Photoaffinity cross-linking of Alzheimer's disease amyloid fibrils reveals interstrand contact regions between assembled beta-amyloid peptide subunits.

The assembly of the beta-amyloid peptide (Abeta) into amyloid fibrils is essential to the pathogenesis of Alzheimer's disease. Detailed structural information about fibrillogenesis has remained elusive due to the highly insoluble, noncrystalline nature of the assembled peptide. X-ray fiber diffraction, infrared spectroscopy, and solid-state NMR studies performed on fibrils composed of Abeta peptides have led to conflicting models of the intermolecular alignment of beta-strands. We demonstrate here the use of photoaffinity cross-linking to determine high-resolution structural constraints on Abeta monomers within amyloid fibrils. A photoreactive Abeta(1-40) ligand was synthesized by substituting L-p-benzoylphenylalanine (Bpa) for phenylalanine at position 4 (Abeta(1-40) F4Bpa). This peptide was incorporated into synthetic amyloid fibrils and irradiated with near-UV light. SDS-PAGE of dissolved fibrils revealed the light-dependent formation of a covalent Abeta dimer. Enzymatic cleavage followed by mass spectrometric analysis demonstrated the presence of a dimer-specific ion at MH(+) = 1825.9, the predicted mass of a fragment composed of the N-terminal Abeta(1-5) F4Bpa tryptic peptide covalently attached to the C-terminal Abeta(29-40) tryptic peptide. MS/MS experiments and further chemical modifications of the cross-linked dimer led to the localization of the photo-cross-link between the ketone of the Bpa4 side chain and the delta-methyl group of the Met35 side chain. The Bpa4-Met35 intermolecular cross-link is consistent with an antiparallel alignment of Abeta peptides within amyloid fibrils.

Exogenous and endogenous catecholamines inhibit the production of macrophage inflammatory protein (MIP) 1 alpha via a beta adrenoceptor mediated mechanism.

Noradrenaline (NA) and adrenaline (Ad) are modulators of cytokine production. Here we investigated the role of these neurotransmitters in the regulation of macrophage inflammatory protein (MIP)-1alpha expression. Pretreatment of RAW 264.7 macrophages with NA or Ad decreased, in a concentration-dependent manner (1 nM-100 microM), MIP-1alpha release induced by bacterial lipopolysaccharide (LPS 10 ng ml(-1) LPS). The effect of NA was reversed by the selective beta-adrenoceptor antagonist propranolol (10 microM), but not by the alpha-adrenoceptor antagonist phentolamine (10 microM). In the concentration range of 10 nM-10 microM, isoproterenol, a beta-adrenoceptor agonist, but not phenylephrine (a selective alpha1-adrenoceptor agonist) or UK-14304 (a selective alpha2-adrenoceptor agonist) mimicked the inhibitory effects of catecholamines on MIP-1alpha production. Increases in intracellular cyclic adenosine monophosphate, elicited either by the selective type IV phosphodiesterase inhibitor rolipram (0.1 - 10 microM), or by prostaglandin E2, (10 nM-10 microM) decreased MIP-1alpha release, suggesting that increased cyclic AMP may contribute to the suppression of MIP-1alpha release by beta-adrenoceptor stimulation. Northern blot analysis demonstrated that NA (100 nM-10 microM), Ad, isoproterenol, as well as rolipram (100 nM-10 microM) decreased LPS-induced MIP-1alpha mRNA accumulation. NA and Ad (1-100 microM) also decreased MIP-1alpha production in thioglycollate-elicited murine peritoneal macrophages. Pretreatment of mice with either isoproterenol (10 mg kg(-1), i.p.) or rolipram (25 mg kg(-1), i.p.) decreased LPS-induced plasma levels of MIP-1alpha, while propranolol (10 mg kg(-1), i.p.) augmented the production of this chemokine, confirming the role of a beta-adrenoceptor mediated endogenous catecholamine action in the regulation of MIP-1alpha production in vivo. Thus, based on our data we conclude that catecholamines are important endogenous regulators of MIP-1alpha expression in inflammation.

Suppression of macrophage inflammatory protein (MIP)-1alpha production and collagen-induced arthritis by adenosine receptor agonists.

1. Ligands of the various adenosine receptor subtypes modulate the production of pro-and anti-inflammatory cytokines. Here we evaluated the effect of adenosine and various ligands of the adenosine receptor subtypes (A1, A2, A3) on the chemokine macrophage inflammatory protein (MIP) 1alpha production in immunostimulated RAW macrophages in vitro. Furthermore, we studied whether a selected A3 adenosine receptor agonist inhibits MIP-1alpha production and affects the course of inflammation in collagen-induced arthritis. 2. In the cultured macrophages, the A3 receptor agonist N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA), and, less potently, the A2 receptor agonist 2-p-(2-carboxyethyl) phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS; 1-200 micro) dose-dependently suppressed the production of MIP-1alpha. The selective A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA, 1-200 microM) was ineffective, and adenosine was a weak inhibitor. The inhibition of MIP-1alpha production by the A3 and A2 agonist was associated with suppression of its steady-state mRNA levels. 3. Based on the in vitro data, we concluded that activation of A3, and to a lesser extent A2 adenosine receptors suppresses MIP-1alpha expression. Since IB-MECA was the most potent inhibitor of MIP-1alpha expression, we next investigated whether it affects the production of other pro-inflammatory mediators. We observed that IB-MECA (1-300 microM) inhibited, in a dose-dependent manner, the production of IL-12, IL-6, and, to a lesser extent, nitric oxide in the immunostimulated cultured macrophages. 4. Since MIP-alpha is a chemokine which enhances neutrophil recruitment into inflammatory sites, we investigated whether the A3 agonist IB-MECA affects the course of inflammation, MIP-alpha production and the degree of neutrophil recruitment in arthritis. In a model of collagen-induced arthritis in mice, IB-MECA (0.5 mg/kg/day) reduced the severity of joint inflammation. IB-MECA inhibited the formation of MIP-1alpha, IL-12 and nitrotyrosine (an indicator of reactive nitrogen species) in the paws, and suppressed neutrophil infiltration. 5. We conclude that adenosine receptor agonists, most notably the A3 agonist IB-MECA suppress the production of MIP-alpha, and exert anti-inflammatory effects. Therefore, stimulation of adenosine receptor subtypes A3 and A2 may be a strategy worthy of further evaluation for the abrogation of acute or chronic inflammatory disorders.

The crucial role of IL-10 in the suppression of the immunological response in mice exposed to staphylococcal enterotoxin B.

Staphylococcal enterotoxin B (SEB), a bacterial superantigen, activates the immune system resulting in a burst of pro- and anti-inflammatory cytokines. A central anti-inflammatory mediator in this process is IL-10. Using IL-10-deficient C57BL/6 (IL-10 KO) mice, we studied the role of endogenous IL-10 in the regulation of the immune response to SEB. SEB (100 microg) induced the release of IL-10 in control C57BL/6 [IL-10 wild type (WT)] mice, but not in their IL-10 KO counterparts. SEB-evoked plasma levels of TNF-alpha, IL-1beta, IL-2, IL-6, IL-12 and IFN-gamma were significantly higher in the IL-10 KO mice than in the WT animals. The release of macrophage inflammatory proteins-1alpha and -2 was also enhanced in the IL-10 KO mice. Further, upon SEB challenge, mice deficient in IL-10 produced higher levels of nitric oxide than the WT animals. IL-10 deficiency resulted in a marked enhancement of the SEB-induced apoptosis of thymocytes. Finally, IL-10 KO mice were more susceptible to SEB-induced lethal shock than their WT controls. These results show that IL-10 plays an important immunoregulatory role in the response to a superantigenic stimulus, by dampening of the shock-inducing inflammatory response and early activation-induced cell death elicited by SEB.

Proton MR spectroscopic characteristics of pediatric pilocytic astrocytomas.

PURPOSE: We report the common characteristics of juvenile pilocytic astrocytomas revealed by proton MR spectroscopy. METHODS: Eight children with pilocytic astrocytomas were studied with proton MR spectroscopy. The selected sampling volume was approximately 4 cm3, obtained from solid tumor. To localize the sampling volume, we used point-resolved spectroscopy (PRESS) and stimulated-echo acquisition mode (STEAM) techniques to acquire long- and short-TE spectra, respectively. Spectra from PRESS and STEAM sequences were processed using Lorentzian-to-Gaussian transformation and exponential apodization, respectively. For PRESS (2000/270) spectra, peaks of creatine, choline, N-acetylaspartate (NAA), and lactate resonances were integrated; for STEAM (2000/20) spectra, we measured the amplitude of the peaks at 3.2, 2.0, 1.3 and 0.9 ppm. RESULTS: An elevated lactate doublet was observed in the PRESS spectra. The choline/NAA ratio was 3.40. The amplitude ratios of the lipid pattern (0.9, 1.3 and 2.0 ppm) to choline were all below one. CONCLUSION: Despite the benign histology of the tumor, which generally lacks necrosis, a lactate signal was detected in all eight patients studied. A dominant lipid pattern was not observed.