Cell migration plays important roles in embryonic development and inflammation, and this process is highly regulated to ensure tissue homeostasis. A number of barriers exist to prevent the inappropriate migration of leukocytes into healthy peripheral tissues, including retention of these cells in the inactive state and maintenance of the integrity and charge of the vascular endothelium. However, active signals also are likely to exist that can repulse cells or abolish existing cell migration. One such paradigm exists in the developing nervous system, where neuronal migration is mediated by a balance between chemoattractive and chemorepulsive signals. The ability of the guidance molecule netrin-1 to repulse or abolish attraction of neuronal cells expressing the UNC5b receptor makes it an attractive candidate for the regulation of inflammatory cell migration. Here, we show that netrin-1 is expressed on vascular endothelium, where it is regulated by infection and inflammatory cytokines. The netrin-1 receptor UNC5b is strongly expressed by leukocytes, upon which netrin-1 acts as a potent inhibitor of migration to different chemotactic stimuli both in vivo and in vitro. These data suggest that endothelial expression of netrin-1 may inhibit basal cell migration into tissues and that its down-regulation with the onset of sepsis͞inflammation may facilitate leukocyte recruitment.chemotaxis ͉ inflammation ͉ guidance cue

Aims Rosuvastatin and pitavastatin have been proposed as probe substrates for the organic anion‐transporting polypeptide (OATP) 1B, but clinical data on their relative sensitivity and selectivity to OATP1B inhibitors are lacking. A clinical study was therefore conducted to determine their relative suitability as OATP1B probes using single oral (PO) and intravenous (IV) doses of the OATP1B inhibitor rifampicin, accompanied by a comprehensive in vitro assessment of rifampicin inhibitory potential on statin transporters. Methods The clinical study comprised of two separate panels of eight healthy subjects. In each panel, subjects were randomized to receive a single oral dose of rosuvastatin (5 mg) or pitavastatin (1 mg) administered alone, concomitantly with rifampicin (600 mg) PO or IV. The in vitro transporter studies were performed using hepatocytes and recombinant expression systems. Results Rifampicin markedly increased exposures of both statins, with greater differential increases after PO vs. IV rifampicin only for rosuvastatin. The magnitudes of the increases in area under the plasma concentration–time curve were 5.7‐ and 7.6‐fold for pitavastatin and 4.4‐ and 3.3‐fold for rosuvastatin, after PO and IV rifampicin, respectively. In vitro studies showed that rifampicin was an inhibitor of OATP1B1 and OATP1B3, breast cancer resistance protein and multidrug resistance protein 2, but not of organic anion transporter 3. Conclusions The results indicate that pitavastatin is a more sensitive and selective and thus preferred clinical OATP1B probe substrate than rosuvastatin, and that a single IV dose of rifampicin is a more selective OATP1B inhibitor than a PO dose.

The transcriptional profiles of yeast cells that have been phagocytosed by either human neutrophils or monocytes were compared by using whole genome arrays. After phagocytosis by neutrophils, both Saccharomyces cerevisiae and Candida albicans respond by inducing genes of the methionine and arginine pathways. Neither of these pathways is induced upon phagocytosis by monocytes. Both fungi show a similar induction of these pathways when transferred from amino acid-rich medium to amino acid-deficient medium. These data suggest that the internal phagosome of the neutrophil is an amino acid-deficient environment. Ingestion of a microorganism by mammalian cells exposes that organism to a novel environment. Transcriptional profiling of a microorganism with a well-annotated genome sequence offers a unique window into the response of that organism to phagocytosis. Used in this way, the microorganism becomes a functional bioprobe of the phagocyte's microenvironment. For example, upon ingestion by macrophages Saccharomyces cerevisiae undergoes a key metabolic shift: the induction of the glyoxylate shunt (1). This pathway is also induced upon ingestion of bacteria (Mycobacterium tuberculosis) (2) and other fungi [Cryptococcus neoformans (3), Candida albicans (1), and Leptosphaeria maculans (4)]. This shift in metabolism has been interpreted as a response to the glucose-poor environment of the macrophage, and the ability to make that shift appears to contribute to the virulence of some pathogens.In this report we use transcriptional profiling of S. cerevisiae to analyze the phagosomal microenvironment of the human neutrophil. The yeast transcriptional response is dominated by induction of the methionine and arginine amino acid biosynthetic genes. This transcriptional response could be mimicked in vitro by transferring yeast from an amino acid-rich environment to one deficient in amino acids. The response of Saccharomyces to neutrophils is distinct from that to monocytes, which do not induce these amino acid pathways. The human pathogen C. albicans also induces the transcription of a similar set of amino acid biosynthetic genes upon exposure to human neutrophils. These data suggest that the human neutrophil phagosome is amino acid-deficient, and indicate an amino acid-deprivation response that is conserved between S. cerevisiae and C. albicans. Materials and MethodsIsolation of Neutrophils and Monocytes and Human Serum Preparation. Using Histopaque 1077 and Histopaque 1119 (Sigma) per manufacturer's instructions, we isolated neutrophils and monocytes from fresh human blood collected from healthy volunteers in accordance with a protocol approved by the Massachusetts Institute of Technology Committee on Use of Humans as Experimental Subjects. Monocytes were separated from lymphocytes on the basis of their differential adherence to plastic. Human serum was prepared from the same blood donors by using Vacutainer SST gel-containing tubes (Becton Dickinson) and following the manufacturer's protocol. Where indicated, human serum was ...

Macrophages interact with other cells and components of the extracellular environment by means of adhesion receptors. Adhesion to artificial substrata in vitro facilitates isolation of macrophages, and has been used to generate antibodies that inhibit their migration in vivo. Unlike other cell types, macrophages attach to tissue culture plastic in the absence of divalent cations. Here we use an adhesion assay exploiting this property to isolate a rat monoclonal antibody, 2F8, which totally inhibits divalent cation-independent adhesion of murine macrophages to tissue culture plastic in the presence of fetal calf serum. Immunoprecipitation from macrophages and stably transfected Chinese hamster ovary cells revealed that the antigen recognized by monoclonal 2F8 is identical to murine macrophage scavenger receptor. We propose a novel function for this molecule, previously described as an endocytic receptor, thus providing a mechanism for mononuclear phagocyte recruitment to and retention in ligand-rich tissues such as in atherosclerotic lesions.

ABSTRACT

Macrophage scavenger receptors are trimeric integral membrane glycoproteins which have been implicated in various macrophage functions including uptake of oxidized lipoprotein and the serum-dependent, divalent cation-independent adhesion of macrophages to tissue culture-treated plastic. In this study we have used a recently defined monoclonal antibody (2F8) which recognizes murine macrophage scavenger receptor, to explore its expression in lymphoid and non-lymphoid organs of the normal adult. Scavenger receptor was detected in the red pulp and marginal zone of normal adult mouse spleen, medulla of the thymus and subcapsular region of lymph nodes. Kupffer cells in the liver, alveolar macrophages in the lung and lamina propria macrophages in the gut all reacted with 2F8 monoclonal antibody. The antigen was not detected on any non-macrophage cells, with the exception of sinusoidal endothelial cells in the liver. In the spleen, lymph node and liver, scavenger receptor antigen expression was associated specifically with phagocytic cells which had taken up colloidal carbon. To examine macrophage adhesion in a context relevant to the interactions occurring within lymphoid and non-lymphoid organs, and the contribution of macrophage scavenger receptor to this adhesion, we designed an assay of macrophage adhesion to frozen tissue sections. Adhesion to most tissues was high and uniform in the absence of any chelating agents. The chelation of Ca2+ and Mg2+ revealed specific patterns of macrophage adhesion in lymphoid and non-lymphoid organs which was completely inhibited by 2F8. The ability of this antibody to block the EDTA-resistant adhesion correlated with tissue expression of the antigen in some tissues. Unlike adhesion to tissue culture-treated plastic, macrophage scavenger receptor-dependent adhesion of macrophages to frozen tissue sections did not exhibit an absolute requirement for exogenous fetal bovine serum indicating the presence of an endogenous ligand for scavenger receptor within the tissues. We propose that macrophage scavenger receptor is a candidate homing or retention molecule for macrophage localization within ligand-rich tissues.

A microdose cocktail containing midazolam, dabigatran etexilate, pitavastatin, rosuvastatin, and atorvastatin has been established to allow simultaneous assessment of a perpetrator impact on the most common drug metabolizing enzyme, cytochrome P450 (CYP)3A, and the major transporters organic anion-transporting polypeptides (OATP)1B, breast cancer resistance protein (BCRP), and MDR1 P-glycoprotein (P-gp). The clinical utility of these microdose cocktail probe substrates was qualified by conducting clinical drug interaction studies with three inhibitors with different in vitro inhibitory profiles (rifampin, itraconazole, and clarithromycin). Generally, the pharmacokinetic profiles of the probe substrates, in the absence and presence of the inhibitors, were comparable to their reported corresponding pharmacological doses, and/or in agreement with theoretical expectations. The exception was dabigatran, which resulted in an approximately twofold higher magnitude for microdose compared to conventional dosing, and, thus, can be used to flag a worst-case scenario for P-gp. Broader application of the microdose cocktail will facilitate a more comprehensive understanding of the roles of drug transporters in drug disposition and drug interactions.

Innate immune responses to bacteria require cooperative interactions between host recognition molecules and phagocytes. The peptidoglycan recognition proteins (PGRPs) are a large group of proteins found in insects and mammals that bind to bacterial peptidoglycan (PGN). PGRP-S is located with other antimicrobial proteins, such as lysozyme, in the granules of human neutrophils. Whereas both PGRP-S and lysozyme recognize PGN, the exact binding specificity of human PGRP-S, its functional activity, and its potential synergy with other neutrophilderived bactericidal proteins such as lysozyme have not been determined. Here we show that human PGRP-S binds to and inhibits the growth of Staphylococcus aureus (containing lysine-type PGN) and Escherichia coli (containing mesodiaminopimelic acid-type PGN). The binding affinity and thus antimicrobial activity of PGRP-S is determined by the third amino acid in the PGN stem peptide. Furthermore, the antimicrobial effect of PGRP-S against E coli is synergistic with lysozyme, and lysozyme and PGRP-S colocalize in neutrophil extracellular traps (NETs), suggesting that these granulederived proteins act together to kill bacteria trapped in the NETs. Taken IntroductionThe innate immune system is a host defense mechanism, evolutionarily conserved from insects to humans, that mediates recognition and control of invading microorganisms. 1,2 The basis of innate immune response lies in the ability of the host to recognize conserved products of microbial metabolism that are unique to microorganisms and are not produced by the host. The best known examples of such molecules, called pathogen-associated molecular patterns (PAMPs), include lipopolysaccharide (LPS) of Gramnegative bacteria, DNA sequences containing unmethylated CpG dinucleotides (CpG DNA), and peptidoglycan (PGN) present in Gram-positive and Gram-negative bacteria. 3 PGN recognition proteins (PGRPs) are a family of patternrecognition receptors (PRRs) that bind to, and in some cases hydrolyze, PGNs of bacterial cell walls. [4][5][6][7][8][9][10] These molecules are highly conserved from insects to mammals, and all share a conserved 160-amino acid domain (the PGRP domain) with notable sequence similarity to N-acetylmuramyl-L-alanine amidases. 11 The essential role of PGRPs in the Drosophila immune response is revealed by characterization of immunodeficient mutants. Two genes that encode PGRPs, PGRP-SA and PGRP-LC, are required in 2 different branches of the immune response. PGRP-SA is a circulating PGRP through which Grampositive organisms activate the Toll pathway, 12 whereas PGRP-LC is a transmembrane PGRP through which Gram-negative organisms activate the Imd/Relish pathway. [13][14][15] Activation of these pathways leads to the production of antimicrobial peptides and to the activation of the phenoloxidase-melanin cascade. A recent study has determined that different forms of PGN (lysine type vs meso-diaminopimelic acid type) confer this Gram-positive versus Gram-negative specificity. 16 Therefore, Drosophila PGRPs mediate d...