Lyme Borreliosis is an infectious disease caused by the spirochete Borrelia burgdorferi that is transmitted through the bite of infected ticks. Both B cell-mediated humoral immunity and T cell immunity develop during natural Borrelia infection. However, compared with humoral immunity, the T cell response to Borrelia infection has not been well elucidated. In this study, a novel T cell-based assay was developed and validated for the sensitive detection of antigen-specific T cell response to B. burgdorferi. Using interferon-g as a biomarker, we developed a new enzyme-linked immunospot method (iSpot Lyme™) to detect Borrelia antigen-specific effector/memory T cells that were activated in vivo by exposing them to recombinant Borrelia antigens ex vivo. To test this new method as a potential laboratory diagnostic tool, we performed a clinical study with a cohort of Borrelia positive patients and healthy controls. We demonstrated that the iSpot Lyme assay has a significantly higher specificity and sensitivity compared with the Western Blot assay that is currently used as a diagnostic measure. A comprehensive evaluation of the T cell response to Borrelia infection should, therefore, provide new insights into the pathogenesis, diagnosis, treatment and monitoring of Lyme disease.
A high correlation was observed between the aryl hydrocarbon hydroxylase activities in short-term lymphocyte cultures of 23 individuals and their plasma half-lives of antipyrine and phenylbutazone. Individuals with low inducibility of aryl hydrocarbon hydroxylase activities had very long plasma half-lives of antipyrine and phenylbutazone, whereas subjects with high inducibility of aryl hydrocarbon hydroxylase activites had relatively short plasma half-lives. Individuals with intermediate aryl hydrocarbon hydroxylase activities displayed intermediate half-lives for both drugs. The observed correlation indicates determinants which are common to the elimination of antipyrine or phenylbutazone, and aryl hydrocarbon hydroxylase metabolism of hydrocarbons. The differences in rates of drug elimination are probably due to genetic differences and may have pharmacological and therapeutic significance.
The inhibitory neurotransmitters GABA, glycine and agmatine and neuromodulators beta-phenylethylamine (beta-PEA) and taurine are important biogenic amines of the sympathetic and parasympathetic nervous systems in the body. Abnormalities in the metabolism of these biomarkers have been implicated in a vast number of neurological diseases. Novel competitive immunoassays, using one unique whole urine derivatization procedure applicable for all five biomarkers, have been developed. The determination of these biomarkers was highly reproducible: the coefficient of variance of inter- and intra-assay variation is between 3.9% and 9.8% for all assays. The assays show a good linearity in urine samples within the range of 100-400 mg Cr/dL and specificity when urine samples are spiked with biogenic amines. The recoveries are between 76 and 154%. The correlation between HPLC and ELISA for glycine and taurine (n = 10) showed regression coefficients of 0.97 and 0.98, respectively. An in vivo study on the urinary clearance of beta-PEA, agmatine and taurine after oral intake by healthy individuals demonstrated the specificity and clinical significance of these new immunoassays. The immunoassays are useful for clinical and basic research where a fast and accurate assay for the screening of biogenic amines in urine is required, without preclearance of the sample.
Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide variety of pathological conditions. However, existing methods lack the sufficient sensitivity and/or specificity necessary to measure the low endogenous level of 3-NT reliably and are too cumbersome for clinical applications. Hence, analytical improvement is urgently needed to accurately quantify the levels of 3-NT and verify the role of 3-NT in pathological conditions. This protocol presents the development of a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) detection combined with a miniaturized solid phase extraction (SPE) for the rapid and accurate measurement of 3-NT in human urine as a non-invasive biomarker for oxidative stress. SPE using a 96-well plate markedly simplified the process by combining sample cleanup and analyte enrichment without tedious derivatization and evaporation steps, reducing solvent consumption, waste disposal, risk of contamination and overall processing time. The employment of 25 mM ammonium acetate (NH4OAc) at pH 9 as the SPE elution solution substantially enhanced the selectivity. Mass spectrometry signal response was improved through adjustment of the multiple reaction monitoring (MRM) transitions. Use of 0.01% HCOOH as additive on a pentafluorophenyl (PFP) column (150 mm x 2.1 mm, 3 µm) improved signal response another 2.5-fold and shortened the overall run time to 7 min. A lower limit of quantitation (LLOQ) of 10 pg/mL (0.044 nM) was achieved, representing a significant sensitivity improvement over the reported assays. This simplified, rapid, selective and sensitive method allows two plates of urine samples (n = 192) to be processed in a 24 h time-period. Considering the markedly improved analytical performance, and non-invasive and inexpensive urine sampling, the proposed assay is beneficial for pre-clinical and clinical studies.
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