Biomarkers of Tuberculosis Severity and Treatment Effect: A Directed Screen of 70 Host Markers in a Randomized Clinical Trial.

More efficacious treatment regimens are needed for tuberculosis, however, drug development is impeded by a lack of reliable biomarkers of disease severity and of treatment effect. We conducted a directed screen of host biomarkers in participants enrolled in a tuberculosis clinical trial to address this need. Serum samples from 319 protocol-correct, culture-confirmed pulmonary tuberculosis patients treated under direct observation as part of an international, phase 2 trial were screened for 70 markers of infection, inflammation, and metabolism. Biomarker assays were specifically developed for this study and quantified using a novel, multiplexed electrochemiluminescence assay. We evaluated the association of biomarkers with baseline characteristics, as well as with detailed microbiologic data, using Bonferroni-adjusted, linear regression models. Across numerous analyses, seven proteins, SAA1, PCT, IL-1ß, IL-6, CRP, PTX-3 and MMP-8, showed recurring strong associations with markers of baseline disease severity, smear grade and cavitation; were strongly modulated by tuberculosis treatment; and had responses that were greater for patients who culture-converted at 8weeks. With treatment, all proteins decreased, except for osteocalcin, MCP-1 and MCP-4, which significantly increased. Several previously reported putative tuberculosis-associated biomarkers (HOMX1, neopterin, and cathelicidin) were not significantly associated with treatment response. In conclusion, across a geographically diverse and large population of tuberculosis patients enrolled in a clinical trial, several previously reported putative biomarkers were not significantly associated with treatment response, however, seven proteins had recurring strong associations with baseline radiographic and microbiologic measures of disease severity, as well as with early treatment response, deserving additional study.

Further biodosimetry investigations using murine partial-body irradiation model.

This study evaluates both the effects of physical restraint and use of candidate biomarkers in a CD2F1 male mouse partial-body irradiation model for biological dosimetry diagnostic assays. Mice were irradiated (6-Gy, 250-kVp X ray) to 3/3rd (total body), 2/3rd (gut and torso), 1/3rd (gut only) and 0/3rd (sham) of total body. Blood was sampled for haematology and blood plasma proteomic biomarkers at 1 and 2 d after exposure. Increases in the body fraction exposed showed progressive decreases in lymphocyte counts and increases in the neutrophil-to-lymphocyte ratios with no significant differences in the neutrophil and platelet counts. The radioresponse for plasma biomarker Flt3L showed proportional increases; however, G-CSF and SAA levels exhibited dramatic and non-proportional increases in levels. Physical restraint at 1 d post-exposure increased lymphocyte counts and SAA, decreased neutrophil-to-lymphocyte ratio and Flt3L and showed no effects on neutrophil and platelet counts or G-CSF.

Screening derivatized peptide libraries for tight binding inhibitors to carbonic anhydrase II by electrospray ionization-mass spectrometry.

This paper describes the use of electrospray ionization-mass spectrometry (ESI-MS) to screen two libraries of soluble compounds to search for tight binding inhibitors for carbonic anhydrase II (EC 4.2.1.1). The two libraries, H2NO2SC6H4C(O)NH-AA1-AA2-C(O)NHCH2CH2CO2H where AA1 and AA2 are L-amino acids (library size: 289 compounds) or D-amino acids (256 compounds), were constructed by attaching tripeptides to the carboxyl group of 4-carboxybenzenesulfonamide. Screening of both libraries yielded, as the tightest binding inhibitor, compound 1 (AA1 = AA2 = L-Leu; binding constant Kb = 1.4 x 10(8) M-1). The ability of ESI-MS to estimate simultaneously the relative binding affinities of a protein to soluble ligands in a library, if general, should be useful in drug development.

A self-assembled monolayer for the binding and study of histidine-tagged proteins by surface plasmon resonance.

This paper reports the generation of a self-assembled monolayer (SAM) that selectively binds proteins whose primary sequence terminates with a His-tag: a stretch of six histidines commonly incorporated in recombinant proteins to simplify purification. The SAM was prepared by the adsorption onto a gold surface of a mixture of two alkanethiols: one thiol that terminated with a nitrilotriacetic acid (NTA) group, a group that forms a tetravalent chelate with Ni(II), and a second thiol that terminated with a tri(ethylene glycol) group, a group that resists protein adsorption. His-tagged proteins bound to the SAM by interaction of the histidines with the two vacant sites on Ni(II) ions chelated to the surface NTA groups. Studies with model proteins showed the binding was specific for His-tagged proteins and required the presence of Ni(II) on the surface. Immobilized His-tagged proteins were kinetically stable in buffered saline at pH 7.2 but could be desorbed by treatment with 200 mM imidazole. Surface plasmon resonance studies for two model systems showed that His-tagged proteins adsorbed on the NTA-SAM retained a greater ability to participate in binding interactions with proteins in solution than protein immobilized in a thin dextran gel layer by covalent coupling.

Contact adhesion of thin gold films on elastomeric supports: cold welding under ambient conditions.

Thin gold films placed in contact on compliant elastomeric poly(dimethylsiloxane) supports weld together. This ;;cold welding'' is remarkable both for the low loads required and for the fact that it occurs under ambient laboratory conditions, conditions in which the gold surfaces are covered with films of weakly adsorbed organic impurities. These impurities are probably displaced laterally during the welding. Welding can be prevented by the presence of a self-assembled gold(I) alkylthiolate monolayer on the gold surfaces. The welded contacts have low electrical resistivity and can be made thin enough to transmit light. This system is a promising one with which to study interaction between interfaces.

Continuous monitoring of receptor-mediated changes in the metabolic rates of living cells.

Activation of beta-adrenergic or muscarinic acetylcholine receptors expressed in transfected cells or epidermal growth factor receptors in human keratinocytes produces 15% to 200% changes in cellular metabolic rates. Changes in cell metabolism were monitored continuously with a previously described silicon-based microphysiometer that detects small changes in extracellular pH. The amplitude and kinetics of the metabolic changes depend upon several factors including pretreatment of the cells prior to receptor stimulation, the dose of hormone/neurotransmitter used, and the receptor complement of the cells. Responses are receptor specific; cells transfected with receptor genes respond only to the appropriate hormone/transmitter, whereas control (nontransfected) cells or cells transfected with different receptors exhibit no response. The specificity of the responses was further documented by using pharmacological antagonists. In Chinese hamster ovary (CHO) cells transfected with human beta 2-adrenergic receptors, isoproterenol produces a 20-60% increase in the rate of extracellular acidification with an EC50 of 4 nM, a response that is competitively antagonized by (-)-propranolol. The EC50 for the isoproterenol response is shifted from 4 nM to 100 nM in the presence of 3 nM (-)-propranolol. The kinetics of the metabolic response induced by beta-adrenergic receptor stimulation are markedly slower than those elicited by muscarinic receptor agonists. The maximal metabolic response in cells transfected with beta-adrenergic receptors peaks at approximately 12 min as compared with less than 30 sec in cells transfected with muscarinic receptors, perhaps reflecting activation of different second-messenger pathways. These findings illustrate an alternative means of studying cellular responses to hormones and neurotransmitters and suggest that metabolic changes will be generally useful for detecting the consequences of receptor-ligand interactions.

Detection of cell-affecting agents with a silicon biosensor.

Cellular metabolism is affected by many factors in a cell's environment. Given a sufficiently sensitive method for measuring cellular metabolic rates, it should be possible to detect a wide variety of chemical and physical stimuli. A biosensor has been constructed in which living cells are confined to a flow chamber in which a potentiometric sensor continually measures the rate of production of acidic metabolites. Exploratory studies demonstrate several applications of the device in basic science and technology.