DNA-directed arrangement of soft synthetic compartments and their behavior in vitro and in vivo.

DNA has been widely used as a key tether to promote self-organization of super-assemblies with emergent properties. However, control of this process is still challenging for compartment assemblies and to date the resulting assemblies have unstable membranes precluding in vitro and in vivo testing. Here we present our approach to overcome these limitations, by manipulating molecular factors such as compartment membrane composition and DNA surface density, thereby controlling the size and stability of the resulting DNA-linked compartment clusters. The soft, flexible character of the polymer membrane and low number of ssDNA remaining exposed after cluster formation determine the interaction of these clusters with the cell surface. These clusters exhibit in vivo stability and lack of toxicity in a zebrafish model. To display the breadth of therapeutic applications attainable with our system, we encapsulated the medically established enzyme laccase within the inner compartment and demonstrated its activity within the clustered compartments. Most importantly, these clusters can interact selectively with different cell lines, opening a new strategy to modify and expand cellular functions by attaching such pre-organized soft DNA-mediated compartment clusters on cell surfaces for cell engineering or therapeutic applications.

Lung Surfactant Lipids Provide Immune Protection Against Haemophilus influenzae Respiratory Infection.

Non-typeable Haemophilus influenzae (NTHi) causes persistent respiratory infections in patients with chronic obstructive pulmonary disease (COPD), probably linked to its capacity to invade and reside within pneumocytes. In the alveolar fluid, NTHi is in contact with pulmonary surfactant, a lipoprotein complex that protects the lung against alveolar collapse and constitutes the front line of defense against inhaled pathogens and toxins. Decreased levels of surfactant phospholipids have been reported in smokers and patients with COPD. The objective of this study was to investigate the effect of surfactant phospholipids on the host-pathogen interaction between NTHi and pneumocytes. For this purpose, we used two types of surfactant lipid vesicles present in the alveolar fluid: (i) multilamellar vesicles (MLVs, > 1 µm diameter), which constitute the tensioactive material of surfactant, and (ii) small unilamellar vesicles (SUVs, 0.1 µm diameter), which are generated after inspiration/expiration cycles, and are endocytosed by pneumocytes for their degradation and/or recycling. Results indicated that extracellular pulmonary surfactant binds to NTHi, preventing NTHi self-aggregation and inhibiting adhesion of NTHi to pneumocytes and, consequently, inhibiting NTHi invasion. In contrast, endocytosed surfactant lipids, mainly via the scavenger receptor SR-BI, did not affect NTHi adhesion but inhibited NTHi invasion by blocking bacterial uptake in pneumocytes. This blockade was made possible by inhibiting Akt phosphorylation and Rac1 GTPase activation, which are signaling pathways involved in NTHi internalization. Administration of the hydrophobic fraction of lung surfactant in vivo accelerated bacterial clearance in a mouse model of NTHi pulmonary infection, supporting the notion that the lipid component of lung surfactant protects against NTHi infection. These results suggest that alterations in surfactant lipid levels in COPD patients may increase susceptibility to infection by this pathogen.

Exploiting scavenger receptors in cancer immunotherapy: Lessons from CD5 and SR-B1.

Scavenger receptors (SRs) are structurally heterogeneous cell surface receptors characterized by their capacity to remove extraneous or modified self-macromolecules from circulation, thus avoiding the accumulation of noxious agents in the extracellular space. This scavenging activity makes SRs important molecules for host defense and homeostasis. In turn, SRs keep the activation of the steady-state immune response in check, and participate as co-receptors in the priming of the effector immune responses when the macromolecules are associated with a threat that might compromise host homeostasis. Therefore, SRs built up sophisticated sensor mechanisms controlling the immune system, which may be exploited to develop novel drugs for cancer immunotherapy. In this review, we focus on the regulation of the anti-tumor immune response by two paradigmatic SRs: the lymphocyte receptor CD5 and the more broadly distributed scavenger receptor class B type 1 (SR-B1). Cancer immunity can be boosted by blockade of SRs working as immune checkpoint inhibitors (CD5) and/or by proper engagement of SRs working as innate danger receptor (SR-B1). Thus, these receptors illustrate both the complexity of targeting SRs in cancer immunotherapy and also the opportunities offered by such an approach.

Structure-Activity Relationship of PEGylated Polylysine Peptides as Scavenger Receptor Inhibitors for Non-Viral Gene Delivery.

PEGylated polylysine peptides of the general structure PEG30 kDa-Cys-Trp-LysN (N = 10 to 30) were used to form fully condensed plasmid DNA (pGL3) polyplexes at a ratio of 1 nmol of peptide per µg of DNA (ranging from N:P 3:1 to 10:1 depending on Lys repeat). Co-administration of 5 to 80 nmols of excess PEG-peptide with fully formed polyplexes inhibited the liver uptake of (125)I-pGL3-polyplexes. The percent inhibition was dependent on the PEG-peptide dose and was saturable, consistent with inhibition of scavenger receptors. The scavenger receptor inhibition potency of PEG-peptides was dependent on the length of the Lys repeat, which increased 10-fold when comparing PEG30 kDa-Cys-Trp-Lys10 (IC50 of 20.2 µM) with PEG30 kDa-Cys-Trp-Lys25 (IC50 of 2.1 µM). We hypothesize that PEG-peptides inhibit scavenger receptors by spontaneously forming small 40 to 60 nm albumin nanoparticles that bind to and saturate the receptor. Scavenger receptor inhibition delayed the metabolism of pGL3-polyplexes, resulting in efficient gene expression in liver hepatocytes following delayed hydrodynamic dosing. PEG-peptides represent a new class of scavenger inhibitors that will likely have broad utility in blocking unwanted liver uptake and metabolism of a variety of nanoparticles.

Phagocytosis of mycobacteria by zebrafish macrophages is dependent on the scavenger receptor Marco, a key control factor of pro-inflammatory signalling.

Scavenger receptors on the cell surface of macrophages play an important role in host defence through their ability to bind microbial ligands and induce phagocytosis. Concurrently, signal transduction pathways are initiated that aid in defence mechanisms against the invading microbe. Here we report on the function of scavenger receptor Marco (Macrophage receptor with collagenous structure) during infection of zebrafish embryos with Mycobacterium marinum, a close relative of M. tuberculosis. Morpholino knockdown demonstrates that Marco is required for the rapid phagocytosis of M. marinum following intravenous infection. Furthermore, gene expression analysis shows that Marco controls the initial transient pro-inflammatory response to M. marinum and remains a determining factor for the immune response signature at later stages of infection. Increased bacterial burden following marco knockdown indicates that this scavenger receptor is important for control of M. marinum growth, likely due to delayed phagocytosis and reduced pro-inflammatory signalling observed under conditions of Marco deficiency.

[The in vitro anti-atherosclerotic activity of compound IMB-1680].

In the previous study, a high-throughput screening method was established to find the antagonists of CD36. In the present study, a new compound named IMB-1680 was found using this method. The anti-atherosclerotic activities of IMB-1680 were then evaluated. Dose-dependent activities of IMB-1680 were detected by using Sf9 [hCD36] and CHO [hCD36] models. Fluorescence microscopic photography and flow cytometry were used to analyze uptake of mLDL. Foam cell test with RAW264.7 macrophages was used to examine lipid accumulation. The results showed that IMB-1680 inhibited CD36 activity with IC50 of 2.80 and 8.79 micromol x L(-1) in Sf9[hCD36] and CHO [hCD36] cells, respectively. Fluorescence microscopic photography and flow cytometry revealed that IMB-1680 could significantly reduce DiI-AcLDL uptake. Meanwhile, IMB-1680 also could reduce lipids accumulation in RAW264.7 macrophages. In all, the data indicated that IMB-1680 might be a potent effective anti-atherosclerotic leading compound.

CXCL16 and CXCR6 are coexpressed in human lung cancer in vivo and mediate the invasion of lung cancer cell lines in vitro.

Despite advances in early diagnosis and multimodality therapy for cancers, most of lung cancer patients have been locally advanced or metastatic at the time of diagnosis, suggesting the highly progressive characteristic of lung cancer cells. The mechanisms underling invasiveness and metastasis of lung cancer are yet to be elucidated. In the present study, immunohistochemistry was performed to detect the expression of CXCL16-CXCR6 in human lung cancer tissues. It was demonstrated that similar to CXCL12 and CXCR4, CXCL16 and CXCR6 were also coexpressed in human primary lung cancer tissues. After confirming the functional existence of CXCL16 and CXCR6 protein in A549, 95D and H292 cells by ELSA and flow cytometry analysis, we further explored the significance of CXCL16-CXCR6 axis in the biological functions of lung cancer cell lines in vitro. It was found that CXCL16 had no effects on the PCNA (proliferating cell nuclear antigen) expression of A549, 95D and H292 cells. However, both exogenous CXCL16 and CM (conditioned medium from A549, 95D or H292) significantly improved the in vitro viability and invasion of three lung cancer cell lines. The neutralizing antibody to CXCL16 or down-regulation of CXCR6 was able to inhibit the increased viability and invasiveness of A549, 95D and H292 cells stimulated by CXCL16 or CM. Our results imply that CXCL16-CXCR6 axis is involved in the regulation of viability and invasion rather than PCNA expression of lung caner cells, which opens the door for better understanding the mechanisms of lung tumor progression and metastasis.

Phenolic acids are in vivo atheroprotective compounds appearing in the serum of rats after blueberry consumption.

Blueberries (BB) have recently been shown to have cardioprotective effects and to prevent atherosclerosis in rodent models. However, the bioactive compounds in BB responsible for these effects have not yet been characterized. Seven phenolic acids (7PA) were identified as metabolites in the serum of rats fed diets supplemented with 10% freeze-dried BB. In this study, 7PA were evaluated for their potential atheroprotective effects in murine macrophage cell line RAW 264.7. 7PA were found to inhibit LPS-induced mRNA expression and protein levels of pro-inflammatory cytokine TNF-α and IL-6 by reducing MAPK JNK, p38, and Erk1/2 phosphorylation. After treatment with 7PA for 2 weeks, mRNA expression and protein levels of scavenger receptor CD36 were decreased (P<0.05), whereas type A scavenger receptor (SR-A) remained unchanged. Moreover, foam cell formation induced by oxLDL and oxLDL binding to macrophages was also inhibited by 7PA. In addition, 7PA increased (P<0.05) expression and protein levels of ATP-binding cassette transporter A1 (ABCA1), which facilitates cholesterol efflux and reduces cholesterol accumulation in macrophages. In summary, the present study demonstrates that certain phenolic acids are potential in vivo atheroprotective compounds following BB consumption in the rodent model. Because BB contain many phytochemicals, other as yet unidentified bioactive compounds may also be important in preventing atherosclerosis in this model and, possibly, in humans.

Oxidized low-density lipoprotein increases interleukin-8 production in human gingival epithelial cell line Ca9-22.

BACKGROUND AND OBJECTIVE: Recent epidemiological studies have shown a correlation between periodontitis and hyperlipidemia. We have found high levels of oxidized low-density lipoprotein (OxLDL) in the gingival crevicular fluid of dental patients. In the present study, we tried to examine the possible role of OxLDL in periodontal inflammation in vitro. MATERIAL AND METHODS: Cells of the human gingival epithelial cell line Ca9-22 were cultured in media containing OxLDL, and the amounts of interleukin-8 (IL-8) and prostaglandin E(2) (PGE(2)) produced were measured using ELISAs. RESULTS: Production of IL-8 by Ca9-22 cells was significantly increased when the cells were treated with OxLDL, but not with native LDL or acetylated LDL. Production of PGE(2) by Ca9-22 cells was enhanced by co-incubation with OxLDL and interleukin-1 beta (IL-1 beta). Scavenger receptor inhibitors, fucoidan and dextran sulfate, inhibited the OxLDL-induced IL-8 and PGE(2) production in the presence of IL-1 beta. The p(38) MAPK inhibitors SB203580 and SB202190 and the ERK inhibitor PD98059 inhibited the OxLDL-induced IL-8 production. Among oxidized lipids and chemically modified LDL, 7-ketocholesterol enhanced IL-8 production. CONCLUSION: This is the first report to show that OxLDL enhances IL-8 production in epithelial cells.

Rapid clearance of the circulating metastatic factor autotaxin by the scavenger receptors of liver sinusoidal endothelial cells.

Autotaxin, also known as NPP2 (nucleotide pyrophosphatase/phosphodiesterase 2), is a secreted lysophospholipase-D that generates lysophosphatidic acid and thereby promotes the metastatic and invasive properties of tumor cell as well as angiogenesis. We show here that, in mice, NPP2 is cleared from the circulation within minutes and is retained by the liver sinusoidal endothelial cells (LSECs). The binding of NPP2 to isolated LSECs resulted in its degradation and could be competed for with ligands of the scavenger receptor family. Our finding that circulating NPP2 has a rapid turnover has important implications for its development as an anti-cancer target.

Scavenger receptor A: a new route for adenovirus 5.

Adenoviruses are common pathogens associated with respiratory diseases, gastrointestinal illnesses and/or conjunctivitis. Currently, this virus is used as a vector in gene therapy trials. The promise of viral gene therapy applications is substantially reduced because the virus is cleared by liver macrophages upon systemic administration. The mechanism underlying adenoviral tropism to and degradation in macrophages is poorly understood. We identified a new adenoviral receptor, the scavenger receptor A (SR-A), responsible for uptake of the virus in macrophages. CHO cells expressing SR-A showed increased viral transgene expression when compared with wild type cells. Preincubation of J774 macrophage cells with SR-A ligands decreased significantly adenoviral uptake. Electron-microscopy analysis of infected J774 cells showed activation of a viral degradation pathway. Infection of mice with adenovirus resulted in a substantial decrease of the virus in liver macrophages when SR-A was blocked. Our data provide a basis for understanding of the adenoviral uptake and degradation mechanism in macrophages in vitro and in vivo. Inhibition of adenoviral SR-A uptake can be utilized in gene therapy applications to increase its efficiency and efficacy.

The role of CXCL16 and its processing metalloproteinases ADAM10 and ADAM17 in the proliferation and migration of human mesangial cells.

In this study, we analyzed the regulation and functional role of CXCL16 in human mesangial cells (hMCs). We can show, that CXCL16 is constitutively expressed in hMCs and is further up-regulated by cytokine mix (IFNgamma, TNFalpha, and IL1beta). The constitutive release of CXCL16 from hMCs was rapidly induced by the stimulation with cytokines. We identified ADAM10 and ADAM17 as being responsible for the cytokine-induced shedding of CXCL16. Notably, targeting ADAM10 and ADAM17 in hMCs decreased the chemotaxis of T-Jurkat cells, whereas the inhibition of CXCL16 had no significant influence. This suggests that both proteases are important players in the recruitment of immune cells into the glomerulus, but other substrates than CXCL16 are involved in this process. Finally, we could show that the inhibition of CXCL16, ADAM10, and ADAM17 led to a strong reduction of cell proliferation and migration of hMCs. This finding could be important to develop novel diagnostic and therapeutic strategies to treat mesangial proliferative kidney diseases.

Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages.

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

In search of antioxidants and anti-atherosclerotic agents from herbal medicines.

Many recent studies have suggested that low-density lipoprotein (LDL) oxidation, endothelial dysfunction, and inflammation are involved in the pathogenesis of atherosclerosis. Herbal regimens in the treatment of blood stasis, a counterpart of atherosclerosis, commonly use medicinal plants of leguminosae and labiatae. We have developed disease-oriented screening methods to search for bioactive components, particularly isoflavones in leguminosae and polyphenols in labiatae from Chinese herbal medicines. Many bioactive components and active fractions capable of inhibiting a. Cu(II)-induced LDL oxidation, b. oxidized LDL-induced endothelial damage, c. uptake of oxidized LDL by macrophages (J774A.1), and d. expression of cell adhesion molecules (CAMs) have been identified. A polyphenol, namely salvianolic acid B from Salvia miltiorrhiza was identified to be a potent antioxidant, endothelial-protecting agent, and an inhibitor to suppress the expression of ICAM and VCAM. This review also briefly describes the strategy for developing herbal medicines as anti-atherosclerotic agents.

Scavenger receptor class-A is a novel cell surface receptor for double-stranded RNA.

Double-stranded RNA (dsRNA) is a potent signal to the host immune system for the presence of an ongoing viral infection. The presence of dsRNA, intracellularly or extracellularly, leads to the induction of innate inflammatory cytokines in many cell types including epithelial cells. However, the cell surface receptor for recognition of extracellular dsRNA is not yet determined. Here, we report that extracellular dsRNA is recognized and internalized by scavenger receptor class-A (SR-A). Treatment of human epithelial cells with specific antagonists of SR-A or with an anti-SR-A antibody significantly inhibited dsRNA induction of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-8, and regulated on activation normal T-cell expressed and secreted (RANTES). Furthermore, intranasal dsRNA treatment of SR-A-deficient (SR-A(-/-)) mice showed a significant decrease in the expression of inflammatory cytokines and a corresponding decrease in the accumulation of polymorphonuclear leukocytes (PMNs) in lungs. These data provide direct evidence that SR-A is a novel cell surface receptor for dsRNA, and therefore, SR-A may play a role in antiviral immune responses.

Adsorbed proteins influence the biological activity and molecular targeting of nanomaterials.

The possible combination of specific physicochemical properties operating at unique sites of action within cells and tissues has led to considerable uncertainty surrounding nanomaterial toxic potential. We have investigated the importance of proteins adsorbed onto the surface of two distinct classes of nanomaterials (single-walled carbon nanotubes [SWCNTs]; 10-nm amorphous silica) in guiding nanomaterial uptake or toxicity in the RAW 264.7 macrophage-like model. Albumin was identified as the major fetal bovine or human serum/plasma protein adsorbed onto SWCNTs, while a distinct protein adsorption profile was observed when plasma from the Nagase analbuminemic rat was used. Damaged or structurally altered albumin is rapidly cleared from systemic circulation by scavenger receptors. We observed that SWCNTs inhibited the induction of cyclooxygenase-2 (Cox-2) by lipopolysaccharide (LPS; 1 ng/ml, 6 h) and this anti-inflammatory response was inhibited by fucoidan (scavenger receptor antagonist). Fucoidan also reduced the uptake of fluorescent SWCNTs (Alexa647). Precoating SWCNTs with a nonionic surfactant (Pluronic F127) inhibited albumin adsorption and anti-inflammatory properties. Albumin-coated SWCNTs reduced LPS-mediated Cox-2 induction under serum-free conditions. SWCNTs did not reduce binding of LPS(Alexa488) to RAW 264.7 cells. The profile of proteins adsorbed onto amorphous silica particles (50-1000 nm) was qualitatively different, relative to SWCNTs, and precoating amorphous silica with Pluronic F127 dramatically reduced the adsorption of serum proteins and toxicity. Collectively, these observations suggest an important role for adsorbed proteins in modulating the uptake and toxicity of SWCNTs and nano-sized amorphous silica.

Downregulation of the hemoglobin scavenger receptor in individuals with diabetes and the Hp 2-2 genotype: implications for the response to intraplaque hemorrhage and plaque vulnerability.

In individuals with diabetes mellitus (DM), the haptoglobin (Hp) genotype is a major determinant of susceptibility to myocardial infarction. We have proposed that this is because of DM and Hp genotype-dependent differences in the response to intraplaque hemorrhage. The macrophage hemoglobin scavenging receptor CD163 plays an essential role in the clearance of hemoglobin released from lysed red blood cells after intraplaque hemorrhage. We sought to test the hypothesis that expression of CD163 is DM and Hp genotype-dependent. CD163 was quantified in plaques by immunohistochemistry, on peripheral blood monocytes (PBMs) by FACS, and as soluble CD163 (sCD163) in plasma by ELISA. In DM plaques, despite an increase in macrophage infiltration, CD163 immunoreactivity was lower, resulting in a dramatic reduction in the percentage of macrophages expressing CD163 (27+/-2% versus 70+/-2%, P=0.0001). In individuals with DM as compared with individuals without DM, the percentage of PBMs expressing CD163 was reduced (3.7+/-0.6% versus 7.1+/-0.9%, P<0.002) whereas soluble plasma CD163 was increased (2.6+/-1.1 microg/mL versus 1.6+/-0.8 microg/mL, P<0.0005). Among DM individuals, the Hp 2-2 genotype was associated with a decrease in the percentage of PBMs expressing CD163 (2.3+/-0.5% versus 5.6+/-1.3%, P=0.01) and an increase in plasma soluble CD163 (3.0+/-0.2 microg/mL versus 2.3+/-0.2 microg/mL, P=0.04). Taken together, these results demonstrate an impaired hemoglobin clearance capacity in Hp 2-2 DM individuals and may provide the key insight explaining the increased incidence of myocardial infarction in this population.