This report describes the first human study of a novel amyloid-imaging positron emission tomography (PET) tracer, termed Pittsburgh Compound-B (PIB), in 16 patients with diagnosed mild AD and 9 controls. Compared with controls, AD patients typically showed marked retention of PIB in areas of association cortex known to contain large amounts of amyloid deposits in AD. In the AD patient group, PIB retention was increased most prominently in frontal cortex (1.94-fold, p = 0.0001). Large increases also were observed in parietal (1.71-fold, p = 0.0002), temporal (1.52-fold, p = 0.002), and occipital (1.54-fold, p = 0.002) cortex and the striatum (1.76-fold, p = 0.0001). PIB retention was equivalent in AD patients and controls in areas known to be relatively unaffected by amyloid deposition (such as subcortical white matter, pons, and cerebellum). Studies in three young (21 years) and six older healthy controls (69.5 +/- 11 years) showed low PIB retention in cortical areas and no significant group differences between young and older controls. In cortical areas, PIB retention correlated inversely with cerebral glucose metabolism determined with 18F-fluorodeoxyglucose. This relationship was most robust in the parietal cortex (r = -0.72; p = 0.0001). The results suggest that PET imaging with the novel tracer, PIB, can provide quantitative information on amyloid deposits in living subjects.

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The near-UV spectroscopic changes induced by the binding of recombinant human cystatin A to papain were appreciably different from those induced by cystatin C, reflecting mainly interactions involving the single tryptophan of cystatin C, Trp-106. Cystatin A bound tightly and rapidly to papain and cathepsin L, with dissociation equilibrium constants of approximately 10(-11)-10(-13) M and association rate constants of 3 x 10(6)-5 x 10(6) M-1.s-1. These affinities are at least 50-100-fold higher than previously reported values. The kinetics of binding to papain were consistent with a simple reversible bimolecular reaction mechanism, indicating that cystatin A, like chicken cystatin and cystatin C, binds to papain with no appreciable conformational adaptation of either reacting protein. Cystatin A bound more weakly to actinidin and cathepsins B, C and H, with dissociation equilibrium constants of 10(-8)-10(-9) M. The weaker binding to cathepsin B was largely due to a considerably reduced association rate constant (approximately 4 x 10(4) M-1.s-1), consistent with the 'occluding loop' of cathepsin B markedly restricting the access of cystatin A to the active site. The lower affinities for actinidin and cathepsins C and H were due partly to lower association rate constants (2 x 10(5)-6 x 10(5) M-1.s-1) but primarily to higher dissociation rate constants. The mode of binding of cystatin A to inactivated papains indicated that there is appreciably less space around the active-site cysteine of papain in the complex with cystatin A than in the complexes with chicken cystatin and cystatin C. An N-terminally truncated form of cystatin A, lacking the first six residues, had considerably lower affinity for papain than the full-length inhibitor, consistent with an intact N-terminal region being of importance for proteinase binding.

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Our results imply that almost 50% of the islets were damaged to the extent that the FDG contained was release within minutes after intraportal transplantation. The distribution of radioactivity without accumulation in the brain indicates that the activity is released from lysed islet cells in the form of [F]FDG-6P rather than native [F]FDG. The presented technique shows promise to become a powerful and quantitative tool, readily available in the clinic, to evaluate initial islet engraftment and survival.

The importance of the evolutionarily conserved Gly-4 residue for the affinity and kinetics of interaction of cystatin A with several cysteine proteinases was assessed by site-directed mutagenesis. Even the smallest replacement, by Ala, resulted in approximately 1000-, approximately 10- and approximately 6000-fold decreased affinities for papain, cathepsin L, and cathepsin B, respectively. Substitution by Ser gave further 3-8-fold reductions in affinity, whereas the largest decreases, >10(5)-fold, were observed for mutations to Arg and Glu. The kinetics of inhibition of papain by the mutants with small side chains, Ala and Ser, were compatible with a one-step bimolecular reaction similar to that with wild-type cystatin A. The decreased affinities of these mutants for papain and cathepsin L were due exclusively to increased dissociation rate constants, but the reduced affinities for cathepsin B were due also to decreased association rate constants. The latter finding indicates that the intact N-terminal region serves as a guide directing cystatin A to the active site of cathepsin B, as has been proposed for cystatin C. The kinetics of binding of the mutants with charged side chains, Arg and Glu, to papain were consistent with a two-step binding mechanism, in which the mutant side chains are accommodated in the complex by a conformational change. The NMR solution structure of the Ala and Trp mutants showed only minor changes compared with wild-type cystatin A, indicating that the large reductions in affinity for proteinases are not due to altered structures of the mutants. Instead, a side chain larger than a hydrogen atom at position 4 affects the interaction with the proteinase most likely by interfering with the binding of the N-terminal region.

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Abstract:The compound {N-methyl-[11 C]}2-(4'-methylaminophenyl)-6-hydroxybenzothiazole, "PIB", measured by positron emission tomography, has been demonstrated to image brain -amyloid deposition in Alzheimer's disease (AD). In the present study the benefit of measuring the PIB accumulation rate together with the unidirectional influx of PIB into the brain was investigated in healthy control subjects and patients with AD. In a monkey changes in the influx rate constant K 1 of PIB closely followed changes in CBF, caused by alteration of Pa CO2 . In addition, K 1 was high both in the monkey and in humans, suggesting that this parameter reflects CBF. Most AD patients studied showed clearly higher accumulation rate for PIB than the controls in cortical brain areas, while a few patients showed as low accumulation as the controls. K 1 did not correlate with the accumulation rate, indicating that K 1 for PIB provides extra information besides the accumulation rate.