RP 67580, a potent and selective substance P non-peptide antagonist]

The pharmacological properties of 7,7-Diphenyl-2 [1-imino-2 (2-methoxy-phenyl)-ethyl] perhydroisoindol-4-one (3 aR, 7 aR) or RP67580 are described. This compound, derived from a novel chemical family, is a potent and selective substance P (SP) antagonist, in vitro and in vivo. In vitro, it inhibited in a competitive manner (IC50 = 10 nM) 3H-SP binding in rat brain (NK1 receptors). It did not interact with the two other tachykinin receptor sites (NK2 and NK3) nor the other receptor sites tested. Moreover, RP67580 competitively antagonized the contractile activity of SP on guinea-pig ileum (pA2 = 7.16); in contrast, it was inactive in rabbit pulmonary artery and in rat portal vein tissues which contain NK2 and NK3 receptors, respectively. In vivo, in the rat, RP67580 inhibited the plasmatic extravasation induced by administration of SP (ED50 = 0.04 mg/kg i.v.) as well as that induced by antidromic stimulation of a peripheral sensory nerve (ED50 = 0.15 mg/kg i.v.). In mice and rats, RP67580, like morphine, potently blocked the nociceptive effects of phenylbenzoquinone and formalin; its antinociceptive effect does not involve opiate receptors since it was not reversed by naloxone. These results indicate that RP67580 is a particularly valuable tool for investigating the physiological and pathological role of SP.     

Mechanisms Controlling Cell Size and Shape during Isotropic Cell Spreading

Cell motility is important for many developmental and physiological processes. Motility arises from interactions between physical forces at the cell surface membrane and the biochemical reactions that control the actin cytoskeleton. To computationally analyze how these factors interact, we built a three-dimensional stochastic model of the experimentally observed isotropic spreading phase of mammalian fibroblasts. The multiscale model is composed at the microscopic levels of three actin filament remodeling reactions that occur stochastically in space and time, and these reactions are regulated by the membrane forces due to membrane surface resistance (load) and bending energy. The macroscopic output of the model (isotropic spreading of the whole cell) occurs due to the movement of the leading edge, resulting solely from membrane force-constrained biochemical reactions. Numerical simulations indicate that our model qualitatively captures the experimentally observed isotropic cell-spreading behavior. The model predicts that increasing the capping protein concentration will lead to a proportional decrease in the spread radius of the cell. This prediction was experimentally confirmed with the use of Cytochalasin D, which caps growing actin filaments. Similarly, the predicted effect of actin monomer concentration was experimentally verified by using Latrunculin A. Parameter variation analyses indicate that membrane physical forces control cell shape during spreading, whereas the biochemical reactions underlying actin cytoskeleton dynamics control cell size (i.e., the rate of spreading). Thus, during cell spreading, a balance between the biochemical and biophysical properties determines the cell size and shape. These mechanistic insights can provide a format for understanding how force and chemical signals together modulate cellular regulatory networks to control cell motility.     

Bone marrow-infiltrating human neuroblastoma cells express high levels of calprotectin and HLA-G proteins

Metastases in the bone marrow (BM) are grim prognostic factors in patients with neuroblastoma (NB). In spite of extensive analysis of primary tumor cells from high- and low-risk NB patients, a characterization of freshly isolated BM-infiltrating metastatic NB cells is still lacking. Our aim was to identify proteins specifically expressed by metastatic NB cells, that may be relevant for prognostic and therapeutic purposes. Sixty-six Italian children over 18 months of age, diagnosed with stage 4 NB, were included in the study. Metastatic NB cells were freshly isolated from patients' BM by positive immunomagnetic bead manipulation using anti-GD2 monoclonal antibody. Gene expression profiles were compared with those obtained from archived NB primary tumors from patients with 5 y-follow-up. After validation by RT-qPCR, expression/secretion of the proteins encoded by the up-regulated genes in the BM-infiltrating NB cells was evaluated by flow cytometry and ELISA. Compared to primary tumor cells, BM-infiltrating NB cells down-modulated the expression of CX3CL1, AGT, ATP1A2 mRNAs, whereas they up-regulated several genes commonly expressed by various lineages of BM resident cells. BM-infiltrating NB cells expressed indeed the proteins encoded by the top-ranked genes, S100A8 and A9 (calprotectin), CD177 and CD3, and secreted the CXCL7 chemokine. BM-infiltrating NB cells also expressed CD271 and HLA-G. We have identified proteins specifically expressed by BM-infiltrating NB cells. Among them, calprotectin, a potent inflammatory protein, and HLA-G, endowed with tolerogenic properties facilitating tumor escape from host immune response, may represent novel biomarkers and/or targets for therapeutic intervention in high-risk NB patients.     

Remodeling the zonula adherens in response to tension and the role of afadin in this response

Morphogenesis requires dynamic coordination between cell–cell adhesion and the cytoskeleton to allow cells to change shape and move without losing tissue integrity. We used genetic tools and superresolution microscopy in a simple model epithelial cell line to define how the molecular architecture of cell–cell zonula adherens (ZA) is modified in response to elevated contractility, and how these cells maintain tissue integrity. We previously found that depleting zonula occludens 1 (ZO-1) family proteins in MDCK cells induces a highly organized contractile actomyosin array at the ZA. We find that ZO knockdown elevates contractility via a Shroom3/Rho-associated, coiled-coil containing protein kinase (ROCK) pathway. Our data suggest that each bicellular border is an independent contractile unit, with actin cables anchored end-on to cadherin complexes at tricellular junctions. Cells respond to elevated contractility by increasing junctional afadin. Although ZO/afadin knockdown did not prevent contractile array assembly, it dramatically altered cell shape and barrier function in response to elevated contractility. We propose that afadin acts as a robust protein scaffold that maintains ZA architecture at tricellular junctions.     

Pneumocystis carinii enhances soluble mannose receptor production by macrophages

Phagocytosis of extracellular organisms in the alveolar spaces of the lungs represents the first-line of host defense against pulmonary pathogens. Disruption of this process is likely to interfere with the generation of appropriate specific immune responses, and lead to a delayed or inefficient clearance of the pathogen. Pneumocystis carinii, an opportunistic pathogen in immunodeficient individuals, is cleared from the lung by alveolar macrophages. In the absence of specific anti-Pneumocystis antibodies, phagocytosis is dependent on the non-opsonic macrophage mannose receptor (MR). Recent studies have demonstrated that alveolar macrophage MR activity is downregulated in individuals infected with HIV, and that functional MR is shed from the macrophage cell surface. Here we report that P. carinii enhances the formation of soluble MR by macrophages in vitro. Soluble MR was detected in cell-free alveolar fluid from humans infected with HIV and/or P. carinii, but not in alveolar fluid from healthy controls. Soluble MR was found in association with extracellular clumps of P. carinii in the lungs of mice with P. carinii pneumonia, and was associated with P. carinii organisms purified from these mice. When purified P. carinii organisms were incubated with soluble MR-containing supernatants, they were phagocytosed less readily by alveolar macrophages than were control organisms. Our results suggest that P. carinii organisms enhance the shedding of MR from the surface of alveolar macrophages, and that the resultant soluble MR binds to intra-alveolar organisms, thereby interfering with their non-opsonic uptake via the macrophage cell surface MR.