Browsing by Subject "Serum"
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Item Development and analytical validation of a gas chromatography-mass spectrometry method for the assessment of gastrointestinal permeability and intestinal absorptive capacity in dogs(2009-05-15) Rodriguez Frausto, HeribertoAssessment of gastrointestinal permeability in vivo is considered a suitable method for the evaluation of gastrointestinal mucosal integrity. Probes commonly used include lactulose (L) and rhamnose (R) for the assessment of intestinal permeability, xylose (X) and 3-O-methylglucose (M) for the evaluation of intestinal absorptive capacity, and sucrose (S) for the assessment of gastric permeability. Traditionally, various methods have been used to quantify these markers in the urine after orogastric administration. However, urine collection is difficult and uncomfortable. A protocol based on the analysis of blood samples would be easier to perform. Thus, the aim of the first part of this project was to develop and validate a new gas chromatography-mass spectrometry (GC-MS) method for the quantification of five sugar probes in canine serum. The method was sensitive, accurate, precise, and reproducible for the simultaneous quantification of 5 sugar probes in serum. The aim of the second part of this project was to assess the kinetic profiles of these 5 sugar probes in serum after orogastric administration in dogs and to determine the optimal time point for sample collection. Dogs received a solution containing L (10 g/L), R (10 g/L), X (10 g/L), M (5 g/L), and S (40 g/L) by orogastric intubation. Baseline blood samples were collected. Subsequent timed blood samples were taken for a 24 hours period. Significant changes in serum concentrations of all 5 sugars were detected after administration of the test dose (p<0.0001 for all 5 probes). Serum concentrations of L and R were significantly different from baseline concentrations from 90 to 240 and from 60 to 300 min post dosing respectively, and those of X, M, and S were significantly different from 30 to 240 min after dosing (p<0.05 for all 5 probes). Variations of the mean sugar concentrations of all dogs at 90, 120, and 180 minutes were analyzed using a Kruskal-Wallis test. Based on the results, only two blood samples, one taken at baseline and a second sample obtained between 90 and 180 after dosing, appear to be sufficient for assessment of intestinal permeability and mucosal absorptive capacity using these sugar probes.Item The USPA2 Protein and Serum Resistance of Moraxella Catarrhalis(2006-05-15) Attia, Ahmed Sherif; Hansen, Eric J.Most isolates of Moraxella (Branhamella) catarrhalis are resistant to the bactericidal activity of normal human serum. Several M. catarrhalis gene products have been linked to the serum-resistant phenotype but none of them was shown to be directly involved in this phenotype. This study provides the first evidence for the direct involvement of the UspA2 protein of several serum-resistant M. catarrhalis strains in the serum-resistant phenotype. This was achieved by using transformation and allelic exchange to introduce hybrid uspA2 genes into M. catarrhalis, together with cloning and expression of different UspA2 proteins in Haemophilus influenzae. Using different types of human sera, it was concluded that serum-sensitive M. catarrhalis strains are killed via the classical complement pathway. Analysis of complement deposition on four different serum-resistant M. catarrhalis strains and their serum-sensitive uspA2 mutants showed similar amounts of early complement components binding to these cells, but a significant reduction occurred in the amount of polymerized C9 on the wild-type strains relative to that on the uspA2 mutants. The binding of the UspA2 proteins of these strains to the complement regulator vitronectin was shown to be responsible for the protection of these strains against complementmediated killing. This represents the first example of vitronectin-mediated serum resistance on a microbe. In contrast, binding of the complement regulator C4BP by the M. catarrhalis strains used in this study did not correlate with serum resistance. Finally, analysis of the untranslated region upstream of the uspA2 open reading frame showed that the presence of a heteropolymeric nucleotide repeat (AGAT) in this region is necessary for both normal expression of the UspA2 protein and serum resistance. Also, it was shown that changes in the number of AGAT repeats affected transcription of the uspA2 gene, with 15-18 AGAT repeats yielding maximal levels of transcription. These results indicate that these AGAT repeats play a regulatory role in the expression of the uspA2 gene.