Functional differences between ATP-gated human and rat P2X3 receptors are caused by critical residues of the intracellular C-terminal domain.

ATP-activated P2X3 receptors of sensory ganglion neurons contribute to pain transduction and are involved in chronic pain signaling. Although highly homologous (97%) in rat and human species, it is unclear whether P2X3 receptors have identical function. Studying human and rat P2X3 receptors expressed in patch-clamped human embryonic kidney (HEK) cells, we investigated the role of non-conserved tyrosine residues in the C-terminal domain (rat tyrosine-393 and human tyrosine-376) as key determinants of receptor function. In comparison with rat P2X3 receptors, human P2X3 receptors were more expressed and produced larger responses with slower desensitization and faster recovery. In general, desensitization was closely related to peak current amplitude for rat and human receptors. Downsizing human receptor expression to the same level of the rat one still yielded larger responses retaining slower desensitization and faster recovery. Mutating phenylalanine-376 into tyrosine in the rat receptor did not change current amplitude; yet, it retarded desensitization onset, demonstrating how this residue was important to functionally link these two receptor states. Conversely, removing tyrosine from position 376 strongly down-regulated human receptor function. The different topology of tyrosine residues in the C-terminal domain has contrasting functional consequences and is sufficient to account for species-specific properties of this pain-transducing channel.

BCG prophylaxis in bladder cancer produces activation of recruited neutrophils.

INTRODUCTION: Bacillus Calmette-Guerin (BCG) is used to treat high risk superficial bladder cancer, but its anti-tumor effect remains incompletely defined. Recently a role for polymophonuclear (PMN) neutrophils has been suggested. To investigate the role of granulocytes, we monitored the activation state of these cells in the urine of BCG-treated patients. MATERIALS AND METHODS: Ten patients with stage T1, grade 3 (T1G3) transitional cell carcinoma received an 8 week course of BCG after undergoing transurethral resection of the bladder. Cytological and enzymatic analyses of urine samples collected before and 2 hours after the physiological expulsion of BCG were performed. The activation state of urine granulocytes and the presence of activating factors within the urine samples were monitored. RESULTS: BCG immunotherapy stimulated, through soluble factors, the activation of PMN neutrophils, which transmigrated into the bladder, and the degree of activation of the PMN neutrophils was related directly to the number of epithelial cells detached from the urothelial layer. CONCLUSIONS: This study suggests that PMN neutrophils can participate in reducing the recurrence of bladder cancer by promoting urothelial cell turnover proportionally to their degree of activation. Our results provide further evidence to support the role of PMN neutrophils in BCG immunotherapy.

Mast cell activation by myelin through scavenger receptor.

A role for mast cells (MC) in the pathogenesis of multiple sclerosis (MS) has been suggested, based on the analysis of human lesions and on an animal model of the disease (EAE). What role MC play in the development of MS is not well understood. We hypothesized that the link connecting MC with demyelinating diseases may be represented by their interaction with myelin. Here we show that myelin can activate mast cells. This process could be a key event in the mast cell function required for inducing EAE in mice and possibly in MS in man.

BCG-induced rabbit alveolar macrophages are endowed with strengthened antioxidant metabolic pathways.

Following i.v. BCG infection, a new population of macrophages are recruited in the rabbit lung. These macrophages, known as activated macrophages, substitute the resident macrophages and can play a key role in the defence against mycobacteria. We report here that BCG-activated alveolar macrophages are equipped with a more active hexose monophosphate pathway, which can maintain an optimal intracellular concentration of NADPH and GSH, and allow to produce mycobactericidal free radicals and to become resistant to mycobacterium-induced programmed cell death. These findings suggest that sustaining the anti-oxidant properties of macrophages could represent a candidate process to be considered as a good therapeutic target in fighting Mycobacterium spp infections.

A procedure for the isolation of asbestos bodies from lung tissue by exploiting their magnetic properties: a new approach to asbestos body study.

The role of asbestos bodies (and associated proteinacious coating) in asbestos associated diseases is not well understood. Currently employed methods of isolation of these bodies employ harsh chemicals that lead to destruction of their proteinacious coating. In this work a method was developed that enabled the purification of whole, integral, unmodified asbestos bodies (AB) by exploiting their magnetic properties. Albumin and ferritin were found to be the major proteins associated with AB isolated from lung tissue of mesothelioma patients. Magnetically isolated AB were shown to be cytotoxic and to activate free radical production from inflammatory cells at a higher extent than that induced by bodies obtained by chemical digestion. The finding that hypochlorite-treated AB induce DNA damage, while AB obtained by the method described in this article failed to do so, together with the differential behavior of these bodies toward inflammatory cells, suggests that native asbestos bodies should be used to investigate the pathogenetic role of these structures.

The role of granulocytes following intravesical BCG prophylaxis.

OBJECTIVES: The antitumour effect of bacillus Calmette-Guérin (BCG) still remains relatively undefined. Most investigations on its mechanism of action have focused on mononuclear cells; little consideration has been given to granulocytes. We analysed urine of patients with bladder cancer during 8 wk of intravesical BCG prophylaxis. The number of polymorphonuclear neutrophils (PMNs) and urothelial cells (UCs) was evaluated. We examined the in vitro response of the T24 UC line to human PMNs after BCG treatment. METHODS: Seventeen patients were enrolled in the study. Cytologic analyses were performed on urine samples collected before each BCG instillation and after 2 h from the first voided urine after BCG instillation. Elastase activity was determined on these samples to evaluate PMN activation. PMN-induced damage was measured on the T24 cell line treated with BCG. RESULTS: After BCG treatment, a large number of PMNs transmigrated through the urothelium and PMNs adherent to detached UCs were found. One patient, who did not respond with significant PMN transmigration, experienced recurrent disease. The number of eosinophils that transmigrated was low, with the exception of three patients with recurrent disease. In vitro, PMNs adhered to BCG-primed T24 cells and damaged the monolayer. CONCLUSIONS: The results agree with recent evidence that PMNs may play an important role in the antitumour action of BCG during the BCG induction period. This role is probably nonspecific because both normal UCs in vivo and tumour cells in vitro appeared to be injured. As suggested by results obtained from a limited number of patients, a high number of eosinophils in the urine may indicate therapy failure.