Cancer therapeutic potential of combinatorial immuno- and vasomodulatory interventions.

Currently, most of the basic mechanisms governing tumour-immune system interactions, in combination with modulations of tumour-associated vasculature, are far from being completely understood. Here, we propose a mathematical model of vascularized tumour growth, where the main novelty is the modelling of the interplay between functional tumour vasculature and effector cell recruitment dynamics. Parameters are calibrated on the basis of different in vivo immunocompromised Rag1(-/-) and wild-type (WT) BALB/c murine tumour growth experiments. The model analysis supports that tumour vasculature normalization can be a plausible and effective strategy to treat cancer when combined with appropriate immunostimulations. We find that improved levels of functional tumour vasculature, potentially mediated by normalization or stress alleviation strategies, can provide beneficial outcomes in terms of tumour burden reduction and growth control. Normalization of tumour blood vessels opens a therapeutic window of opportunity to augment the antitumour immune responses, as well as to reduce intratumoral immunosuppression and induced hypoxia due to vascular abnormalities. The potential success of normalizing tumour-associated vasculature closely depends on the effector cell recruitment dynamics and tumour sizes. Furthermore, an arbitrary increase in the initial effector cell concentration does not necessarily imply better tumour control. We evidence the existence of an optimal concentration range of effector cells for tumour shrinkage. Based on these findings, we suggest a theory-driven therapeutic proposal that optimally combines immuno- and vasomodulatory interventions.

Design of Gram-negative selective antimicrobial peptides.

Lipopolysaccharide (LPS), a major component of Gram-negative bacteria, signals bacterial invasion and triggers defensive host responses. However, excessive responses also lead to the serious pathophysiological consequence of septic shock. To develop Gram-negative selective compounds that can inhibit the effects of LPS-induced sepsis, we have designed constrained cyclic antimicrobial peptides based on a cystine-stabilized beta-stranded framework mimicking the putative LPS-binding sites of the LPS-binding protein family. Our prototype termed R4A, c(PACRCRAG-PARCRCAG), consists of an eight amino acid degenerated repeat constrained by a head-to-tail cyclic peptide backbone and two cross-bracing disulfides. NMR study of K4A, an R4A analogue with four Arg --> Lys replacements, confirmed the amphipathic design elements with four Lys on one face of the antiparallel beta-strand and two hydrophobic cystine pairs plus two Ala on the opposite face. K4A and R4A displayed moderate microbicidal potency and Gram-negative selectivity. However, R4A analogues with single or multiple replacements of Ala and Gly with Arg or bulky hydrophobic amino acids displayed increased potency and selectivity in both low- and high-salt conditions. Analogues R5L and R6Y containing additional cationic and bulky hydrophobic amino acids proved the best mimics of the amphipathic topology of the "active-site" beta-strands of LPS-binding proteins. They displayed potent activity against Gram-negative E. coli with a minimal inhibitory concentration of 20 nM and a >200-fold selectivity over Gram-positive S. aureus. Our results suggest that an LPS-targeted design may present an effective approach for preparing selective peptide antibiotics.

A modular approach to structurally diverse bidentate chelate ligands for transition metal catalysis

A modular approach to a new class of structurally diverse bidentate P/N, P/P, P/S, and P/Se chelate ligands has been developed. Starting from hydroquinone, various ligands were synthesized in a divergent manner via orthogonally bis-protected bromohydroquinones as the central building block. The first donor functionality (L1) is introduced to the aromatic (hydroquinone) ligand backbone either by Pd-catalyzed cross-coupling (Suzuki coupling) with hetero-aryl bromides, by Pd-catalyzed amination, or by lithiation and subsequent treatment with electrophiles (e.g., chlorophosphanes, disulfides, diselenides, or carbamoyl chlorides). After selective deprotection, the second ligand tooth (L2) is attached by reaction of the phenolic OH functionality with a chlorophosphane, a chlorophosphite, or a related reagent. Some of the resulting chelate ligands were converted into the respective PdX2 complexes (X = Cl, I), two of which were characterized by X-ray crystallography. The methodology developed opens an access to a broad variety of new chiral and achiral transition metal complexes and is generally suited for the solid-phase synthesis of combinatorial libraries, as will be reported separately.

[Consequences of the latest Euratom directive 96/29 ("basic standard") regarding radiotherapy]. / Auswirkungen der neuen Euratom-Richtlinie 96/29 ("Grundnorm") im Bereich der Strahlentherapie.

AIM: The Council Directive 96/26 will be brought into force by the Member States of the EC until the year 2000. What practical consequences on both mounting and holding radiotherapeutical facilities should be considered? METHODS: Regarding the Council Directive, facts on workers' actual occupational exposure in medicine and representative calculations on structural shielding the consequences will be evaluated. RESULTS: Increasing both total number of occupationally exposed workers and number of Category A workers but also enforced structural shielding should be expected. Working in any protected area during pregnancy will be strictly prohibited now. CONCLUSIONS: With respect to the generally sufficient structural shielding no restrictions for existing radiotherapeutical facilities are to be expected. But it is emphasized to carry out further mountings on base of the latest Council Directive 96/26 Euratom.

Misincorporation of nucleotides opposite five-membered exocyclic ring guanine derivatives by escherichia coli polymerases in vitro and in vivo: 1,N2-ethenoguanine, 5,6,7,9-tetrahydro-9-oxoimidazo[1, 2-a]purine, and 5,6,7,9-tetrahydro-7-hydroxy-9-oxoimidazo[1, 2-a]purine.

A variety of exocyclic modified bases have been shown to be formed in DNA from various procarcinogens (e.g., acrolein, malonaldehyde, vinyl chloride, urethan) and are also found in untreated animals and humans, presumably arising as a result of lipid peroxidation. 1, N2-Ethenoguanine (1,N2-epsilon-Gua), a product known to be formed from several 2-carbon electrophiles, was placed in a known site (6256) in bacteriophage M13MB19 and mutations were analyzed in Escherichia coli, with 2.05% G-->A, 0.74% G-->T, and 0.09% G-->C changes found in uvrA- bacteria. 5,6,7, 9-Tetrahydro-7-hydroxy-9-oxoimidazo[1,2-a]purine (HO-ethanoGua), formally the hydrated derivative of 1,N2-epsilon-Gua, is a stable DNA product also derived from vinyl halides. When this base was placed in the same context, the mutation rate was 0.007-0.19% for G-->A, C, or T changes. The saturated etheno ring derivative of 1, N2-epsilon-Gua, 5,6,7,9-tetrahydro-9-oxoimidazo[1,2-a]purine (ethanoGua) produced G-->A and G-->T mutations (0.71% each). All mutants were SOS-dependent and were attenuated by uvrA activity in E. coli. In vitro studies with four polymerases showed strong blocks to addition beyond the adduct site in the order ethanoGua > HO-ethanoGua > 1,N2-epsilon-Gua. Both E. coli polymerases (pol) I exo- and II exo- and bacteriophage pol T7 exo- showed extensive misincorporation opposite ethanoGua in vitro, with pol I exo- incorporating G and T, pol II exo- incorporating A, and pol T7 exo- incorporating A and G. All modified bases reduced the use of the minus strand bearing the modified guanine in E. coli cells. It is of interest that even though the normal base pairing site of guanine is completely blocked, all of the five-membered ring derivatives incorporate the normal base (C) in >80% of the replication events in E. coli. Major differences in blockage and misincorporation are seen due to what might appear to be relatively modest structural differences, and polymerases can differ dramatically in their selectivities.