Krill-Oil-Dependent Increases in HS-Omega-3 Index, Plasma Choline and Antioxidant Capacity in Well-Conditioned Power Training Athletes.

There is evidence that both omega-3 polyunsaturated fatty acids (n-3 PUFAs) and choline can influence sports performance, but information establishing their combined effects when given in the form of krill oil during power training protocols is missing. The purpose of this study was therefore to characterize n-3 PUFA and choline profiles after a one-hour period of high-intensity physical workout after 12 weeks of supplementation. Thirty-five healthy power training athletes received either 2.5 g/day of Neptune krill oilTM (550 mg EPA/DHA and 150 mg choline) or olive oil (placebo) in a randomized double-blind design. After 12 weeks, only the krill oil group showed a significant HS-Omega-3 Index increase from 4.82 to 6.77% and a reduction in the ARA/EPA ratio (from 50.72 to 13.61%) (p < 0.001). The krill oil group showed significantly higher recovery of choline concentrations relative to the placebo group from the end of the first to the beginning of the second exercise test (p = 0.04) and an 8% decrease in total antioxidant capacity post-exercise versus 21% in the placebo group (p = 0.35). In conclusion, krill oil can be used as a nutritional strategy for increasing the HS-Omega-3 Index, recover choline concentrations and address oxidative stress after intense power trainings.

Wnt5a is a TLR2/4-ligand that induces tolerance in human myeloid cells.

Innate immune responses are rapid, dynamic and highly regulated to avoid overt reactions. This regulation is executed by innate immune tolerance mechanisms that remain obscure. Wnt5a is a signalling protein mainly involved in developmental processes and cancer. The effect of Wnt5a on inflammatory myeloid cells is controversial. Here, we combine primary cell cultures, in vitro binding studies, mass spectrometry and Drosophila protein modelling to show that Wnt5a is a direct ligand of toll-like receptor (TLR) 2 and 4. The binding promotes a MyD88-non-canonical nuclear factor of kappa B (NFκB) and AP-1 signalling cascade, with contradictory profiles in mouse (pro-inflammatory) and human (anti-inflammatory) myeloid immune cells. These data reveal that the true nature of Wnt5a in inflammatory cells, is to regulate TLR signals, and in human myeloid cells it acts as an endogenous, tolerance-associated molecular pattern (TAMP), inducing IL-10 and innate immune tolerance.

CD14 Is a Co-Receptor for TLR4 in the S100A9-Induced Pro-Inflammatory Response in Monocytes.

The cytosolic Ca2+-binding S100A9 and S100A8 proteins form heterodimers that are primarily expressed in human neutrophils and monocytes. We have recently shown that S100A9 binds to TLR4 in vitro and induces TLR4-dependent NF-κB activation and a pro-inflammatory cytokine response in monocytes. In the present report we have further investigated the S100A9-mediated stimulation of TLR4 in monocytes. Using transmission immunoelectron microscopy, we detected focal binding of S100A9 to monocyte membrane subdomains containing the caveolin-1 protein and TLR4. Furthermore, the S100A9 protein was detected in early endosomes of the stimulated cells, indicating that the protein could be internalized by endocytosis. Although stimulation of monocytes with S100A9 was strictly TLR4-dependent, binding of S100A9 to the plasma membrane and endocytosis of S100A9 was still detectable and coincided with CD14 expression in TLR4-deficient cells. We therefore investigated whether CD14 would be involved in the TLR4-dependent stimulation and could show that the S100A9-induced cytokine response was inhibited both in CD14-deficient cells and in cells exposed to CD14 blocking antibodies. Further, S100A9 was not internalized into CD14-deficient cells suggesting a direct role of CD14 in endocytosis of S100A9. Finally, we could detect satiable binding of S100A9 to CD14 in surface plasmon resonance experiments. Taken together, these results indicate that CD14 is a co-receptor of TLR4 in the S100A9-induced cytokine response.

Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells.

We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway.

The tumor targeted superantigen ABR-217620 selectively engages TRBV7-9 and exploits TCR-pMHC affinity mimicry in mediating T cell cytotoxicity.

The T lymphocytes are the most important effector cells in immunotherapy of cancer. The conceptual objective for developing the tumor targeted superantigen (TTS) ABR-217620 (naptumomab estafenatox, 5T4Fab-SEA/E-120), now in phase 3 studies for advanced renal cell cancer, was to selectively coat tumor cells with cytotoxic T lymphocytes (CTL) target structures functionally similar to natural CTL pMHC target molecules. Here we present data showing that the molecular basis for the anti-tumor activity by ABR-217620 resides in the distinct interaction between the T cell receptor ß variable (TRBV) 7-9 and the engineered superantigen (Sag) SEA/E-120 in the fusion protein bound to the 5T4 antigen on tumor cells. Multimeric but not monomeric ABR-217620 selectively stains TRBV7-9 expressing T lymphocytes from human peripheral blood similar to antigen specific staining of T cells with pMHC tetramers. SEA/E-120 selectively activates TRBV7-9 expressing T lymphocytes resulting in expansion of the subset. ABR-217620 selectively triggers TRBV7-9 expressing cytotoxic T lymphocytes to kill 5T4 positive tumor cells. Furthermore, ABR-217620 activates TRBV7-9 expressing T cell line cells in the presence of cell- and bead-bound 5T4 tumor antigen. Surface plasmon resonance analysis revealed that ABR-217620 binds to 5T4 with high affinity, to TRBV7-9 with low affinity and to MHC class II with very low affinity. The T lymphocyte engagement by ABR-217620 is constituted by displaying high affinity binding to the tumor cells (KD approximately 1 nM) and with the mimicry of natural productive immune TCR-pMHC contact using affinities of around 1 µM. This difference in kinetics between the two components of the ABR-217620 fusion protein will bias the binding towards the 5T4 target antigen, efficiently activating T-cells via SEA/E-120 only when presented by the tumor cells.

Common interactions between S100A4 and S100A9 defined by a novel chemical probe.

S100A4 and S100A9 proteins have been described as playing roles in the control of tumor growth and metastasis. We show here that a chemical probe, oxyclozanide (OX), selected for inhibiting the interaction between S100A9 and the receptor for advanced glycation end-products (RAGE) interacts with both S100A9 and S100A4. Furthermore, we show that S100A9 and S100A4 interact with RAGE and TLR4; interactions that can be inhibited by OX. Hence, S100A4 and S100A9 display similar functional elements despite their primary sequence diversity. This was further confirmed by showing that S100A4 and S100A9 dimerize both in vitro and in vivo. All of these interactions required levels of Zn++ that are found in the extracellular space but not intracellularly. Interestingly, S100A4 and S100A9 are expressed by distinct CD11b+ subpopulations both in healthy animals and in animals with either inflammatory disease or tumor burden. The functions of S100A9 and S100A4 described in this paper, including heterodimerization, may therefore reflect S100A9 and S100A4 that are released into the extra-cellular milieu.

Tasquinimod Is an Allosteric Modulator of HDAC4 survival signaling within the compromised cancer microenvironment.

Tasquinimod is an orally active antiangiogenic drug that is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer. However, the target of this drug has remained unclear. In this study, we applied diverse strategies to identify the histone deacetylase HDAC4 as a target for the antiangiogenic activity of tasquinimod. Our comprehensive analysis revealed allosteric binding (Kd 10-30 nmol/L) to the regulatory Zn(2+) binding domain of HDAC4 that locks the protein in a conformation preventing HDAC4/N-CoR/HDAC3 complex formation. This binding inhibited colocalization of N-CoR/HDAC3, thereby inhibiting deacetylation of histones and HDAC4 client transcription factors, such as HIF-1α, which are bound at promoter/enhancers where epigenetic reprogramming is required for cancer cell survival and angiogenic response. Through this mechanism, tasquinimod is effective as a monotherapeutic agent against human prostate, breast, bladder, and colon tumor xenografts, where its efficacy could be further enhanced in combination with a targeted thapsigargin prodrug (G202) that selectively kills tumor endothelial cells. Together, our findings define a mechanism of action of tasquinimod and offer a perspective on how its clinical activity might be leveraged in combination with other drugs that target the tumor microenvironment. Cancer Res; 73(4); 1386-99. ©2012 AACR.

S100A9 interaction with TLR4 promotes tumor growth.

By breeding TRAMP mice with S100A9 knock-out (S100A9(-/-)) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b(+) S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68(+) macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9(-/-) and TLR4(-/-), but not in RAGE(-/-) animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFß expression in splenic CD11b(+) cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies.

Polypeptide-guided assembly of conducting polymer nanocomposites.

A strategy for fabrication of electroactive nanocomposites with nanoscale organization, based on self-assembly, is reported. Gold nanoparticles are assembled by a polypeptide folding-dependent bridging. The polypeptides are further utilized to recruit and associate with a water soluble conducting polymer. The polymer is homogenously incorporated into the nanocomposite, forming conducting pathways which make the composite material highly conducting.

Supramolecular assembly of designed α-helical polypeptide-based nanostructures and luminescent conjugated polyelectrolytes.

Designed polypeptides with controllable folding properties are utilized as supramolecular templates for fabrication of ordered nanoscale molecular and fibrous assemblies of LCPs. The properties of the LCPs as well as the three dimensional conformation of the polypeptide-scaffold determine how the polymers are arranged in the supramolecular construct, which highly affects the properties of the hybrid material. The ability to control the polypeptide conformation and assembly into fibers provides a promising route for tuning the optical properties of LCPs and for fabrication of complex functional supramolecules with well defined structural properties.

Identification of human S100A9 as a novel target for treatment of autoimmune disease via binding to quinoline-3-carboxamides.

Despite more than 25 years of research, the molecular targets of quinoline-3-carboxamides have been elusive although these compounds are currently in Phase II and III development for treatment of autoimmune/inflammatory diseases in humans. Using photoaffinity cross-linking of a radioactively labelled quinoline-3-carboxamide compound, we could determine a direct association between human S100A9 and quinoline-3-carboxamides. This interaction was strictly dependent on both Zn++ and Ca++. We also show that S100A9 in the presence of Zn++ and Ca++ is an efficient ligand of receptor for advanced glycation end products (RAGE) and also an endogenous Toll ligand in that it shows a highly specific interaction with TLR4/MD2. Both these interactions are inhibited by quinoline-3-carboxamides. A clear structure-activity relationship (SAR) emerged with regard to the binding of quinoline-3-carboxamides to S100A9, as well as these compounds potency to inhibit interactions with RAGE or TLR4/MD2. The same SAR was observed when the compound's ability to inhibit acute experimental autoimmune encephalomyelitis in mice in vivo was analysed. Quinoline-3-carboxamides would also inhibit TNFalpha release in a S100A9-dependent model in vivo, as would antibodies raised against the quinoline-3-carboxamide-binding domain of S100A9. Thus, S100A9 appears to be a focal molecule in the control of autoimmune disease via its interactions with proinflammatory mediators. The specific binding of quinoline-3-carboxamides to S100A9 explains the immunomodulatory activity of this class of compounds and defines S100A9 as a novel target for treatment of human autoimmune diseases.

Conjugated polythiophene probes target lysosome-related acidic vacuoles in cultured primary cells.

Conformation-sensitive optical probes for studying biological processes and structures are of great interest. The present work shows for the first time that conjugated polyelectrolyte (CPE) probes can be used for specific targeting of chromatin, nuclear and cytoplasmatic vesicles, and cytoskeletal components in a complex system of cultured cells. One of the probes could also be used for vital staining of live cells. When bound to different entities, the polythiophene derivative probes emitted light with different colors due to the unique spectral properties of these conformation sensitive probes. The physical pre-requisites for binding could also be exploited for characterization of the target. Unexpectedly, lysosome-related acidic vacuoles were targeted in cultured primary cells by both anionic, cationic, and zwitter-ionic polythiophene derivatives. Pre-treatment with Bafilomycin A1, a specific inhibitor of vacuolar-type H(+)-ATPase, caused redistribution of the staining. The targeting of lysosome-related acidic vesicles could not be demonstrated in transformed cells (melanoma, neuroblastoma, and prostate cancer cell lines), indicating a difference in the localization, structure, accessibility, or quantity of the target in cultured normal cells as compared with the malignant cell lines. The chemical nature of the conjugated polyelectrolyte complex in the cytoplasmatic vacuoles remains elusive.

Discovery of selective small-molecule CD80 inhibitors.

Protein-protein interactions are widely found in biological systems controlling diverse cellular events. Because these interactions are implicated in many diseases such as autoimmunity and cancer, regulation of protein-protein interactions provides ideal targets for drug intervention. The CD80-CD28 costimulatory pathway plays a critical role in regulation of the immune response and thus constitutes an attractive target for therapeutic manipulation of autoimmune diseases. The objective of this study is to identify small compounds disrupting these pivotal protein-protein interactions. Compounds that specifically blocked binding of CD80 to CD28 were identified using a strategy involving a cell-based scintillation proximity assay as the initial step. Secondary screening (e.g., by analyzing the direct binding of these compounds to the target immobilized on a biosensor surface) revealed that these compounds are highly selective CD80 binders. Screening of structurally related derivatives led to the identification of the chemical features required for inhibition of the CD80-CD28 interaction. In addition, the optimization process led to a 10-fold increase in binding affinity of the CD80 inhibitors. Using this approach, the authors identify low-molecular-weight compounds that specifically and with high potency inhibit the interaction between CD80 and CD28. These compounds serve as promising starting points for further development of CD80 inhibitors as potential immunomodulatory drugs.

Identification of protein-protein interfaces implicated in CD80-CD28 costimulatory signaling.

The B7 ligands CD80 and CD86 on APCs deliver either costimulatory or inhibitory signals to the T cell when interacting with their counter-receptors CD28 and CD152 (CTLA-4) on the T cell surface. Although crucial for lymphocyte regulation, the structural basis of these interactions is still not completely understood. Using multivalent presentation and conditions mimicking clustering, believed to be essential for signaling through these receptors, and by applying a combined differential mass spectrometry and structural mapping approach to these conditions, we were able to identify a putative contact area involving hydrophilic regions on both CD28 and CD80 as well as a putative CD28 oligomerization interface induced by B7 ligation. Analysis of the CD80-CD28 interaction site reveals a well-defined interface structurally distinct from that of CD80 and CD152 and thus provides valuable information for therapeutic intervention targeted at this pathway, suggesting a general approach for other receptors.