Amphitrite ornata dehaloperoxidase (DHP) is a heme enzyme with a globin structure, which is capable of oxidizing para-halogenated phenols to the corresponding quinones. Cloning, high-level expression, and purification of recombinant DHP are described. Recombinant DHP was assayed by stopped-flow experiments for its ability to oxidatively debrominate 2,4,6-tribromophenol (TBP). The enzymatic activity of the ferric form of recombinant DHP is intermediate between that of a typical peroxidase (horseradish peroxidase) and a typical globin (horse heart myoglobin). The present study shows that, unlike other known peroxidases, DHP activity requires the addition of substrate, TBP, prior to the cosubstrate, peroxide. The presence of a substrate-binding site in DHP is consistent with a two-electron oxidation mechanism and an obligatory order for activation of the enzyme by addition of the substrate prior to the cosubstrate.

Cells of the mononuclear phagocytic system including monocytes and macrophages (e.g., pooled human monocytes, bone marrow-derived macrophages, etc.) are often employed for in vitro assessment of novel biomaterials and to assay anti-inflammatory drug activity. In this context, numerous macrophage cells are treated interchangeably in the literature despite a lack of demonstrated equivalence among immortalized cell lines and further, between cell lines and primary-derived macrophages of different species. Three murine (monocyte-) macrophage cell lines (IC-21, J774A.1, and RAW 264.7), commonly utilizedin biomaterial and pharmaceutical screening research, have been compared with primary-derived murine bone marrow macrophages. Significant differences were discovered in the expression of cell surface proteins requisite for cell adhesion and activation among cell lines and primary-derived cells as well as between the different cell lines. Results demonstrate activation but with reduced cytokine expression to chemical stimulus (lipopolysaccharide) by cell lines compared with that of primary-derived macrophages. Limited correlation between cultured primary and immortalized cells in cytokine production, phenotype and intrinsic activation states has relevance to fidelity for in vitro testing. These differences warrant justification for selection of various cell lines for specific assay purposes, and merit caution if comparisons to primary cell types (i.e., for biocompatibility) are required.

Plasma-polymerized films deposited from AlAm, HxAm, NVP, NVFA, AA and FC were compared to TCPS and PS surfaces in supporting cellular attachment, viability, and proliferation in serum-based culture in vitro for extended periods of time (>7 d). Surface patterns were created using multi-step depositions with physical masks. Cell adhesion in the presence of serum was compared for (monocyte-) macrophage and fibroblast cell lines. Cellular response was tracked over time, reporting adhesive behavior, proliferative rates, and morphological changes as a function of surface chemistry. Micropatterned surfaces containing different surface chemistries and functional groups (e.g. -NH(2), -COOH, -CF(3)) produced differential cell adhesive patterns for NIH 3T3 fibroblasts compared to J774A.1, RAW 264.7 or IC-21 (monocyte-) macrophage cell types. Significantly, macrophage adhesion is substantial on surfaces where fibroblasts do not adhere under identical culture conditions.

The Rho GTPase cellular signaling cascade was investigated in pro-monocyte and (monocyte-) macrophage cells by examining GTPase expression and activation in serum-containing cultures on model biomaterials. Abundance of Rho GDI and the Rho GTPase proteins RhoA, Cdc42 and Rac1 was determined in cells grown on tissue culture polystyrene, polystyrene, poly-L-lactide and Teflon ® AF surfaces. Protein expression was compared based on cell maturity (pro-monocyte to monocyte to macrophage lineages) and by model surface chemistry: Rho proteins were present in the majority of macrophage cells tested on model surfaces suggesting that a pool of Rho proteins is readily available for signaling events in response to numerous activating cues, including biomaterials surface encounter. Rho GTPase activation profiles in these cell lines indicate active Cdc42 and Rho proteins in RAW 264.7, Rac1 and Rho in J774A.1, and Cdc42 and Rac1 in IC-21 cell lines, respectively. Collectively, these proteins are known to play critical roles in all actin-based cytoskeletal rearrangement necessary for cell adhesion, spreading and motility, and remain important to establishing cellular responses required for foreign body reactions in vivo. Differences in Rho GTPase protein expression levels based on cell sourcing (primary versus secondary-derived cell source), or as a function of surface chemistry were insignificant. Rho GTPase expression profiles varied between pro-monocytic non-adherent precursor cells and mature adherent monocyte/ macrophage cells. The active GTP-bound forms of the Rho GTPase proteins were detected from monocyte-macrophage cell lines RAW 264.7 and J774A.1 on all polymer surfaces, suggesting that while these proteins are central to cell adhesive behavior, differences in surface chemistry are insufficient to differentially regulate GTPase activation in these cell types. Active Cdc42 was detected from cells cultured on the more-polar tissue culture polystyrene and poly-L-lactide surfaces after several days, but absent from those grown on apolar polystyrene and Teflon ® AF, indicating some surface influence on this GTPase in serum-containing cultures.

Monocyte/macrophage adhesion to biomaterials, correlated with foreign body response, occurs through protein-mediated surface interactions. Albumin-selective perfluorocarbon (FC) biomaterials are generally poorly cell-conducive due to insufficient receptor-mediated surface interactions, but macrophages bind to albumin-coated substrates and also preferentially to highly hydrophobic fluorinated surfaces. Bone marrow macrophages (BMMO) and IC-21, RAW 264.7 and J774A.1 monocyte/macrophage cells were cultured on FC surfaces. Protein deposition onto two distinct FC surfaces from complex and single-component solutions was tracked using fluorescence and time-of-flight secondary ion mass spectrometry (ToF-SIMS) methods. Cell adhesion and growth on protein pre-treated substrates were compared by light microscopy. Flow cytometry and integrin-directed antibody receptor blocking assessed integrins critical for monocyte/macrophage adhesion in vitro. Albumin predominantly adsorbs onto both FC surfaces from 10% serum. In cultures pre-adsorbed with albumin or serum-dilutions, BMMO responded similar to IC-21 at early time points. Compared to Teflon® AF, plasma-polymerized FC was less permissive to extended cell proliferation. The β2 integrins play major roles in macrophage adhesion to FC surfaces: antibody blocking significantly disrupted cell adhesion. Albumin-mediated cell adhesion mechanisms to FC surfaces could not be clarified. Primary BMMO and secondary IC-21 macrophages behave similarly on FC surfaces, regardless of pre-adsorbed protein biasing, with respect to adhesion, cell morphology, motility and proliferation.