Cytokine expanded myeloid precursors function as regulatory antigen presenting cells and induce tolerance through IL-10 producing regulatory T cells

The initiation of graft vs. host disease (GVHD) after stem cell transplantation is dependent on direct antigen presentation by host antigen presenting cells (APC) while the effect of indirect antigen presentation by donor APC is unknown. We have studied the role of indirect antigen presentation in allogenic responses by adding populations of cytokine-expanded donor APC to haematopoietic grafts that would otherwise induce lethal GVHD. Progenipoietin-1 (a synthetic G-CSF/Flt-3 L molecule) and G-CSF expanded myeloid DC, plasmacytoid DC and a novel granulocyte-monocyte precursor population (GM) that differentiate into class IIpos, CD80/CD86pos, CD40neg APC during GVHD. Whereas addition of plasmacytoid and myeloid donor DC augmented GVHD, GM cells induced transplant tolerance via MHC class II restricted generation of IL-10-secreting regulatory T cells. Thus a population of cytokine expanded granulocyte-monocyte precursors function as regulatory antigen presenting cells, suggesting that G-CSF derivatives may have application in disorders characterised by a loss of self-tolerance.

2-Methoxy-6-methyl-3-nitro-4-(2-nitroprop-1-enyl)phenyl acetate

In the title compound, C13H14N2O7, steric crowding around the aromatic ring results in significant out-of-plane twisting of the nitro, methoxy, acetoxy and 2-nitropropenyl functional groups. These distortions are explained by comparison with less congested substituted benzene analogues.

Synthesis of alpha-aspartyl-containing cyclic peptides

alpha-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.

Chemistry of 5-oxodihydroisoxazoles .17. Acylation of 5-oxodihydroisoxazoles

2-Unsubstituted isoxazol-5(4H)-ones and -5(2N)-ones may be acylated by acid chlorides, anhydrides or carboxylic acids in the presence of carbodiimides, to give O- and N-acylated products, The solvent, the presence of base and the temperature are found to alter the product ratios dramatically, but the substituents present at C-3 have the greatest effect, Aliphatic acid anhydrides and chlorides generally react at nitrogen, but aroyl halides give significant proportions of O-acylated products, Limited success in converting O-aroyl to N-aroyl isoxazolones is reported.

Bioactivation of Phenytoin by Human Cytochrome P450: Characterization of the Mechanism and Targets of Covalent Adduct Formation

The cytochrome P450-dependent covalent binding of radiolabel derived fi om phenytoin (DPH) and its phenol and catechol metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) and 5-(3',4'-dihydroxyphenyl)-5-phenylhydantoin (CAT), was examined in liver microsomes. Radiolabeled HPPH and CAT and unlabeled CAT were obtained from microsomal incubations and isolated by preparative HPLC. NADPH-dependent covalent binding was demonstrated in incubations of human liver microsomes with HPPH. When CAT was used as substrate, covalent adduct formation was independent of NADPH, was enhanced in the presence of systems generating reactive oxygen species, and was diminished under anaerobic conditions or in the presence of cytoprotective reducing agents. Fluorographic analysis showed that radiolabel derived from DPH and HPPH was selectively associated with proteins migrating with approximate relative molecular weights of 57-59 kDa and at the dye front (molecular weights < 23 kDa) on denaturing gels. Lower levels of radiolabel were distributed throughout the molecular weight range. In contrast, little selectivity was seen in covalent adducts formed from CAT. HPPH was shown to be a mechanism-based inactivator of P450, supporting the contention that a cytochrome P450 is one target of covalent binding. These results suggest that covalent binding of radiolabel derived from DPH in rat and human Liver microsomes occurs via initial P450-dependent catechol formation followed by spontaneous oxidation to quinone and semiquinone derivatives that ultimately react with microsomal protein. Targets for covalent binding may include P450s, though the catechol appears to be sufficiently stable to migrate out of the P450 active site to form adducts with other proteins. In conclusion, we have demonstrated that DPH can be bioactivated in human liver to metabolites capable of covalently binding to proteins. The relationship of adduct formation to DPH-induced hypersensitivity reactions remains to be clarified.

Photodegradation of irinotecan (CPT-11) in aqueous solutions: Identification of fluorescent products and influence of solution composition

The photodegradation of irinotecan (CPT-11), the semisynthetic derivative of the antitumor alkaloid 20(S)-camptothecin, has been investigated. The drug was exposed to laboratory light for up to 5 days in 0.9% saline solution (pH 8.5). Five significant photodegradation products were observed and a high-performance liquid chromatography (HPLC) assay was employed to isolate them from CPT-11 using gradient conditions. The structures were elucidated by nuclear magnetic resonance spectroscopy and tandem mass spectrometry and shown to be the result of extensive modifications of the lactone ring of CPT-11. Three of the compounds were found to belong to the mappicine group of alkaloids. In addition, the effect of light on the stability of CPT-11 in aqueous solutions and biological fluids was also assessed, Potassium phosphate buffers (0.05 M, pH 5.0-8.2) and saline, plasma, urine, and bile solutions containing 20 mu M CPT-11 were equilibrated in the dark for 24 h before being exposed to laboratory light for up to 171 h at ambient temperature. Four of the five identified photodegradation products were observed and quantitated by isocratic HPLC, using a different detection mode (fluorescence) than the one used for gradient elution, In general, CPT-11 was found to be unstable under neutral and alkaline conditions for all solutions investigated, with the exception of bile. We conclude that CPT-11 is photolabile and that care should be taken to protect samples, particularly those intended for the isolation and identification of novel metabolites of CPT-11.