lipid-a and Pneumonia

This study aimed to explore mechanism of colistin resistance amongst Klebsiella pneumoniae isolates through plasmid mediated mcr-1 gene in Pakistan. Carbapenem and Colistin resistant K. pneumoniae isolates (n  = 34) stored at - 80 °C as part of the Aga Khan University Clinical Laboratory strain bank were randomly selected and subjected to mcr-1 gene PCR. To investigate mechanisms of resistance, other than plasmid mediated mcr-1 gene, whole genome sequencing was performed on 8 clinical isolates, including 6 with colistin resistance (MIC  >  4 μg/ml) and 2 with intermediate resistance to colistin (MIC  >  2 μg/ml).. RT-PCR conducted revealed absence of mcr-1 gene in all isolates tested. Whole genome sequencing results revealed modifications in Lipid A-Ara4N pathway. Modifications in Lipid A-Ara4N pathway were detected in ArnA_ DH/FT, UgdH, ArnC and ArnT genes. Mutation in ArnA_ DH/FT gene were detected in S3, S5, S6 and S7 isolates. UgdH gene modifications were found in all isolates except S3, mutations in ArnC were present in all except S1, S2 and S8 and ArnT were detected in all except S4 and S7. In the absence of known mutations linked with colistin resistance, lipid pathway modifications may possibly explain the phenotype resistance to colistin, but this needs further exploration.

Topics: Anti-Bacterial Agents; Bacterial Proteins; Colistin; Drug Resistance, Bacterial; Humans; Klebsiella pneumoniae; Laboratories, Clinical; Lipid A; Microbial Sensitivity Tests; Pakistan; Plasmids; Pneumonia

Allergen-specific immunotherapy (SIT) effectively alleviates type I allergic diseases characterized by T helper (Th)2-type immunity. Our recent studies have shown that a synthetic trivalent glycocluster, triacedimannose (TADM), suppresses the Th2-type allergic inflammation. The aim of this study was to compare TADM with two well-known adjuvants, unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG) and monophosphoryl lipid A (MPLA) in a grass allergen-induced chronic allergic inflammation model in mice.. Female BALB/c mice were intranasally sensitized with 50 μL of timothy grass pollen extract (TE) twice a week for a period of 15 weeks. Therapeutic intranasal treatments were then performed once a week after the tenth intranasal TE instillation using TADM (10 or 25 μg/50 μL), CpG-ODN (20 μg/50 μL) or MPLA (2 μg/50 μL). Groups of 9-10 animals per treatment were killed 24 hours after the last timothy dosage. Blood, bronchoalveolar lavage (BAL) fluids and lung biopsies were taken for subsequent analysis.. When mice were repeatedly exposed to TE for 15 weeks, the number of eosinophils and lymphocytes increased in the BAL fluids. The eosinophil and lymphocyte counts decreased dose-dependently and were practically abolished in the mice treated with TADM. Treatments with MPLA or CpG significantly increased the numbers of neutrophils, while CpG nonsignificantly decreased eosinophilia compared to timothy exposure.. A novel synthetic glycocluster molecule inhibited the development of grass-induced eosinophilic pulmonary inflammation in mice when administrated in the airways. This compound could be a candidate to be used either as an adjuvant in SIT or as a topical anti-inflammatory treatment.

Topics: Adjuvants, Immunologic; Allergens; Animals; Bronchoalveolar Lavage Fluid; Desensitization, Immunologic; Disaccharides; Disease Models, Animal; Eosinophils; Female; Hypersensitivity; Lipid A; Lymphocyte Count; Mannans; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Phleum; Plant Extracts; Pneumonia; Pollen; Statistics, Nonparametric

Pulmonary immunization poses the unique challenge of balancing vaccine efficacy with minimizing inflammation in the respiratory tract. While previous studies have shown that mice immunized intranasally with F1-V-loaded polyanhydride nanoparticles are protected from a lethal challenge with Yersinia pestis, little is known about the initial interaction between the nanoparticles and immune cells following intranasal administration. Here, the deposition within the lung and internalization by phagocytic cells of polyanhydride nanovaccines encapsulating F1-V are compared with that of soluble F1-V alone or F1-V adjuvanted with monophosphoryl lipid A (MPLA). Encapsulation of F1-V into polyanhydride nanoparticles prolonged its presence while F1-V administered with MPLA is undetectable within 48 h. The inflammation induced by the polyanhydride nanovaccine is mild compared with the marked inflammation induced by the MPLA-adjuvanted F1-V. Even though F1-V delivered with saline is detected in the lung 48 h after administration, it is known that this regimen does not elicit a protective immune response. The prolonged F1-V presence in the lung in concert with the mild inflammatory response provided by the nanovaccine provides new insights into the development of protective immune responses with a single intranasal dose.

Topics: Adjuvants, Immunologic; Animals; Female; Immunization; Lipid A; Lung; Mice; Mice, Inbred C57BL; Nanostructures; Plague; Pneumonia; Polyanhydrides; Vaccines; Yersinia pestis

Haemorrhage-induced immunosuppression has been linked to nosocomial infections. We assessed the impact of monophosphoryl lipid A, a Toll/interleukin-1 receptor-domain-containing adaptor protein inducing interferon-biased Toll-like receptor-4 agonist currently used as a vaccine adjuvant in humans, on post-haemorrhage susceptibility to infection. We used a mouse model of post-haemorrhage pneumonia induced by methicillin-susceptible Staphylococcus aureus. Monophosphoryl lipid A was administered intravenously after haemorrhage and before pneumonia onset. Haemorrhage altered survival rate, increased lung damage (neutrophil accumulation, oedema and cytokine release) and altered the functions of dendritic and natural killer cells. Here, we show that monophosphoryl lipid A decreased systemic dissemination of S. aureus and dampened inflammatory lung lesions. Monophosphoryl lipid A partially restored the capacity for antigen presentation and the transcriptional activity in dendritic cells. Monophosphoryl lipid A did not restore the interferon-γ mRNA but prevented interleukin-10 mRNA overexpression in natural killer cells compared with untreated mice. Ex vivo monophosphoryl lipid A-stimulated dendritic cells or natural killer cells harvested from haemorrhaged animals were adoptively transferred into mice undergoing post-haemorrhage pneumonia. Stimulated dendritic cells (but not stimulated natural killer cells) improved the survival rate compared with mice left untreated. In vivo depletion of natural killer cells decreased survival rate of monophosphoryl lipid A-treated mice. Dendritic and natural killer cells are critically involved in the beneficial effects of monophosphoryl lipid A within post-haemorrhage pneumonia.

Topics: Animals; Bronchoalveolar Lavage; Dendritic Cells; Endothelial Cells; Hemorrhage; Immunocompromised Host; Immunosuppression Therapy; Inflammation; Interferon-gamma; Interleukin-10; Killer Cells, Natural; Lipid A; Lung; Male; Mice; Mice, Inbred BALB C; Peroxidase; Phenotype; Pneumonia; Spleen; Staphylococcus aureus; Toll-Like Receptor 4

Lipopolysaccharide (LPS) induces acute lung injury (ALI) via Toll-like receptor 4 (TLR4)-mediated MAPK activation. The lipid A fraction of LPS is considered to be the active moiety, but whether the lipid A-TLR4 interaction accounts completely for ALI-associated MAPK activation in vivo has not been determined. The lipid A fraction of LPS induces a discrete MAPK activation pattern in murine ALI. Mice (C57BL/6J, C3H/HeJ) were treated with intratracheal instillations of purified lipid A or LPS (10, 30, and 100 microg per mouse) or vehicle. ALI was assessed by histology. Chromogenic myeloperoxidase (MPO) activity was measured in lung homogenates. MAPK expression was quantified by immunoblotting. In vitro ERK inhibitor studies using thioglycollate-elicited macrophages were also performed. MPO increased in a dose- and time-responsive fashion. Notably, MPO was 2.4-fold greater after lipid A compared with LPS and vehicle at 6 h after instillation (lipid A, 0.88 +/- 0.25 vs. LPS, 0.37 +/- 0.21 optical density units.min(-1).mg(-1); P < 0.05). However, ALI scores were comparable at 6 and 24 h between LPS and lipid A. MPO was also comparable in vehicle-treated or C3H/HeJ mice treated with LPS or lipid A at 6 and 24 h. Phospho-ERK activation was pronounced at 6 and 24 h after lipid A but not LPS treatment. In vitro studies confirmed the relationship between phospho-ERK activation and cytokine expression in macrophage stimulated with either LPS or lipid A. Compared with whole LPS, the lipid A fraction is associated with amplified whole lung MPO and ERK activation 6 h after intratracheal instillation in mice.

Topics: Animals; Cytokines; Enzyme Inhibitors; Flavonoids; Lipid A; Lipopolysaccharides; Lung; Macrophages; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Peroxidase; Pneumonia; Respiratory Distress Syndrome

In this study, we sought the possibility of a new therapeutic strategy for dampening endotoxin-induced inflammation using soluble form of extracellular rTLR4 domain (sTLR4) and soluble form of rMD-2 (sMD-2). Addition of sTLR4 plus sMD-2 was significantly effective in inhibiting LPS-elicited IL-8 release from U937 cells and NF-kappaB activation in the cells transfected with TLR4 and MD-2 when compared with a single treatment with sTLR4 or sMD-2. Thus, we investigated the role of the extracellular TLR4 domain in interaction of lipid A with MD-2. Biotinylated sTLR4 failed to coprecipitate [(3)H]lipid A when it was sedimented with streptavidin-agarose, demonstrating that the extracellular TLR4 domain does not directly bind lipid A by itself. The amounts of lipid A coprecipitated with sMD-2 significantly increased when coincubated with sTLR4, and sTLR4 increased the affinity of lipid A for the binding to sMD-2. Soluble CD14 is required for the sTLR4-stimulated increase of lipid A binding to sMD-2. We also found that addition of sTLR4 plus sMD-2 inhibited the binding of Alexa-conjugated LPS to the cells expressing TLR4 and MD-2. Murine lungs that had received sTLR4 plus sMD-2 with LPS did not show any findings indicative of interstitial edema, neutrophil flux, and hemorrhage. Co-instillation of sTLR4 plus sMD-2, but not sTLR4 or sMD-2 alone, significantly decreased neutrophil infiltration and TNF-alpha levels in bronchoalveolar lavage fluids from LPS-treated mice. This study provides novel usage of sTLR4 and sMD-2 as an antagonist against endotoxin-induced pulmonary inflammation.

Topics: Animals; Cell Line; Cell Membrane; Extracellular Fluid; Female; Interleukin-8; Lipid A; Lipopolysaccharide Receptors; Lipopolysaccharides; Lymphocyte Antigen 96; Mice; Mice, Inbred BALB C; NF-kappa B; Pneumonia; Protein Binding; Recombinant Proteins; Toll-Like Receptor 4

Although chronic inhalation of endotoxin or lipopolysaccharide (LPS) causes all of the classic features of asthma, including airway hyperreactivity, airway inflammation, and airway remodeling, the mechanisms involved in this process are not clearly understood. The objective of this study was to determine whether intratracheal treatment with LPS antagonist (E5564, a lipid A analog) prevented the development of chronic endotoxin-induced airway disease in a mouse model of environmental airway disease. Pretreatment with 10 and 100 microg of E5564 was found to inhibit the airway response (hyperreactivity and inflammation) for up to 48 h after the administration of the compound. Repeated dosing with 50 microg of E5564 intratracheally did not cause any measurable toxicity. Therefore, in a chronic experiment, mice were treated with either E5564 (50 microg) or vehicle three times weekly for 5 wk and simultaneously daily exposed to either LPS (4.65 +/- 0.30 microg/m3) or saline aerosol. E5564 was effective in decreasing the airway hyperreactivity to methacholine, the air space neutrophilia, the interleukin-6 in the lung lavage fluid, and the neutrophil infiltration of the airways 36 h after 5 wk of LPS inhalation. Less collagen deposition was observed in the airways of E5564-treated mice compared with vehicle-treated mice after a 4-wk recovery period. Our results indicate that E5564, a Toll-like receptor 4 antagonist, minimizes the physiological and biological effects of chronic LPS inhalation, suggesting a therapeutic role for competitive LPS antagonists in preventing or reducing endotoxin-induced environmental airway disease.

Topics: Administration, Inhalation; Animals; Endotoxins; Escherichia coli; Lipid A; Lipopolysaccharides; Male; Mice; Mice, Inbred C3H; Pneumonia; Receptors, Immunologic; Respiratory Hypersensitivity; Toll-Like Receptor 4

A cross-sectional study was performed on the occurrence of IgG antibodies to lipid A of the Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) on serum of 2272 cattle distributed on 19 Danish dairy herds. The relationship between the concentration of antibodies to lipid A (ALI) and age, herd, pregnancy rate and occurrence of mastitis, bovine virus diarrhoea (BVD), reproductive and digestive disorders, diarrhoea, pneumonia, foot disorders, various infections and traumatic udder lesions was investigated. ALI generally was low in calves and increased during their first 1.5 years of life to a steady state, which could be altered by the occurrence of disease. There were significant differences in the mean ALI among the herds (P < 0.001). High ALI was associated with a low herd pregnancy rate, to preceding occurrence of mastitis (P < 0.048), BVD (P < 0.01), reproduction diseases (P < 0.041) and digestion disorders (P < 0.064) in animals older than 2 years. The calf mortality rate was not associated to ALI and there was no correlation between the ALI in calves and their dams. The occurrence of high ALI levels on a herd basis may be an indication of increased challenge or enhanced immunological defense to Gram-negative bacteria or endotoxin.

Topics: Aging; Animals; Antibodies, Bacterial; Bovine Virus Diarrhea-Mucosal Disease; Cattle; Cattle Diseases; Cross-Sectional Studies; Denmark; Diarrhea; Endotoxins; Female; Gram-Negative Bacteria; Gram-Negative Bacterial Infections; Immunoglobulin G; Lipid A; Mastitis, Bovine; Pneumonia; Pregnancy; Pregnancy Rate

A high rate of phenotypic variation in the lipooligosaccharide (LOS) electrophoretic profile of Haemophilus somnus occurred in most isolates obtained at approximately weekly intervals from three calves intrabronchially challenged with a cloned isolate of H. somnus 2336. Daily subculturing for 2 weeks resulted in at least one major alteration in the LOS electrophoretic profiles for strain 2336 and both additional disease isolates examined, but no change occurred in the LOS electrophoretic profiles for any of three commensal isolates examined. None of the LOSs from any of the postchallenge intrabronchial isolates reacted with rabbit antiserum to the challenge strain LOS in immunoblotting, but LOSs from two nasopharyngeal isolates did. Antigenic variation in the extracted LOSs of most of the isolates was supported by the results of an enzyme-linked immunosorbent assay. Preimmune serum from each of the calves did not react with any of the isolates or the challenge strain, whereas sera obtained 35 days after challenge reacted with the challenge strain and zero to five additional isolates and sera obtained 74 days after challenge reacted with two to six additional isolates. Recognition of LOSs from isolates obtained near the end of the 70-day experiment by day-74 sera was related to clearance of the bacteria from the lungs. Isolates demonstrating major electrophoretic changes showed variations in the composition of the oligosaccharide, but not lipid A, moiety of their LOSs. The oligosaccharide of the LOS of each isolate was composed predominantly of glucose but varied substantially in the contents of galactose, arabinose, xylose, mannose, and 3-deoxy-D-manno-octulosonic acid. Therefore, the LOS of H. somnus is capable of undergoing compositional and antigenic variations, which may act as an important virulence mechanism for evading host immune defense mechanisms.

Topics: Animals; Antigenic Variation; Bronchi; Cattle; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Haemophilus; Immunization, Passive; Immunoblotting; Lipid A; Lipopolysaccharides; Male; Nasopharynx; Phenotype; Pneumonia; Time Factors

Gram-negative bacillary pneumonias represent a major problem in hospitalized patients. A major research interest has been towards the development of rapid and accurate diagnostic procedures for these infections. An ELISA procedure for the detection of lipid A in pulmonary lavage fluid has been developed. Using the quantitation of lipid A, an antigen common to all clinically important gram-negative bacilli, as a marker for gram-negative bacilli, the presence or absence of pneumonia caused by these organisms could be demonstrated in laboratory animals. Additionally, by quantitating the lipid A associated with the lavageable pulmonary cell population, a measure of bacterial lung clearance could be obtained.

Topics: Animals; Enzyme-Linked Immunosorbent Assay; Female; Lipid A; Models, Biological; Pneumonia; Pulmonary Alveoli; Rabbits; Therapeutic Irrigation