The determination of budesonide and fluticasone in human sputum samples collected from COPD patients using LC-MS/MS.

A bioanalytical method for the quantitative determination of budesonide and fluticasone in human sputum was developed. Sputolysin(®) Reagent was added to the sputum samples. After incubation (37°C; 60-70 min under shaking) and automated solid phase extraction the extracts were analysed using LC-MS/MS. Budesonide and fluticasone showed good linearity (r>0.99) over the range 0.1-100 nM in the first and second validation batch, and over the range 0.25-10,000 nM in the third and fourth validation batch. The lower limit of quantification (LLOQ) achieved was 5 nM for budesonide and fluticasone in 100 µL human sputum. Intra-run and inter-run RSD for four quality control levels (5-100 nM) were within 6.9% (budesonide) and 8.0% (fluticasone). The accuracy ranged from -11.4% to -1.6% (budesonide), and from -11.8% to 0.4% (fluticasone). The validated method was applied to clinical sputum samples from COPD patients.

Quantitative analysis of Tenecteplase in rat plasma samples using LC-MS/MS as an alternative for ELISA.

An LC-MS/MS method has been developed for the quantitative determination of a protein drug (Tenecteplase; M(W) 58,777 Da) in rat plasma. The protein was digested with trypsin without prior clean-up of the plasma sample, without the use of a label nor internal standard. A limited validation was performed to assess the linearity, the sensitivity and the specificity of the method. In addition, the developed method was applied to the quantitative analysis of Tenecteplase in rat plasma samples originating from a single-dose study in rats.

Construction of acetate auxotrophs of Neisseria meningitidis to study host-meningococcal endotoxin interactions.

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. < or =10 ng/ml), we have generated acetate auxotrophs NMBACE1 from encapsulated Neisseria meningitidis (serogroup B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking the polysialic acid capsule. Growth of the auxotrophs in medium containing [(14)C]acetate yielded (14)C-lipooligosaccharides containing approximately 600 cpm/ng. Gel sieving resolved 14C-lipooligosaccharide-containing aggregates with an estimated molecular mass of > or =20 x 10(6) Da (peak A) and approximately 1 x 10(6) Da (peak B) from both strains. Lipooligosaccharides in peaks A and B had the same fatty acid composition and SDS-polyacrylamide gel electrophoresis profile. 14C-Labeled capsule copurified with (14)C-lipooligosaccharides in peak B from NMBACE1, whereas the other aggregates contained only 14C-lipooligosaccharide. For all aggregates, lipopolysaccharide-binding protein and soluble CD14-induced delivery of lipooligosaccharides to endothelial cells and cell activation correlated with disaggregation of lipooligosaccharides. These processes were inhibited by the presence of capsule but unaffected by the size of the aggregates. In contrast, endotoxin activation of cells containing membrane CD14 was unaffected by capsule but diminished when endotoxin was presented in larger aggregates. These findings demonstrate that the physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.

Treatment of leg telangiectasia by using a long-pulse dye laser at 595 nm with and without dynamic cooling device.

BACKGROUND AND OBJECTIVE: The long-pulse dye laser (PDL) at 595 nm and pulse duration of 1.5 msec has been shown to improve clearance of larger vessels such as those seen in leg telangiectasia. The objectives of this study are twofold. First, to determine the effect of the dynamic cooling device (DCD) in clearance of leg telangiectasia by using a long-pulse PDL at 595 nm. Next, to determine the effect of the DCD in reducing transient discomfort associated with treatment and in reducing epidermal damage (blistering, hyper/hypopigmentation, scarring) caused by the laser. STUDY DESIGN/MATERIALS AND METHODS: Matched treatment sites were compared at energy densities of 20 and 24 J/cm(2) with and without the use of the cryogen spray in 18 patients. In areas treated without the DCD, the laser pulse was delivered through a single layer of Spenco Second Skin. Patients received two treatments 6 weeks apart. Discomfort ratings, clearance of leg telangiectasia, and complications were assessed at 6 weeks, 12 weeks, and 6 months. RESULTS: A reduction in discomfort ratings was found in most patients using the DCD. Six-month follow-up data revealed at the 20 J/cm(2) treatment sites, with or without the DCD, 76.9% showed greater than 50% clearance. At the 24 J/cm(2) treatment sites, with or without the DCD, 84.6% showed greater than 50% clearance. CONCLUSION: The long-pulse dye laser at 595 nm with a 1.5-msec pulse duration cleared leg telangiectasia an average of 67.5% with two treatments at 6 months. The major effect of the DCD was on pain reduction. There was no difference in clearance rates when using the DCD vs. cooled Second Skin. Further studies with longer cooling times with the DCD are needed to optimize treatment parameters.

Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor.

Adherence and invasion are thought to be key events in the pathogenesis of non-typeable Haemophilus influenzae (NTHi). The role of NTHi lipooligosaccharide (LOS) in adherence was examined using an LOS-coated polystyrene bead adherence assay. Beads coated with NTHi 2019 LOS adhered significantly more to 16HBE14 human bronchial epithelial cells than beads coated with truncated LOS isolated from an NTHi 2019 pgmB:ermr mutant (P = 0.037). Adherence was inhibited by preincubation of cell monolayers with NTHi 2019 LOS (P = 0.0009), but not by preincubation with NTHi 2019 pgmB:ermr LOS. Competitive inhibition studies with a panel of compounds containing structures found within NTHi LOS suggested that a phosphorylcholine (ChoP) moiety was involved in adherence. Further experiments revealed that mutations affecting the oligosaccharide region of LOS or the incorporation of ChoP therein caused significant decreases in the adherence to and invasion of bronchial cells by NTHi 2019 (P < 0.01). Analysis of infected monolayers by confocal microscopy showed that ChoP+ NTHi bacilli co-localized with the PAF receptor. Pretreatment of bronchial cells with a PAF receptor antagonist inhibited invasion by NTHi 2109 and two other NTHi strains expressing ChoP+ LOS glycoforms exhibiting high reactivity with an anti-ChoP antibody on colony immunoblots. These data suggest that a particular subset of ChoP+ LOS glycoforms could mediate NTHi invasion of bronchial cells by means of interaction with the PAF receptor.

Infection of primary human bronchial epithelial cells by Haemophilus influenzae: macropinocytosis as a mechanism of airway epithelial cell entry.

Nontypeable Haemophilus influenzae is an exclusive human pathogen which infects the respiratory epithelium. We have initiated studies to explore the interaction of the nontypeable H. influenzae strain 2019 with primary human airway epithelial cells by electron and confocal microscopy. Primary human airway cell cultures were established as monolayers on glass collagen-coated coverslips or on semipermeable membranes at an air-fluid interface. Scanning electron microscopy indicated that bacteria adhered to nonciliated cells in the population. The surface of infected cells showed evidence of cytoskeletal rearrangements manifested by microvilli and lamellipodia extending toward and engaging bacteria. Confocal microscopic analysis demonstrated that infection induced actin polymerization with an increase in cortical actin as well as evidence of actin strands around the bacteria. Transmission electron microscopic analysis showed lamellipodia and microvilli surrounding organisms, as well as organisms adherent to the cell surface. These studies also demonstrated the presence of bacteria within vacuoles inside of airway cells. Confocal microscopic studies with Texas red-labeled dextran (molecular weight, 70,000) indicated that H. influenzae cells were entering cells by the process of macropinocytosis. These studies indicate that nontypeable H. influenzae can initiate cytoskeletal rearrangement within human airway epithelium, resulting in internalization of the bacteria within nonciliated human airway epithelial cells by the process of macropinocytosis.

Determination of 1,2,6-inositol trisphosphate (derivatives) in plasma using iron(III)-loaded adsorbents and capillary zone electrophoresis-(indirect) UV detection.

A method for the determination of 1,2,6-inositol trisphosphate (IP3) and derivatives in plasma by capillary zone electrophoresis with (indirect) UV detection has been developed. The sample pretreatment is based on the selective isolation after complexation of inositol phosphates with iron(III) loaded on an adsorbent. Plasma protein denaturation was performed with sodium dodecyl sulfate. The selectivity of the method is demonstrated with the analysis of phenylacetate-IP3. The recoveries amount to 65% and 88% in plasma and in water, respectively.