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1.  [No title available] 
This Round Table will host an open discussion about NIH support for shared resource facilities. NIH recently implemented a reorganization plan that eliminated the National Center for Research Resources (NCRR). Programs formerly administered by NCRR have been reassigned to other NIH Institutes and Centers. The Shared Instrumentation Grant (SIG) and High End Instrumentation (HEI) programs are now part of the NIH Office of the Director in DPCPSI, directed by James M. Anderson, MD, PhD. Dr. Anderson will present an overview of the role of his division and of NIH in supporting shared resource facilities. Before joining NIH, Dr. Anderson was Chair of the Department of Cell and Molecular Physiology in the School of Medicine of the University of North Carolina at Chapel Hill. He was responsible for the operation and management of a number of research core labs at UNC. He is a new voice of support for shared resource facilities that support NIH research. Abraham Levy, PhD, is the newly appointed Program Director responsible for the SIG and HEI programs. Dr. Levy worked for Dr. Marjorie Tingle in the NCRR SIG office for many years. He is highly qualified to be the new director of these important programs. Dr. Levy will discuss how SIG and HEI will be administered in their new home in DPCPSI. Time will be allowed for questions and discussion following the presentations of Drs. Anderson and Levy.
PMCID: PMC3630669
2.  Instability of Self-Assembled Monolayers (SAM) as a Model Material System for Macrophage/FBGC Cellular Behavior 
Novel self-assembled monolayers (SAMs) designed to present homogenous surface chemistries were utilized to further investigate the material surface chemistry dependent macrophage and foreign body giant cell (FBGC) behaviors including macrophage adhesion, fusion, and apoptosis. Contact angle analysis revealed instabilities in the –CH3 and –COOH terminate SAM surfaces upon incubation in serum-free media at 37oC or under dry, room temperatureconditions. Further analysis indicated that the –CH3 terminated SAM surface degraded rapidly within 2 hours and loss of sufficient SAM units to be comparable to the gold (Au) control surface within 24 hours of incubation in serum-free media (SFM) at 37oC. After 5days of incubation in SFM at 37oC, the contact angles for the –COOH terminated SAMsurfaces increased markedly. AFM analysis confirmed the desorption of –CH3 terminated SAM molecules from the surface with increased roughness and marked appearance of peaks andvalleys within 2 hours. A decrease in the thickness of the –COOH terminated SAM surface also suggests molecular desorption over time. No significant changes in contact angle or AFM analyses were observed on the –OH terminated SAM surfaces. Cellular adhesion decreased morerapidly on the Au control and –CH3 terminated SAM surfaces in comparison to the other surfaces. However by day 10, cellular adhesion, fusion, and apoptosis were comparable on all SAM surfaces and the Au control . These studies suggest that SAM surfaces may not be suitable for long-term studies where material dependent properties are investigated.
PMCID: PMC4017672  PMID: 18412136
self-assembled monolayers; instability; macrophage; foreign body giant cell; contact angles
3.  Lymphocyte/Macrophage Interactions: Biomaterial Surface-Dependent Cytokine, Chemokine, and Matrix Protein Production 
Journal of biomedical materials research. Part A  2008;87(3):10.1002/jbm.a.31630.
The role of lymphocytes in the biological response to synthetic polymers is poorly understood despite the transient appearance of lymphocytes at the biomaterial implant site. To investigate cytokines, chemokines, and extracellular matrix (ECM) proteins produced by lymphocytes and macrophages in response to biomaterial surfaces, human peripheral blood monocytes and lymphocytes were co-cultured on polyethylene terephthalate (PET)-based material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic chemistries. Antibody array screening showed the majority of detected proteins are inflammatory mediators that guide the early inflammatory phases of wound healing. Proteomic ELISA quantification and adherent cell analysis were performed after 3, 7, and 10 days of culture. IL-2 and IFN-γ were not detected in any co-cultures suggesting lack of lymphocyte activation. The hydrophilic/neutral surfaces increased IL-8 relative to the hydrophobic PET surface (p<0.05). The hydrophilic/anionic surfaces promoted increased TNF-α over hydrophobic and cationic surfaces and increased MIP-1β compared to hydrophobic surfaces (p<0.05). Since enhanced macrophage fusion was observed on hydrophilic/anionic surfaces, the production of these cytokines likely plays an important role in the fusion process. The hydrophilic/cationic surface promoted IL-10 production and increased matrix metalloproteinase (MMP)-9/tissue inhibitor of MMP (TIMP) relative to hydrophilic/neutral and anionic surfaces (p<0.05). These results suggest hydrophilic/neutral and anionic surfaces promote pro-inflammatory responses and reduced degradation of the ECM, whereas the hydrophilic/cationic surfaces induce an anti-inflammatory response and greater MMP-9/TIMP with an enhanced potential for ECM breakdown. The study also underscores the usefulness of protein arrays in assessing the role of soluble mediators in the inflammatory response to biomaterials.
PMCID: PMC3867010  PMID: 18200554
PET biomaterials; lymphocytes; macrophages; cytokines; matrix metalloproteinases
4.  The Foreign Body Reaction in T cell deficient mice 
Journal of biomedical materials research. Part A  2009;90(1):10.1002/jbm.a.32050.
The role/s of T lymphocytes in the foreign body response has not been thoroughly elucidated. Lymphocytes are known to augment macrophage adhesion and fusion in vitro. Furthermore T lymphocytes are a possible source of the cytokines, IL-4 and IL-13 which induce macrophage fusion. In this study we used BALB/c mice and BALB/c (nu/nu) nude mice to investgate foreign body giant cell (FBGC) formation in a T cell deficient setting. Mice were implanted with Elasthane 80A (PEU), silicone rubber (SR) or poly(ethylene terephthalate) (PET) for 7, 14, or 21 days using the cage implant system. Exudate cells and IL-4 and IL-13 levels in exudate supernatants were analyzed by flow cytometry and a multiplex immunoassay, respectively, at days 7, 14, and 21. Macrophage adhesion and fusion on material surfaces were analyzed using optical microscopy. T cell deficient mice had lower total leukocyte concentrations at the biomaterial implant site at all time points. Adherent cell density was comparable between normal and T cell deficient mice except in the PEU group at day 21. However, percent fusion, average nuclei per FBGC, and FBGC morphology was comparable between normal and T cell deficient mice. IL-4 was not detected in any samples, but IL-13 levels were also comparable between normal and T cell deficient mice indicating Th2 polarized T cells are not the sole source of this cytokine. We have shown that there are pathways that do not require thymus-matured T lymphocytes which lead to a normal foreign body response to biomaterials in a murine model.
PMCID: PMC3864678  PMID: 18491378
foreign body response; IL-4; IL-13; T lymphocyte; biomaterials
5.  Paracrine and Juxtacrine Lymphocyte Enhancement of Adherent Macrophage and Foreign Body Giant Cell Activation 
Journal of biomedical materials research. Part A  2009;89(2):10.1002/jbm.a.31981.
Lymphocytes have been shown to be involved in modulating monocyte and macrophage behavior in the foreign body reaction. Lymphocyte effects on biomaterial-adherent macrophage and foreign body giant cell (FBGC) behavior were further investigated by culturing monocytes alone or together with lymphocytes, either in direct co-cultures or indirectly in transwells, on a series of polyethylene terephthalate (PET)-based photograft co-polymerized material surfaces displaying distinct hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic chemistries. After periods of 3, 7, and 10 days, cytokine production was quantified by ELISA and normalized to adherent macrophage/FBGC density to yield a measure of adherent macrophage/FBGC activation. Interactions with lymphocytes enhanced adherent macrophage and FBGC production of pro-inflammatory IL-1β, TNF-α, IL-6, IL-8, and MIP-1β on the hydrophobic and hydrophilic/cationic surfaces but had no effect on anti-inflammatory IL-10 production indicating lymphocytes promote a pro-inflammatory response to biomaterials. Lymphocytes also did not significantly influence MMP-9, TIMP-1, and TIMP-2 production. Interactions through indirect (paracrine) signaling showed a significant effect in enhancing adherent macrophage/FBGC activation at early time points while interactions via direct (juxtacrine) mechanisms dominated at later time points. Biomaterial surface chemistries differentially affected the observed responses as hydrophilic/neutral and hydrophilic/anionic surfaces evoked the highest levels of activation relative to the other surfaces but did not facilitate lymphocyte enhancement of adherent macrophage/FBGC activation.
PMCID: PMC3864690  PMID: 18437695
biomaterials; foreign body reaction; lymphocytes; macrophages; activation
6.  Quantitative in vivo Cytokine Analysis at Synthetic Biomaterial Implant Sites 
Journal of biomedical materials research. Part A  2009;89(1):10.1002/jbm.a.31939.
In order to further elucidate the foreign body reaction, investigation of cytokines at biomaterial implant sites was carried out using a multiplex immunoassay and ELISA. Macrophage activation cytokines (IL-1β, IL-6, TNFα), cytokines important for macrophage fusion (IL-4, IL-13), anti-inflammatory cytokines (IL-10, TGFβ), chemokines (GRO/KC, MCP-1), and the T cell activation cytokine IL-2 were quantified at biomaterial implant sites. Empty cages (controls) or cages containing synthetic biomedical polymer (Elasthane 80A (PEU), Silicone rubber (SR), or polyethylene terephthalate (PET)) were implanted subcutaneously in Sprague Dawley rats for 4, 7, or 14 days, and cytokines in exudate supernatants and macrophage surface adhesion and fusion were quantified. The presence of a polymer implant did not affect the levels of IL-1β, TGFβ, and MCP-1 in comparison to the control group. IL-2 was not detected in virtually any of the samples. Although the levels of IL-4, IL-13, IL-10, and GRO/KC were affected by polymer implantation, but not dependent on a specific polymer, IL-6 and TNFα were significantly greater in those animals implanted with PEU and SR, materials that do not promote fusion. The results indicate differential material dependent cytokine profiles are produced by surface adherent macrophages and foreign body giant cells in vivo.
PMCID: PMC3864694  PMID: 18431759
cage implant system; multiplex immunoassay; cytokines; synthetic biomaterials
7.  Quality Improvement Initiative to Reduce Serious Safety Events and Improve Patient Safety Culture 
Pediatrics  2012;130(2):e423-e431.
Many thousands of patients die every year in the United States as a result of serious and largely preventable safety events or medical errors. Safety events are common in hospitalized children. We conducted a quality improvement initiative to implement cultural and system changes with the goal of reducing serious safety events (SSEs) by 80% within 4 years at our large, urban pediatric hospital.
A multidisciplinary SSE reduction team reviewed the safety literature, examined recent SSEs, interviewed internal leaders, and visited other leading organizations. Senior hospital leaders provided oversight, monitored progress, and helped to overcome barriers. Interventions focused on: (1) error prevention; (2) restructuring patient safety governance; (3) a new root cause analysis process and a common cause database; (4) a highly visible lessons learned program; and (5) specific tactical interventions for high-risk areas. Our outcome measures were the rate of SSEs and the change in patient safety culture.
SSEs per 10 000 adjusted patient-days decreased from a mean of 0.9 at baseline to 0.3 (P < .0001). The days between SSEs increased from a mean of 19.4 at baseline to 55.2 (P < .0001). After a worsening of patient safety culture outcomes in the first year of intervention, significant improvements were observed between 2007 and 2009.
Our multifaceted approach was associated with a significant and sustained reduction of SSEs and improvements in patient safety culture. Multisite studies are needed to better understand contextual factors and the significance of specific interventions.
PMCID: PMC3408689  PMID: 22802607
patient safety; quality improvement; safety culture
8.  Growth and Maximum Size of Tiger Sharks (Galeocerdo cuvier) in Hawaii 
PLoS ONE  2014;9(1):e84799.
Tiger sharks (Galecerdo cuvier) are apex predators characterized by their broad diet, large size and rapid growth. Tiger shark maximum size is typically between 380 & 450 cm Total Length (TL), with a few individuals reaching 550 cm TL, but the maximum size of tiger sharks in Hawaii waters remains uncertain. A previous study suggested tiger sharks grow rather slowly in Hawaii compared to other regions, but this may have been an artifact of the method used to estimate growth (unvalidated vertebral ring counts) compounded by small sample size and narrow size range. Since 1993, the University of Hawaii has conducted a research program aimed at elucidating tiger shark biology, and to date 420 tiger sharks have been tagged and 50 recaptured. All recaptures were from Hawaii except a single shark recaptured off Isla Jacques Cousteau (24°13′17″N 109°52′14″W), in the southern Gulf of California (minimum distance between tag and recapture sites  =  approximately 5,000 km), after 366 days at liberty (DAL). We used these empirical mark-recapture data to estimate growth rates and maximum size for tiger sharks in Hawaii. We found that tiger sharks in Hawaii grow twice as fast as previously thought, on average reaching 340 cm TL by age 5, and attaining a maximum size of 403 cm TL. Our model indicates the fastest growing individuals attain 400 cm TL by age 5, and the largest reach a maximum size of 444 cm TL. The largest shark captured during our study was 464 cm TL but individuals >450 cm TL were extremely rare (0.005% of sharks captured). We conclude that tiger shark growth rates and maximum sizes in Hawaii are generally consistent with those in other regions, and hypothesize that a broad diet may help them to achieve this rapid growth by maximizing prey consumption rates.
PMCID: PMC3885620  PMID: 24416287
9.  Controlling fibrous capsule formation through long-term down-regulation of collagen type I (COL1A1) expression by nanofiber-mediated siRNA gene silencing 
Acta biomaterialia  2012;9(1):4513-4524.
The foreign body reaction often interferes with the long-term functionality and performance of implanted biomedical devices through fibrous capsule formation. While many implant modification techniques have been adopted in attempts to control fibrous encapsulation, the outcomes remained sub-optimal. Nanofiber scaffold-mediated RNA interference may serve as an alternative approach through the localized and sustained delivery of siRNA at implant sites. In this study, we investigated the efficacy of siRNA-PCLEEP (poly(caprolactone-co-ethylethylene phosphate) nanofibers in controlling fibrous capsule formation through the down-regulation of Collagen type I (COL1A1) in vitro and in vivo. By encapsulating complexes of COL1A1 siRNA with a transfection reagent (Transit TKO) or cell penetrating peptides (CPPs), CADY or MPG, within the nanofibers (550–650 nm in diameter), a sustained release of siRNA was obtained for at least 28 days (loading efficiency ~ 60–67%). Scaffold-mediated transfection significantly enhanced cellular uptake of oligonucleotides and prolonged in vitro gene silencing duration by at least 2–3 times as compared to conventional bolus delivery of siRNA (14 days vs 5–7 days by bolus delivery). In vivo subcutaneous implantation of siRNA scaffolds revealed a significant decrease in fibrous capsule thickness at weeks 2 and 4 as compared to plain nanofibers (p < 0.05). Taken together, the results demonstrated the efficacy of scaffold-mediated siRNA gene-silencing in providing effective long-term control of fibrous capsule formation.
PMCID: PMC3523808  PMID: 23036951
RNA interference; Gene knockdown; Scaffold-mediated transfection; Cell penetrating peptides; Electrospinning
10.  Zonula Occludens-1 and -2 Are Cytosolic Scaffolds That Regulate the Assembly of Cellular Junctions 
The integrity of the tight junction barrier in epithelial and endothelial cells is critical to human health, but we still lack a detailed mechanistic knowledge of how the barrier is formed during development or responds to pathological and pharmacological insults. This limits our understanding of barrier dysfunction in disease and slows the development of therapeutic strategies. Recent studies confirm the long-maintained but previously unsupported view that the zonula occludens (ZO) proteins ZO-1 and ZO-2 are critical determinants of barrier formation. However, ZO proteins can also be components of adherens junctions, and recent studies suggest that ZO proteins may also promote the assembly and function of these junctions during epithelial morphogenesis. We review these studies and outline several recent observations that suggest that one role of ZO proteins is to regulate cytoskeletal dynamics at cell junctions. Finally, we propose a model by which the functional activities of ZO proteins in the adherens junction and tight junction are differentiated by a novel regulatory motif known as the U6 or acidic motif.
PMCID: PMC3759978  PMID: 19538295
tight junction; adherens junction; zonula occludens; ZO-1; ZO-2; E-cadherin; cytoskeleton; MAGUK; PDZ; scaffold; permeability; epithelia; morphogenesis
11.  Targeted Delivery of Vancomycin to Staphylococcus epidermidis Biofilms Using a Fibrinogen-Derived Peptide 
This study reports on the use of a fibrinogen-derived peptide for the specific targeting and delivery of vancomycin to Staphylococcus epidermidis biofilms. One method by which S. epidermidis initially adheres to biomaterials uses the plasma protein fibrinogen as an intermediary, where the S. epidermidis surface protein SdrG binds to a short amino acid sequence near the amino terminus of the Bβ chain of fibrinogen. We mimicked this binding interaction and demonstrated the use of a synthetic fibrinogen-based β6-20 peptide to target and deliver vancomycin to S. epidermidis in vitro. The β6-20 peptide was synthesized and labeled with a nanogold probe, and its targeting capabilities were examined through the use of scanning electron microscopy. The Nanogold component was then replaced by vancomycin, utilizing a flexible, variable length poly(ethylene glycol) linker between the peptide and antibiotic to create the targeted vancomycin products, β6-20-PEGx-VAN. Initial binding to surface adherent S. epidermidis was increased in a concentration-dependent manner relative to vancomycin for all equivalent concentrations ≥4 μg/ml, with targeted vancomcyin content up to 22.9 times that of vancomycin alone. Retention of the targeted antibiotics was measured after an additional 24 hour incubation period, revealing levels 1.3 times that of vancomycin. The results demonstrate the improved targeting and retention of vancomycin within a biofilm due to the incorporation of a specific targeting motif.
PMCID: PMC3461832  PMID: 22623343
Staphylococcus epidermidis; SdrG; β6-20 peptide; Targeted Delivery; Vancomycin
12.  Tricellulin deficiency affects tight junction architecture and cochlear hair cells 
The Journal of Clinical Investigation  2013;123(9):4036-4049.
The two compositionally distinct extracellular cochlear fluids, endolymph and perilymph, are separated by tight junctions that outline the scala media and reticular lamina. Mutations in TRIC (also known as MARVELD2), which encodes a tricellular tight junction protein known as tricellulin, lead to nonsyndromic hearing loss (DFNB49). We generated a knockin mouse that carries a mutation orthologous to the TRIC coding mutation linked to DFNB49 hearing loss in humans. Tricellulin was absent from the tricellular junctions in the inner ear epithelia of the mutant animals, which developed rapidly progressing hearing loss accompanied by loss of mechanosensory cochlear hair cells, while the endocochlear potential and paracellular permeability of a biotin-based tracer in the stria vascularis were unaltered. Freeze-fracture electron microscopy revealed disruption of the strands of intramembrane particles connecting bicellular and tricellular junctions in the inner ear epithelia of tricellulin-deficient mice. These ultrastructural changes may selectively affect the paracellular permeability of ions or small molecules, resulting in a toxic microenvironment for cochlear hair cells. Consistent with this hypothesis, hair cell loss was rescued in tricellulin-deficient mice when generation of normal endolymph was inhibited by a concomitant deletion of the transcription factor, Pou3f4. Finally, comprehensive phenotypic screening showed a broader pathological phenotype in the mutant mice, which highlights the non-redundant roles played by tricellulin.
PMCID: PMC3754262  PMID: 23979167
Foreign body-type multinucleated giant cells (FBGC), formed by macrophage fusion, are a prominent cell type on implanted biomaterials, although the roles they play at these and other sites of chronic inflammation are not understood. Why lymphocytes are present in this scenario and the effects of fusing macrophages/FBGC on subsequent lymphocyte responses are also unclear. To address the physiological significance of FBGC in this regard, we employed our in vitro system of interleukin (IL)-4-induced human monocyte-derived macrophage fusion/FBGC formation. Initially, we pursued the identities of lymphocyte co-stimulatory molecules on fusing macrophages/FBGC. In addition, we further compared the FBGC phenotype to that currently associated with osteoclasts and dendritic cells using recognized markers. Immunoblotting of cell lysates and immunochemistry of macrophages/FBGC in situ, revealed that IL-4-induced macrophages/FBGC strongly express HLA-DR, CD98, B7-2 (CD86), and B7-H1 (PD-L1), but not B7-1 (CD80) or B7-H2 (B7RP-1). Furthermore, molecules currently recognized to be expressed on osteoclasts (calcitonin receptor, tartrate-resistant acid phosphatase, RANK) or dendritic cells (CD1a, CD40, CD83, CD95/fas) are undetectable. In contrast, fusing macrophages/FBGC strongly express the macrophage markers αX integrin (CD11c), CD68, and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), whereas CD14 is completely down-modulated with IL-4-induced macrophage fusion. These novel data demonstrate that IL-4-induction of macrophage multinucleation/FBGC formation features the acquisition of a CD14-negative phenotypic profile which is distinguishable from that of dendritic cells and osteoclasts, yet potentially exhibits multiple capacities for lymphocyte interactions with resultant lymphocyte down-modulation.
PMCID: PMC3220734  PMID: 21798256
Biocompatibility; chronic inflammation; lymphocyte; macrophage; multinucleated giant cell
14.  Evaluation of Clinical Biomaterial Surface Effects on T Lymphocyte Activation 
Previous in vitro studies in our laboratory have shown that lymphocytes can influence macrophage adhesion and fusion on biomaterial surfaces. However, few studies have evaluated how material adherent macrophages can influence lymphocyte behavior, specifically T cells. In this study, we cultured human peripheral blood mononuclear cells from healthy donors on three synthetic non-biodegradable biomedical polymers: Elasthane 80A (PEU), Silicone rubber (SR), or polyethylene terephthalate (PET) and tissue culture polystyrene (TCPS). Upregulation of T cell surface activation markers (CD69 and CD25), lymphocyte proliferation, and interleukin-2 (IL-2) and interferon-γ (IFNγ) concentrations were evaluated by flow cytometry, carboxy-fluorescein diacetate, succinimydyl ester (CFSE) incorporation, and multiplex cytokine immunoassay, respectively, to assess T cell activation. Following 3 and 7 days of culture, CD4+ helper T cells from cultures of any of the material groups did not express the activation markers CD69 and CD25 and lymphocyte proliferation was not present. IL-2 and IFNγ levels were produced, but dependent on donor. These data indicate that T cells are not activated in response to clinically relevant synthetic biomaterials. The data also suggest that lymphocyte subsets exclusive of T cells are the source of the lymphokines, IL-2 and IFN-γ, in certain donors.
PMCID: PMC2788042  PMID: 19172618
Lymphocyte activation; biomaterials; interferonγ; Interleukin-2
15.  Lymphocyte Adhesion and Interactions with Biomaterial Adherent Macrophages and Foreign Body Giant Cells 
To characterize the effects of adherent macrophages and biomaterial surface chemistries on lymphocyte adhesion and activation, lymphocytes were co-cultured with monocytes alone and together, directly and separated by a porous membrane transwell on hydrophobic, hydrophilic/neutral, hydrophilic/anionic, and hydrophilic/cationic biomaterial surfaces. Surface adherent cells were quantitatively analyzed after 3 days utilizing immunofluorescence and phase contrast imaging. After periods of 3, 7, and 10 days, secreted interferon-γ (IFN-γ) was quantified by ELISA. Limited direct biomaterial-adherent lymphocytes were identified regardless of the presence of macrophages or foreign body giant cells (FBGC). The majority of adherent lymphocytes, which were T cells (> 95%) rather than natural killer cells, predominantly interacted with adherent macrophages and FBGCs; greater than 90% were interacting on surfaces with higher levels of adherent macrophages and FBGCs and greater than 55% were interacting on surfaces with lower levels of macrophages and FBGCs. The hydrophilic/anionic surface promoted higher levels of macrophage- and FBGC-adherent lymphocytes but was nonselective for lymphocyte subtype interactions. The hydrophilic/neutral surface was selective for CD4+ T lymphocyte interactions while the hydrophobic surface was selective for CD8+ T lymphocyte interactions. IFN-γ was produced in direct and indirect co-cultures but not in lymphocyte- and monocyte-only cultures suggesting that lymphocytes are activated via macrophage-derived cytokines rather than direct biomaterial contact. Direct lymphocyte interactions with adherent macrophages/FBGCs enhanced IFN-γ production relative to indirect co-cultures. These results suggest that lymphocytes prefer interactions with adherent macrophages and FBGCs, resulting in lymphocyte activation, and these interactions can be influenced by biomaterial surface chemistries.
PMCID: PMC2783376  PMID: 19148923
biomaterials; lymphocyte activation; adhesion; macrophages and foreign body giant cells; cytokines
16.  Zonula occludens-1 and -2 regulate apical cell structure and the zonula adherens cytoskeleton in polarized epithelia 
Molecular Biology of the Cell  2012;23(4):577-590.
ETOC: Our study reveals that ZO proteins in fully polarized cells regulate the assembly and contractility of the perijunctional actomyosin ring associated with the adherens junction.
The structure and function of both adherens (AJ) and tight (TJ) junctions are dependent on the cortical actin cytoskeleton. The zonula occludens (ZO)-1 and -2 proteins have context-dependent interactions with both junction types and bind directly to F-actin and other cytoskeletal proteins, suggesting ZO-1 and -2 might regulate cytoskeletal activity at cell junctions. To address this hypothesis, we generated stable Madin-Darby canine kidney cell lines depleted of both ZO-1 and -2. Both paracellular permeability and the localization of TJ proteins are disrupted in ZO-1/-2–depleted cells. In addition, immunocytochemistry and electron microscopy revealed a significant expansion of the perijunctional actomyosin ring associated with the AJ. These structural changes are accompanied by a recruitment of 1-phosphomyosin light chain and Rho kinase 1, contraction of the actomyosin ring, and expansion of the apical domain. Despite these changes in the apical cytoskeleton, there are no detectable changes in cell polarity, localization of AJ proteins, or the organization of the basal and lateral actin cytoskeleton. We conclude that ZO proteins are required not only for TJ assembly but also for regulating the organization and functional activity of the apical cytoskeleton, particularly the perijunctional actomyosin ring, and we speculate that these activities are relevant both to cellular organization and epithelial morphogenesis.
PMCID: PMC3279387  PMID: 22190737
Current opinion in hematology  2009;16(1):53-57.
This review is intended to provide insight into the current state of understanding regarding the molecular and cellular mechanisms underlying the formation and function of various types of multinucleated giant cells.
Recent Findings
Recent studies involving mainly osteoclasts and foreign body giant cells have revealed a number of common factors, e.g., vitronectin, an adhesion protein, dendritic cell-specific transmembrane protein (DC-STAMP), a fusion factor, and macrophage fusion receptor (MFR), that contribute to giant cell formation and function. Insight into common molecules, receptors, and mediators of the adhesion and fusion mechanisms of giant cell formation have been complicated by the wide diversity of species, models, and cell types utilized in these studies.
These recently identified factors together with the well-known osteoclast receptor, αvβ3, may serve as potential therapeutic targets for the modulation and inhibition of multinucleated giant cell formation and function. Further studies on intracellular and intercellular signaling mechanisms modulating multinucleated giant cell formation and function are necessary for the identification of therapeutic targets as well as a better understanding of giant cell biology.
PMCID: PMC2679387  PMID: 19057205
Multinucleated Giant Cell; Foreign Body Giant Cell; Macrophage; Osteoclast
Seminars in immunology  2007;20(2):86-100.
The foreign body reaction composed of macrophages and foreign body giant cells is the end-stage response of the inflammatory and wound healing responses following implantation of a medical device, prosthesis, or biomaterial. A brief, focused overview of events leading to the foreign body reaction is presented. The major focus of this review is on factors that modulate the interaction of macrophages and foreign body giant cells on synthetic surfaces where the chemical, physical, and morphological characteristics of the synthetic surface are considered to play a role in modulating cellular events. These events in the foreign body reaction include protein adsorption, monocyte/macrophage adhesion, macrophage fusion to form foreign body giant cells, consequences of the foreign body response on biomaterials, and cross-talk between macrophages/foreign body giant cells and inflammatory/wound healing cells. Biomaterial surface properties play an important role in modulating the foreign body reaction in the first two to four weeks following implantation of a medical device, even though the foreign body reaction at the tissue/material interface is present for the in vivo lifetime of the medical device. An understanding of the foreign body reaction is important as the foreign body reaction may impact the biocompatibility (safety) of the medical device, prosthesis, or implanted biomaterial and may significantly impact short- and long-term tissue responses with tissue-engineered constructs containing proteins, cells, and other biological components for use in tissue engineering and regenerative medicine. Our perspective has been on the inflammatory and wound healing response to implanted materials, devices, and tissue-engineered constructs. The incorporation of biological components of allogeneic or xenogeneic origin as well as stem cells into tissue-engineered or regenerative approaches opens up a myriad of other challenges. An in depth understanding of how the immune system interacts with these cells and how biomaterials or tissue-engineered constructs influences these interactions may prove pivotal to the safety, biocompatibility, and function of the device or system under consideration.
PMCID: PMC2327202  PMID: 18162407
Foreign body reaction; Macrophages; Foreign body giant cells; Biodegradation; Cytokines; Biomaterials
19.  Phenotypic Dichotomies in the Foreign Body Reaction 
Biomaterials  2007;28(34):5114-5120.
To better understand the relationship between macrophage/foreign body giant cell adhesion and activation on surface-modified biomaterials, quantitative assessment of adherent cell density (cells per mm²) and cytokine production (pgs per mL) were determined by ELISA. Further analysis to identify cellular activation was carried out by normalizing the cytokine concentration data to provide a measure of cellular activation. This method of analysis demonstrated that hydrophobic surfaces provided statistically significantly greater adherent cell densities than hydrophilic/neutral surfaces. However, when cell activation parameters were determined by normalization to the adherent cell density, the hydrophilic/neutral surfaces demonstrated statistically significantly greater levels of activation and production of IL-10, IL-1β, IL-6, IL-8, and MIP-1β. With increasing time, production of the anti-inflammatory cytokine IL-10 increased, whereas IL-1β, IL-6, and IL-8 decreased and MIP-1β was relatively constant over the culture time period. This observed dichotomy or disparity between adhesion and activation may be related to surface-induced adherent cell apoptosis. Further evaluation of macrophage activation on biomaterial surfaces indicated that an apparent phenotypic switch in macrophage phenotype occurred over the course of the in vitro culture. Analysis of cytokine/chemokine profiles with surface-modified biomaterials revealed similarities between the classically activated macrophages and the biomaterial-adherent macrophages early (day 3) in culture, while at later timepoints the biomaterial-adherent macrophages produced profiles similar to alternatively activated macrophages. Classically activated macrophages are those commonly activated by LPS (lipopolysaccharide) or IFN-γ (interferon-γ) and alternatively activated macrophages are those activated by IL-4/IL-13 or IL-10. Surface modification of biomaterials offers an opportunity to control cellular activation and cytokine profiles in the phenotypic switch, and may provide a means by which macrophages can be induced to regulate particular secretory proteins that direct inflammation, the foreign body reaction, wound healing, and ultimately biocompatibility.
PMCID: PMC2248374  PMID: 17706278
macrophages; adhesion; activation; hydrophilic, hydrophobic, and ionic surface chemistry; foreign body reaction
20.  A PDZ-Binding Motif Controls Basolateral Targeting of Syndecan-1 Along the Biosynthetic Pathway in Polarized Epithelial Cells 
Traffic (Copenhagen, Denmark)  2008;9(11):1915.
The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1’s arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.
PMCID: PMC2820280  PMID: 18764819
basolateral; epithelia; PDZ; polarity; syndecan-1
21.  ZO-1 Stabilizes the Tight Junction Solute Barrier through Coupling to the Perijunctional Cytoskeleton 
Molecular Biology of the Cell  2009;20(17):3930-3940.
ZO-1 binds numerous transmembrane and cytoplasmic proteins and is required for assembly of both adherens and tight junctions, but its role in defining barrier properties of an established tight junction is unknown. We depleted ZO-1 in MDCK cells using siRNA methods and observed specific defects in the barrier for large solutes, even though flux through the small claudin pores was unaffected. This permeability increase was accompanied by morphological alterations and reorganization of apical actin and myosin. The permeability defect, and to a lesser extent morphological changes, could be rescued by reexpression of either full-length ZO-1 or an N-terminal construct containing the PDZ, SH3, and GUK domains. ZO-2 knockdown did not replicate either the permeability or morphological phenotypes seen in the ZO-1 knockdown, suggesting that ZO-1 and -2 are not functionally redundant for these functions. Wild-type and knockdown MDCK cells had differing physiological and morphological responses to pharmacologic interventions targeting myosin activity. Use of the ROCK inhibitor Y27632 or myosin inhibitor blebbistatin increased TER in wild-type cells, whereas ZO-1 knockdown monolayers were either unaffected or changed in the opposite direction; paracellular flux and myosin localization were also differentially affected. These studies are the first direct evidence that ZO-1 limits solute permeability in established tight junctions, perhaps by forming a stabilizing link between the barrier and perijunctional actomyosin.
PMCID: PMC2735491  PMID: 19605556
22.  Physiology and Function of the Tight Junction 
Understanding of tight junctions has evolved from their historical perception as inert solute barriers to recognition of their physiological and biochemical complexity. Many proteins are specifically localized to tight junctions, including cytoplasmic actin-binding proteins and adhesive transmembrane proteins. Among the latter are claudins, which are critical barrier proteins. Current information suggests that the paracellular barrier is most usefully modeled as having two physiologic components: a system of charge-selective small pores, 4 Å in radius, and a second pathway created by larger discontinuities in the barrier, lacking charge or size discrimination. The first pathway is influenced by claudin expression patterns and the second is likely controlled by different proteins and signals. Recent information on claudin function and disease-causing mutations have led to a more complete understanding of their role in barrier formation, but progress is impeded by lack of high resolution structural information.
Claudin proteins in tight junctions form a barrier to diffusion between epithelial cells. The permeability of the epithelium depends on the isoforms expressed.
PMCID: PMC2742087  PMID: 20066090
23.  Domain Swapping within PDZ2 Is Responsible for Dimerization of ZO Proteins*s 
The Journal of biological chemistry  2007;282(52):37710-37716.
ZO-1 is a multidomain protein involved in cell-cell junctions and contains three PDZ domains, which are necessary for its function in vivo. PDZ domains play a central role in assembling diverse protein complexes through their ability to recognize short peptide motifs on other proteins. We determined the structure of the second of the three PDZ domains of ZO-1, which is known to promote dimerization as well as bind to C-terminal sequences on connexins. The dimer is stabilized by extensive symmetrical domain swapping of β-strands, which is unlike any other known mechanism of PDZ dimerization. The canonical peptide-binding groove remains intact in both subunits of the PDZ2 dimer and is created by elements contributed from both monomers. This unique structure reveals an additional example of how PDZ domains dimerize and has multiple implications for both peptide binding and oligomerization in vivo.
PMCID: PMC2661428  PMID: 17928286
Multinucleated giant cells are a classic cellular feature of chronic inflammation, although the mechanism of macrophage fusion leading to their formation is not well understood. Here, we investigate the participation of protein kinase C (PKC) in the interleukin (IL)-4-induced fusion of human monocyte-derived macrophages and foreign body giant cell (FBGC) formation in vitro. The PKC inhibitors H-7 and calphostin C attenuated macrophage fusion, whereas H-8, which is more selective for PKA and PKG, did not. Macrophage fusion was also prevented by the phospholipase C inhibitor, Et-18-OCH3, the PKC isoform inhibitors GO6983 or rottlerin and by peptide inhibitors for PKC (20-28), PKCβ, or PKCζ but not by HBDDE or peptide inhibitors for PKCε or PKA. In cultures of fusing macrophages/FBGC, we detected only PKCα, β, δ, and ζ by immunoprecipitation and immunoblotting, and we also observed strong expression of these isoforms by immunocytochemistry. Our collective results suggest that the γ, ε, η, μ, θ, or ι PKC isoforms are not required in the mechanism of IL-4-induced macrophage fusion; whether PKCα is required is unclear. However, new evidence is provided that FBGC formation is supported by PKCβ, PKCδ, and PKCζ in combined diacylglycerol-dependent (PKCβ and PKCδ) and - independent (PKCζ) signaling pathways.
PMCID: PMC2275167  PMID: 18067888
diacylglycerol; foreign body giant cell; interleukin-4; monocyte; macrophage; fusion; multinucleated giant cell; protein kinase C
25.  The Unique-5 and -6 Motifs of ZO-1 Regulate Tight Junction Strand Localization and Scaffolding Properties 
Molecular Biology of the Cell  2007;18(3):721-731.
The proper cellular location and sealing of tight junctions is assumed to depend on scaffolding properties of ZO-1, a member of the MAGUK protein family. ZO-1 contains a conserved SH3-GUK module that is separated by a variable region (unique-5), which in other MAGUKs has proven regulatory functions. To identify motifs in ZO-1 critical for its putative scaffolding functions, we focused on the SH3-GUK module including unique-5 (U5) and unique-6 (U6), a motif immediately C-terminal of the GUK domain. In vitro binding studies reveal U5 is sufficient for occludin binding; U6 reduces the affinity of this binding. In cultured cells, U5 is required for targeting ZO-1 to tight junctions and removal of U6 results in ectopically displaced junction strands containing the modified ZO-1, occludin, and claudin on the lateral cell membrane. These results provide evidence that ZO-1 can control the location of tight junction transmembrane proteins and reveals complex protein binding and targeting signals within its SH3-U5-GUK-U6 region. We review these findings in the context of regulated scaffolding functions of other MAGUK proteins.
PMCID: PMC1805089  PMID: 17182847

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