.

Prophylactic effect of liposomal N-acetylcysteine against LPS-induced liver injuries.

The aim of this study was to evaluate and compare the effectiveness of N-acetylcysteine (NAC) and liposomally-encapsulated NAC (L-NAC) in ameliorating the hepatotoxic effects of lipopolysaccharide (LPS). LPS, a major cell wall molecule of Gram-negative bacteria and the principal initiator of septic shock, causes liver injury in vivo that is dependent on neutrophils, platelets, and several inflammatory mediators, including tumour necrosis factor-alpha (TNF-alpha). Male Sprague-Dawley rats were pretreated intravenously with saline, plain liposomes (dipalmitoylphosphatidylcholine [DPPC]), NAC (25 mg/kg body weight), or L-NAC (25 mg/kg NAC body weight) and 4 h later were challenged intravenously with LPS (Escherichia coli O111:B4, 1.0 mg/kg body weight); animals were killed 20 h post-LPS challenge. Hepatic cell injury was evaluated by measuring the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities in plasma. LPS-induced activation of the inflammatory response was evaluated by measuring the levels of myeloperoxidase activity and chloramine concentration in liver homogenates as well as TNF-alpha levels in plasma. The hepatic levels of lipid peroxidation products and non-protein thiols (NPSH) were used to assess the extent of involvement of oxidative stress mechanisms. In general, challenge of animals with LPS resulted in hepatic injuries, activation of the inflammatory response, decreases in NPSH levels and increases in the levels of lipid peroxidation products (malondialdehyde and 4-hydroxyalkenals). Pretreatment of animals with NAC or empty liposomes did not have any significant protective effect against LPS-induced hepatotoxicity. On the other hand, pretreatment of animals with an equivalent dose of L-NAC conferred protection against the liver injuries induced following LPS challenge. These data suggest that NAC when delivered as a liposomal formulation is a potentially more effective prophylactic pharmacological agent in alleviating LPS-induced liver injuries.