Citations

AbstractThis unit describes three methods for the detection of caspase activation as cells undergo apoptosis. Simple and relatively quantitative enzymatic assays are provided using suitable substrates. Because the various low‐molecular‐weight substrates available for these assays are not selective, however, the assays do not accurately distinguish between various caspases. Immunoblotting is described for following the activation of specific caspases. When coupled with subcellular fractionation, this method can provide large amounts of temporal and spatial information about caspase activation. Finally, affinity labeling protocols are provided for detecting active caspases in whole‐cell lysates or subcellular fractions.

Homeostasis in the intact organism is achieved implicitly by repeated incremental feedback (inhibitory) and feedforward (stimulatory) adjustments enforced via intermittent signal exchange. In separated systems, neurohormone signals act deterministically on target cells via quantifiable effector-response functions. On the other hand, in vivo interglandular signaling dynamics have not been estimable to date. Indeed, experimentally isolating components of an interactive network definitionally disrupts time-sensitive linkages. We implement and validate analytical reconstruction of endogenous effector-response properties via a composite model comprising ( i ) a deterministic basic feedback and feedforward ensemble structure; ( ii ) judicious statistical allowance for possible stochastic variability in individual biologically interpretable dose–response properties; and ( iii ) the sole data requirement of serially observed concentrations of a paired signal (input) and response (output). Application of this analytical strategy to a prototypical neuroendocrine axis in the conscious uninjected horse, sheep, and human ( i ) illustrates probabilistic estimation of endogenous effector dose–response properties; and ( ii ) unmasks statistically vivid (2- to 5-fold) random fluctuations in inferred target-gland responsivity within any given pulse train. In conclusion, balanced mathematical formalism allows one to ( i ) reconstruct deterministic properties of interglandular signaling in the intact mammal and ( ii ) quantify apparent signal-response variability over short time scales in vivo . The present proof-of-principle experiments introduce a previously undescribed means to estimate time-evolving signal-response relationships without isotope infusion or pathway disruption.

Giant cell arteritis (GCA) is a granulomatous and occlusive vasculitis that causes blindness, stroke, and aortic aneurysm. CD4+ T cells are selectively activated in the adventitia of affected arteries. In human GCA artery–severe combined immunodeficiency (SCID) mouse chimeras, depletion of CD83+ dendritic cells (DCs) abrogated vasculitis, suggesting that DCs are critical antigen-presenting cells in GCA. Healthy medium-size arteries possessed an indigenous population of DCs at the adventitia–media border. Adoptive T cell transfer into temporal artery–SCID mouse chimeras demonstrated that DCs in healthy arteries were functionally immature, but gained T cell stimulatory capacity after injection of lipopolysaccharide. In patients with polymyalgia rheumatica (PMR), a subclinical variant of GCA, adventitial DCs were mature and produced the chemokines CCL19 and CCL21, but vasculitic infiltrates were lacking. Human histocompatibility leukocyte antigen class II–matched healthy arteries, PMR arteries, and GCA arteries were coimplanted into SCID mice. Immature DCs in healthy arteries failed to stimulate T cells, but DCs in PMR arteries could attract, retain, and activate T cells that originated from the GCA lesions. We propose that in situ maturation of DCs in the adventitia is an early event in the pathogenesis of GCA. Activation of adventitial DCs initiates and maintains T cell responses in the artery and breaks tissue tolerance in the perivascular space.

Fluorodeoxyglucose positron emission tomography (PET) studies have found that patients with Alzheimer's dementia (AD) have abnormally low rates of cerebral glucose metabolism in posterior cingulate, parietal, temporal, and prefrontal cortex. We previously found that cognitively normal, late-middle-aged carriers of the apolipoprotein E ε4 allele, a common susceptibility gene for late-onset Alzheimer's dementia, have abnormally low rates of glucose metabolism in the same brain regions as patients with probable AD. We now consider whether ε4 carriers have these regional brain abnormalities as relatively young adults. Apolipoprotein E genotypes were established in normal volunteers 20–39 years of age. Clinical ratings, neuropsychological tests, magnetic resonance imaging, and PET were performed in 12 ε4 heterozygotes, all with the ε3/ε4 genotype, and 15 noncarriers of the ε4 allele, 12 of whom were individually matched for sex, age, and educational level. An automated algorithm was used to generate an aggregate surface-projection map that compared regional PET measurements in the two groups. The young adult ε4 carriers and noncarriers did not differ significantly in their sex, age, educational level, clinical ratings, or neuropsychological test scores. Like previously studied patients with probable AD and late-middle-aged ε4 carriers, the young ε4 carriers had abnormally low rates of glucose metabolism bilaterally in the posterior cingulate, parietal, temporal, and prefrontal cortex. Carriers of a common Alzheimer's susceptibility gene have functional brain abnormalities in young adulthood, several decades before the possible onset of dementia.

A relationship between gout and Kienböck’s disease has been proposed on the basis of a previously published case report. A review of patients at our institution identified a single case with the combination of both Kienböck’s disease and gout. We believe that the association is coincidental.

Cross-sectional positron emission tomography (PET) studies find that cognitively normal carriers of the apolipoprotein E (APOE) ɛ4 allele, a common Alzheimer's susceptibility gene, have abnormally low measurements of the cerebral metabolic rate for glucose (CMRgl) in the same regions as patients with Alzheimer's dementia. In this article, we characterize longitudinal CMRgl declines in cognitively normal ɛ4 heterozygotes, estimate the power of PET to test the efficacy of treatments to attenuate these declines in 2 years, and consider how this paradigm could be used to efficiently test the potential of candidate therapies for the prevention of Alzheimer's disease. We studied 10 cognitively normal ɛ4 heterozygotes and 15 ɛ4 noncarriers 50–63 years of age with a reported family history of Alzheimer's dementia before and after an interval of approximately 2 years. The ɛ4 heterozygotes had significant CMRgl declines in the vicinity of temporal, posterior cingulate, and prefrontal cortex, basal forebrain, parahippocampal gyrus, and thalamus, and these declines were significantly greater than those in the ɛ4 noncarriers. In testing candidate primary prevention therapies, we estimate that between 50 and 115 cognitively normal ɛ4 heterozygotes are needed per active and placebo treatment group to detect a 25% attenuation in these CMRgl declines with 80% power and P = 0.005 in 2 years. Assuming these CMRgl declines are related to the predisposition to Alzheimer's dementia, this study provides a paradigm for testing the potential of treatments to prevent the disorder without having to study thousands of research subjects or wait many years to determine whether or when treated individuals develop symptoms.