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Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid-based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water ([.sup.2][H.sub.2]O), distinct, newly synthesized [.sup.2]H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct [.sup.2]H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected [.sup.2]H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After [.sup.2][H.sub.2]O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson's disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects., Introduction Biomarkers that provide insight into the pathophysiology, progression, and treatment response of neurodegenerative diseases would represent a major advance for disease research, clinical management, and drug development. Currently, in [...]

Abstract: Objective: Reverse cholesterol transport (RCT) can be defined as a pathway of flux of cholesterol from peripheral tissues to the liver for potential excretion into feces. This prospective, placebo-controlled, double-blind crossover study assessed the effect of ezetimibe on several RCT parameters in hyperlipidemic patients. Methods: Following 7 weeks of treatment (ezetimibe 10 mg/day or placebo), 26 patients received 24-h continuous IV infusions of [3,4-13C2]-cholesterol, then took heavy water (2H2O) by mouth. Cholesterol excretion was measured by quantification of neutral/acid sterols in stool and blood samples during 7 days post-infusion with continued treatment. Plasma de novo cholesterol synthesis was assessed by 2H-labeling from 2H2O. Results: Ezetimibe significantly reduced levels of low-density lipoprotein cholesterol (22%, P < 0.001) without significant changes in triglycerides and high-density lipoprotein cholesterol and significantly increased the flux of plasma-derived cholesterol into fecal neutral sterols by 52% (P = 0.04) without change in flux into fecal bile acids. Total fecal neutral sterol output increased by 23% (P = 0.02). Plasma de novo cholesterol synthesis increased by 57% (P < 0.001). The fractional clearance rate (FCR) of plasma cholesteryl-ester trended higher (7%; P = 0.055) with a reduction in absolute cholesteryl-ester production rate (9%, P < 0.01). Whole-body free cholesterol efflux rate from extra-hepatic tissues into plasma was not measurably changed by ezetimibe. Conclusion: Ezetimibe treatment approximately doubled the flux of plasma-derived cholesterol into fecal neutral sterols, in association with increases in total fecal neutral sterol excretion, FCR of plasma cholesterol ester, and plasma de novo cholesterol synthesis. These effects are consistent with increased cholesterol transport through the plasma compartment and excretion from the body, in response to ezetimibe treatment in hyperlipidemic humans. Clintrials.gov: NCT00701727. [Copyright &y& Elsevier]

Background: Reverse cholesterol transport from peripheral tissues is considered the principal atheroprotective mechanism of high-density lipoprotein, but quantifying reverse cholesterol transport in humans in vivo remains a challenge. We describe here a method for measuring flux of cholesterol though 3 primary components of the reverse cholesterol transport pathway in vivo in humans: tissue free cholesterol (FC) efflux, esterification of FC in plasma, and fecal sterol excretion of plasma-derived FC., Methods and Results: A constant infusion of [2,3-(13)C(2)]-cholesterol was administered to healthy volunteers. Three-compartment SAAM II (Simulation, Analysis, and Modeling software; SAAM Institute, University of Washington, WA) fits were applied to plasma FC, red blood cell FC, and plasma cholesterol ester (13)C-enrichment profiles. Fecal sterol excretion of plasma-derived FC was quantified from fractional recovery of intravenous [2,3-(13)C(2)]-cholesterol in feces over 7 days. We examined the key assumptions of the method and evaluated the optimal clinical protocol and approach to data analysis and modeling. A total of 17 subjects from 2 study sites (n=12 from first site, age 21 to 75 years, 2 women; n=5 from second site, age 18 to 70 years, 2 women) were studied. Tissue FC efflux was 3.79±0.88 mg/kg per hour (mean ± standard deviation), or ≍8 g/d. Red blood cell-derived flux into plasma FC was 3.38±1.10 mg/kg per hour. Esterification of plasma FC was ≍28% of tissue FC efflux (1.10±0.38 mg/kg per hour). Recoveries were 7% and 12% of administered [2,3-(13)C(2)]-cholesterol in fecal bile acids and neutral sterols, respectively., Conclusions: Three components of systemic reverse cholesterol transport can be quantified, allowing dissection of this important function of high-density lipoprotein in vivo. Effects of lipoproteins, genetic mutations, lifestyle changes, and drugs on these components can be assessed in humans. (J Am Heart Assoc. 2012;1:e001826 doi: 10.1161/JAHA.112.001826.).

Progressive HIV disease has been associated with loss of memory T cell responses to Ag. To better characterize and quantify long-lived memory T cells in vivo, we have refined an in vivo labeling technique to study the kinetics of phenotypically distinct, low-frequency CD8(+) T cell subpopulations in humans. HIV-negative subjects and antiretroviral-untreated HIV-infected subjects in varying stages of HIV disease were studied. After labeling the DNA of dividing cells with deuterated water ((2)H(2)O), (2)H-label incorporation and die-away kinetics were quantified using a highly sensitive FACS/mass spectrometric method. Two different populations of long-lived memory CD8(+) T cells were identified in HIV-negative subjects: CD8(+)CD45RA(-)CCR7(+)CD28(+) central memory (T(CM)) cells expressing IL-7Ralpha and CD8(+)CD45RA(+)CCR7(-)CD28(-) RA effector memory (T(EMRA)) cells expressing CD57. In pilot studies in HIV-infected subjects, T(CM) cells appeared to have a shorter half-life and reduced abundance, particularly in those with high viral loads; T(EMRA) cells, by contrast, retained a long half-life and accumulated in the face of progressive HIV disease. These data are consistent with the hypothesis that IL-7Ralpha(+) T(CM) cells represent true memory CD8(+) T cells, the loss of which may be responsible in part for the progressive loss of T cell memory function during progressive HIV infection.