ABSTRACT
BACKGROUND AND PURPOSE: P2X7 is a membrane receptor for extracellular ATP which is highly expressed in dendritic cells, macrophages and microglia where it mediates pro-inflammatory responses. The antibiotic polymyxin B, which binds to and neutralizes the toxic residue of bacterial lipopolysaccharide, greatly amplifies cellular responses mediated by the P2X7 receptor. However, the molecular mechanism involved is so far unknown. EXPERIMENTAL APPROACH: We investigated the effects of polymyxin B and polymyxin B nonapeptide (PMBN) which is the deacylated amino derivative of polymyxin B lacking the N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue, in human macrophages and HEK293 cells stably expressing the human P2X7 receptor (HEK293-hP2X7). Differences between the two antibiotics were assessed by monitoring the following: nucleotide-induced cytoplasmic free Ca2+ concentration changes, plasma membrane permeability changes, lactate dehydrogenase activity, cell morphology changes. Western blot and microscopic analyses of P2X7GFP-expressing cells were also performed. KEY RESULTS: In contrast to polymyxin B, the polymyxin B nonapeptide was unable to potentiate: a) the ATP-induced Ca2+ increase, b) pore formation and consequently ATP-mediated plasma membrane permeabilization; c) ATP-dependent cytotoxicity. Moreover, in contrast to polymyxin B, polymyxin B nonapeptide did not affect aggregation of the P2X7 receptor subunits and it did not potentiate P2X7-dependent cell fusion. CONCLUSIONS AND IMPLICATIONS: The effects of polymyxin B depended on the presence of its N-terminal fatty amino acid 6-methylheptanoic/octanoic-Dab residue as deletion of this residue abolished polymyxin B-dependent modulation of ATP-triggered responses. These findings are important in the search for allosteric modulators of the P2X7 receptor.
Subject(s)
Anti-Bacterial Agents/pharmacology , Polymyxin B/pharmacology , Receptors, Purinergic P2/drug effects , Adenosine Triphosphate/physiology , Anti-Bacterial Agents/chemistry , Blotting, Western , Calcium/metabolism , Cell Fusion , Cell Line , Cell Membrane/drug effects , Cell Membrane/metabolism , Cell Membrane Permeability/drug effects , Cell Shape/drug effects , Cytoplasm/drug effects , Cytoplasm/metabolism , Green Fluorescent Proteins/metabolism , Humans , L-Lactate Dehydrogenase/metabolism , Macrophages/drug effects , Polymyxin B/analogs & derivatives , Polymyxin B/chemistry , Porosity , Receptors, Purinergic P2X7 , Structure-Activity RelationshipABSTRACT
After a brief review of cryptococcosis in dogs and cats from the literature from 1980 to 92, three cases in dogs (two epidemiologically strictly connected) and two in cats are reported. In the three dogs and in one of the two cats lesions were seen in many sites, but only one dog and one cat had a central nervous system localization. The cutis was affected in the three dogs and in one cat; in two of the dogs it was probably the primary lesion, and in the cat it was the only lesion. An unidentified genetic defect was probably the predisposing factor in two of the dogs of the same litter, exposed to soil cryptococcal contamination (from pigeon guano); a third dog, of different breeding, was not infected. Steroid treatment was predisposing in one cat. Flucytosine treatment was initially successful in two dogs, but in both relapses were reported; in one dog fluconazole treatment was successful. The isolates, before and after treatment, demonstrated a large increase in flucytosine MIC, strictly connected with the relapse.
Subject(s)
Cat Diseases , Cryptococcosis/veterinary , Dog Diseases , Animals , Antifungal Agents/therapeutic use , Cat Diseases/drug therapy , Cat Diseases/epidemiology , Cat Diseases/microbiology , Cats , Cryptococcosis/drug therapy , Cryptococcosis/epidemiology , Cryptococcosis/microbiology , Cryptococcus neoformans/drug effects , Cryptococcus neoformans/isolation & purification , Dog Diseases/drug therapy , Dog Diseases/epidemiology , Dog Diseases/microbiology , Dogs , Italy/epidemiologyABSTRACT
In restorative dentistry, any technique that employs retentive endoradicular pins can be associated with a simple technique that includes ceramization of the metallic surface of the pin, producing great adhesion to a composite resin by interposition of a silane coupling agent. The strong bond obtained using this technique between pin and filling material surrounding it prevents a dangerous concentration of mechanical forces around the pin itself and reduces the probability of breaks and consequent failure of the restoration.
