Interactions between Siglec-7/9 receptors and ligands influence NK cell-dependent tumor immunosurveillance.

Alteration of the surface glycosylation pattern on malignant cells potentially affects tumor immunity by directly influencing interactions with glycan-binding proteins (lectins) on the surface of immunomodulatory cells. The sialic acid-binding Ig-like lectins Siglec-7 and -9 are MHC class I-independent inhibitory receptors on human NK cells that recognize sialic acid-containing carbohydrates. Here, we found that the presence of Siglec-9 defined a subset of cytotoxic NK cells with a mature phenotype and enhanced chemotactic potential. Interestingly, this Siglec-9+ NK cell population was reduced in the peripheral blood of cancer patients. Broad analysis of primary tumor samples revealed that ligands of Siglec-7 and -9 were expressed on human cancer cells of different histological types. Expression of Siglec-7 and -9 ligands was associated with susceptibility of NK cell-sensitive tumor cells and, unexpectedly, of presumably NK cell-resistant tumor cells to NK cell-mediated cytotoxicity. Together, these observations have direct implications for NK cell-based therapies and highlight the requirement to consider both MHC class I haplotype and tumor-specific glycosylation.

Weaker ligands can dominate an odor blend due to syntopic interactions.

Most odors in natural environments are mixtures of several compounds. Perceptually, these can blend into a new "perfume," or some components may dominate as elements of the mixture. In order to understand such mixture interactions, it is necessary to study the events at the olfactory periphery, down to the level of single-odorant receptor cells. Does a strong ligand present at a low concentration outweigh the effect of weak ligands present at high concentrations? We used the fruit fly receptor dOr22a and a banana-like odor mixture as a model system. We show that an intermediate ligand at an intermediate concentration alone elicits the neuron's blend response, despite the presence of both weaker ligands at higher concentration, and of better ligands at lower concentration in the mixture. Because all of these components, when given alone, elicited significant responses, this reveals specific mixture processing already at the periphery. By measuring complete dose-response curves we show that these mixture effects can be fully explained by a model of syntopic interaction at a single-receptor binding site. Our data have important implications for how odor mixtures are processed in general, and what preprocessing occurs before the information reaches the brain.

The T-cell receptor is not hardwired to engage MHC ligands.

The bias of αβ T cells for MHC ligands has been proposed to be intrinsic to the T-cell receptor (TCR). Equally, the CD4 and CD8 coreceptors contribute to ligand restriction by colocalizing Lck with the TCR when MHC ligands are engaged. To determine the importance of intrinsic ligand bias, the germ-line TCR complementarity determining regions were extensively diversified in vivo. We show that engagement with MHC ligands during thymocyte selection and peripheral T-cell activation imposes remarkably little constraint over TCR structure. Such versatility is more consistent with an opportunist, rather than a predetermined, mode of interface formation. This hypothesis was experimentally confirmed by expressing a hybrid TCR containing TCR-γ chain germ-line complementarity determining regions, which engaged efficiently with MHC ligands.

Ligands for pheromone-sensing neurons are not conformationally activated odorant binding proteins.

Pheromones form an essential chemical language of intraspecific communication in many animals. How olfactory systems recognize pheromonal signals with both sensitivity and specificity is not well understood. An important in vivo paradigm for this process is the detection mechanism of the sex pheromone (Z)-11-octadecenyl acetate (cis-vaccenyl acetate [cVA]) in Drosophila melanogaster. cVA-evoked neuronal activation requires a secreted odorant binding protein, LUSH, the CD36-related transmembrane protein SNMP, and the odorant receptor OR67d. Crystallographic analysis has revealed that cVA-bound LUSH is conformationally distinct from apo (unliganded) LUSH. Recombinantly expressed mutant versions of LUSH predicted to enhance or diminish these structural changes produce corresponding alterations in spontaneous and/or cVA-evoked activity when infused into olfactory sensilla, leading to a model in which the ligand for pheromone receptors is not free cVA, but LUSH that is "conformationally activated" upon cVA binding. Here we present evidence that contradicts this model. First, we demonstrate that the same LUSH mutants expressed transgenically affect neither basal nor pheromone-evoked activity. Second, we compare the structures of apo LUSH, cVA/LUSH, and complexes of LUSH with non-pheromonal ligands and find no conformational property of cVA/LUSH that can explain its proposed unique activated state. Finally, we show that high concentrations of cVA can induce neuronal activity in the absence of LUSH, but not SNMP or OR67d. Our findings are not consistent with the model that the cVA/LUSH complex acts as the pheromone ligand, and suggest that pheromone molecules alone directly activate neuronal receptors.

Guiding ligands to nuclear receptors.

Retinoic acid-the active metabolite of vitamin A-influences biological processes by activating the retinoic acid receptor (RAR). In this issue, Schug et al. (2007) demonstrate that retinoic acid also activates the peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta). Remarkably, retinoic acid signaling through RAR or PPARbeta/delta-which depends on cytoplasmic retinoic acid transporters-commits the cell to opposite fates, apoptosis or survival, respectively.

Nano-particle vaccination combined with TLR-7 and -9 ligands triggers memory and effector CD8⁺ T-cell responses in melanoma patients.

Optimal vaccine strategies must be identified for improving T-cell vaccination against infectious and malignant diseases. MelQbG10 is a virus-like nano-particle loaded with A-type CpG-oligonucleotides (CpG-ODN) and coupled to peptide(16-35) derived from Melan-A/MART-1. In this phase IIa clinical study, four groups of stage III-IV melanoma patients were vaccinated with MelQbG10, given (i) with IFA (Montanide) s.c.; (ii) with IFA s.c. and topical Imiquimod; (iii) i.d. with topical Imiquimod; or (iv) as intralymph node injection. In total, 16/21 (76%) patients generated ex vivo detectable Melan-A/MART-1-specific T-cell responses. T-cell frequencies were significantly higher when IFA was used as adjuvant, resulting in detectable T-cell responses in all (11/11) patients, with predominant generation of effector-memory-phenotype cells. In turn, Imiquimod induced higher proportions of central-memory-phenotype cells and increased percentages of CD127(+) (IL-7R) T cells. Direct injection of MelQbG10 into lymph nodes resulted in lower T-cell frequencies, associated with lower proportions of memory and effector-phenotype T cells. Swelling of vaccine site draining lymph nodes, and increased glucose uptake at PET/CT was observed in 13/15 (87%) of evaluable patients, reflecting vaccine triggered immune reactions in lymph nodes. We conclude that the simultaneous use of both Imiquimod and CpG-ODN induced combined memory and effector CD8(+) T-cell responses.

An update of the mechanisms of resistance to EGFR-tyrosine kinase inhibitors in breast cancer: gefitinib (Iressatrade mark)-induced changes in the expression and nucleo-cytoplasmic trafficking of HER-ligands (Review)

Intrinsic resistance to the epidermal growth factor receptor (EGFR; HER1) tyrosine kinase inhibitor (TKI) gefitinib, and more generally to EGFR TKIs, is a common phenomenon in breast cancer. The availability of molecular criteria for predicting sensitivity to EGFR-TKIs is, therefore, the most relevant issue for their correct use and for planning future research. Though it appears that in non-small-cell lung cancer (NSCLC) response to gefitinib is directly related to the occurrence of specific mutations in the EGFR TK domain, breast cancer patients cannot be selected for treatment with gefitinib on the same basis as such EGFR mutations have beenreported neither in primary breast carcinomas nor in several breast cancer cell lines. Alternatively, there is a generalagreement on the hypothesis that the occurrence of molecular alterations that activate transduction pathways downstreamof EGFR (i.e., MEK1/MEK2 - ERK1/2 MAPK and PI-3'K - AKT growth/survival signaling cascades) significantly affect the response to EGFR TKIs in breast carcinomas. However,there are no studies so far addressing a role of EGF-related ligands as intrinsic breast cancer cell modulators of EGFR TKIefficacy. We recently monitored gene expression profiles andsub-cellular localization of HER-1/-2/-3/-4 related ligands (i.e., EGF, amphiregulin, transforming growth factor-α, ß-cellulin,epiregulin and neuregulins) prior to and after gefitinib treatment in a panel of human breast cancer cell lines. First, gefitinibinduced changes in the endogenous levels of EGF-related ligands correlated with the natural degree of breast cancer cellsensitivity to gefitinib. While breast cancer cells intrinsically resistant to gefitinib (IC50 ≥15 μM) markedly up-regulated(up to 600 times) the expression of genes codifying for HERspecific ligands, a significant down-regulation (up to 106 times)of HER ligand gene transcription was found in breast cancer cells intrinsically sensitive to gefitinib (IC50 ≤1 μM). Second,loss of HER1 function differentially regulated the nuclear trafficking of HER-related ligands. While gefitinib treatment induced an active import and nuclear accumulation of the HER ligand NRG in intrinsically gefitinib-resistant breastcancer cells, an active export and nuclear loss of NRG was observed in intrinsically gefitinib-sensitive breast cancer cells.In summary, through in vitro and pharmacodynamic studies we have learned that, besides mutations in the HER1 gene,oncogenic changes downstream of HER1 are the key players regulating gefitinib efficacy in breast cancer cells. It now appears that pharmacological inhibition of HER1 functionalso leads to striking changes in both the gene expression and the nucleo-cytoplasmic trafficking of HER-specific ligands,and that this response correlates with the intrinsic degree of breast cancer sensitivity to the EGFR TKI gefitinib. Therelevance of this previously unrecognized intracrine feedback to gefitinib warrants further studies as cancer cells could bypassthe antiproliferative effects of HER1-targeted therapeutics without a need for the overexpression and/or activation of other HER family members and/or the activation of HER-driven downstream signaling cascades

Surface functionalization of alumina ceramic foams with organic ligands.

Different anchoring groups have been studied with the aim of covalently binding organic linkers to the surface of alumina ceramic foams. The results suggested that a higher degree of functionalization was achieved with a pyrogallol derivative - as compared to its catechol analogue - based on the XPS analysis of the ceramic surface. The conjugation of organic ligands to the surface of these alumina materials was corroborated by DNP-MAS NMR measurements.

ErbB Ligands in Angiogenesis

The formation of new blood vessels, i.e. angiogenesis, is an important phenomenon during normal development and wound repair, as well as during various pathological processes, such as tumor growth and metastasis. Specific growth factors regulate angiogenesis by modulating the different cellular functions of endothelial cells (EC), and periendothelial cells, such as pericytes (PC) and smooth muscle cells (SMC), which interact with ECs in a paracrine manner. ErbB receptors form a subgroup of transmembrane receptor tyrosine kinases that interact with growth factors of the epidermal growth factor (EGF) family. ErbB receptors regulate behaviour of a variety of normal as well as tumor cell types. Cancer drugs that target epidermal growth factor receptor (EGFR, ErbB1) or ErbB2 receptor have been approved for clinical use. It has been speculated that part of the antitumor activity of ErbB inhibitor compounds result from an antiangiogenic mechanism. The results presented here indicate a role for endothelial-derived EGF-like growth factors heparin binding EGF-like growth factor (HB-EGF) and neuregulin-1 (NRG-1) in the paracrine regulation of angiogenesis. HB-EGF, EGFR and ErbB2 are shown to mediate interaction between ECs and SMCs in vitro, and gefitinib, an inhibitor of EGFR kinase activity, suppresses recruitment of PCs/SMCs in vivo. NRG-1 is shown to regulate EC functions in vitro and angiogenesis in vivo by indirect mechanisms involving vascular endothelial growth factor-A (VEGF-A) and VEGF receptor-2 (VEGFR-2). Furthermore, EGFR activity is demonstrated to regulate recruitment of bone marrow-derived perivascular cells during tumor neovascularization in vivo. These results indicate that ErbB signaling is involved in the cellular processes of new blood vessel formation. This study gives new information about the role of ErbB ligands and receptors in angiogenesis and vasculogenesis and about the mechanisms by which ErbB inhibitor drugs such as gefitinib affect tumor growth.

Estudio epidemiológico de úlceras por presión en tobillo-pie (UPP-TP) en el lesionado medular ingresado

Objetivo general: Determinar los indicadores de incidencia y prevalencia de úlceras por presión en tobillo-pie en lesionados medulares ingresados. Material y métodos: Ensayo clínico controlado de 549 lesionados medulares, durante 48 meses. Resultados: La incidencia promedio de pacientes con UPP-TP de todo el periodo estudiado fue de: 1"178 pac./mes. La incidencia promedio de UPP-TP de todo el periodo estudiado fue de: 1"789 UPP-TP/mes. La prevalencia promedio de pacientes con UPP-TP de todo el periodo estudiado, respecto al total de pacientes fue de: 7"75%. La prevalencia promedio de todo el periodo de las UPP-TP, respecto al total de UPP fue de: 39"28%. La prevalencia promedio de todo el periodo estudiado de las UPP-TP, respecto al total de heridas (UPP+Traumáticas) fue de: 24"39%. Todos los pacientes crónicos con UPP-TP presentaban LM completa, por tanto ningún pac. crónico incompleto presento UPP-TP. Un 39"66% (23 pac.) presentaron más de 1 lesión. El 47"72% de las UPP-TP se presentaron en talón y un 19"32 en maléolo ext, siendo respectivamente las zonas de mayor localización de las lesiones. Un 13"63% de las UPP-TP se presentaron en dedos a pesar de la no deambulación de estos y de los 6 pac. que tuvieron la lesión en 1º dedo 4 pac. padecieron onicocriptosis. En el 19"23% (5 pac.) de los pac. crónicos sus UPPTP presentaron estadio IV, 3 pac. la presentaron en su domicilio y 2 pac. en ULM. El 25"86% de los pac. cicatrizaron sus lesiones dentro del 1º mes. Conclusiones: Teniendo en cuenta que el 29"30% de los pacientes, la incidencia de su UPP-TP, tuvo origen en el domicilio habitual, que de estos el 94% eran crónicos con LM completa y que el 34"48% del total de pacientes afectos de UPP-TP del estudio fueron dados de alta a su domicilio sin la curación de su úlcera, es notoria la importancia del seguimiento domiciliario de proximidad, la educación sanitaria de pacientes y familiarescuidadores, así como el uso de guías clínicas preventivas en nuestra asistencia podológica, especialmente en pacientes con lesión medular completa.

Platinum (II) and palladium (II) complexes with (N,N') and (C,N,N') ligands derived from pyrazole as anticancer and antimalarial agents: synthesis, characterization and in vitro activities

The study of the reactivity of three 1-(2-dimethylaminoethyl)-1H-pyrazole derivatives of general formula [1-(CH2)2NMe2}-3,5-R2-pzol] {where pzol represents pyrazole and Rdouble bond; length as m-dashH (1a), Me (1b) or Ph (1c)} with [MCl2(DMSO)2] (Mdouble bond; length as m-dashPt or Pd) under different experimental conditions allowed us to isolate and characterize cis-[M{κ2-N,N′-{[1-(CH2)2NMe2}-3,5-R2-pzol])}Cl2] {MMdouble bond; length as m-dashPtPt (2a-2c) or Pd (3a-3c)} and two cyclometallated complexes [M{κ3-C,N,N′-{[1-(CH2)2NMe2}-3-(C5H4)-5-Ph-pzol])}Cl] {Mdouble bond; length as m-dashPt(II) (4c) or Pd(II) (5c)}. Compounds 4c and 5c arise from the orthometallation of the 3-phenyl ring of ligand 1c. Complex 2a has been further characterized by X-ray crystallography. Ligands and complexes were evaluated for their in vitro antimalarial against Plasmodium falciparum and cytotoxic activities against lung (A549) and breast (MDA MB231 and MCF7) cancer cellular lines. Complexes 2a-2c and 5c exhibited only moderate antimalarial activities against two P. falciparum strains (3D7 and W2). Interestingly, cytotoxicity assays revealed that the platinacycle 4c exhibits a higher toxicity than cisplatin in the three human cell lines and that the complex 2a presents a remarkable cytotoxicity and selectivity in lung (IC50 = 3 μM) versus breast cancer cell lines (IC50 > 20 μM). Thus, complexes 2c and 4c appear to be promising leads, creating a novel family of anticancer agents. Electrophoretic DNA migration studies in presence of the synthesized compounds have been performed, in order to get further insights into their mechanism of action.

Nanocarriers for DNA Vaccines: Co-Delivery of TLR-9 and NLR-2 Ligands Leads to Synergistic Enhancement of Proinflammatory Cytokine Release

Adjuvants enhance immunogenicity of vaccines through either targeted antigen delivery or stimulation of immune receptors. Three cationic nanoparticle formulations were evaluated for their potential as carriers for a DNA vaccine, and muramyl dipeptide (MDP) as immunostimulatory agent, to induce and increase immunogenicity of Mycobacterium tuberculosis antigen encoding plasmid DNA (pDNA). The formulations included (1) trimethyl chitosan (TMC) nanoparticles, (2) a squalene-in-water nanoemulsion, and (3) a mineral oil-in-water nanoemulsion. The adjuvant effect of the pDNA-nanocomplexes was evaluated by serum antibody analysis in immunized mice. All three carriers display a strong adjuvant effect, however, only TMC nanoparticles were capable to bias immune responses towards Th1. pDNA naturally contains immunostimulatory unmethylated CpG motifs that are recognized by Toll-like receptor 9 (TLR-9). In mechanistic in vitro studies, activation of TLR-9 and the ability to enhance immunogenicity by simultaneously targeting TLR-9 and NOD-like receptor 2 (NLR-2) was determined by proinflammatory cytokine release in RAW264.7 macrophages. pDNA in combination with MDP was shown to significantly increase proinflammatory cytokine release in a synergistic manner, dependent on NLR-2 activation. In summary, novel pDNA-Ag85A loaded nanoparticle formulations, which induce antigen specific immune responses in mice were developed, taking advantage of the synergistic combinations of TLR and NLR agonists to increase the adjuvanticity of the carriers used.

rDock: A Fast, Versatile and Open Source Program for Docking Ligands to Proteins and Nucleic Acids

Identification of chemical compounds with specific biological activities is an important step in both chemical biology and drug discovery. When the structure of the intended target is available, one approach is to use molecular docking programs to assess the chemical complementarity of small molecules with the target; such calculations provide a qualitative measure of affinity that can be used in virtual screening (VS) to rank order a list of compounds according to their potential to be active. rDock is a molecular docking program developed at Vernalis for high-throughput VS (HTVS) applications. Evolved from RiboDock, the program can be used against proteins and nucleic acids, is designed to be computationally very efficient and allows the user to incorporate additional constraints and information as a bias to guide docking. This article provides an overview of the program structure and features and compares rDock to two reference programs, AutoDock Vina (open source) and Schrodinger's Glide (commercial). In terms of computational speed for VS, rDock is faster than Vina and comparable to Glide. For binding mode prediction, rDock and Vina are superior to Glide. The VS performance of rDock is significantly better than Vina, but inferior to Glide for most systems unless pharmacophore constraints are used; in that case rDock and Glide are of equal performance. The program is released under the Lesser General Public License and is freely available for download, together with the manuals, example files and the complete test sets, at http://rdock.sourceforge.net/

Terutroban, A TP-receptor antagonist, reduces portal pressure in cirrhotic rats.

Increased production of vasoconstrictive prostanoids, such as thromboxane A2 (TXA2 ), contributes to endothelial dysfunction and increased hepatic vascular tone in cirrhosis. TXA2 induces vasoconstriction by way of activation of the thromboxane-A2 /prostaglandin-endoperoxide (TP) receptor. This study investigated whether terutroban, a specific TP receptor blocker, decreases hepatic vascular tone and portal pressure in rats with cirrhosis due to carbon tetrachloride (CCl4 ) or bile duct ligation (BDL). Hepatic and systemic hemodynamics, endothelial dysfunction, liver fibrosis, hepatic Rho-kinase activity (a marker of hepatic stellate cell contraction), and the endothelial nitric oxide synthase (eNOS) signaling pathway were measured in CCl4 and BDL cirrhotic rats treated with terutroban (30 mg/kg/day) or its vehicle for 2 weeks. Terutroban reduced portal pressure in both models without producing significant changes in portal blood flow, suggesting a reduction in hepatic vascular resistance. Terutroban did not significantly change arterial pressure in CCl4 -cirrhotic rats but decreased it significantly in BDL-cirrhotic rats. In livers from CCl4 and BDL-cirrhotic terutroban-treated rats, endothelial dysfunction was improved and Rho-kinase activity was significantly reduced. In CCl4 -cirrhotic rats, terutroban reduced liver fibrosis and decreased alpha smooth muscle actin (α-SMA), collagen-I, and transforming growth factor beta messenger RNA (mRNA) expression without significant changes in the eNOS pathway. In contrast, no change in liver fibrosis was observed in BDL-cirrhotic rats but an increase in the eNOS pathway. CONCLUSION: Our data indicate that TP-receptor blockade with terutroban decreases portal pressure in cirrhosis. This effect is due to decreased hepatic resistance, which in CCl4 -cirrhotic rats was linked to decreased hepatic fibrosis, but not in BDL rats, in which the main mediator appeared to be an enhanced eNOS-dependent vasodilatation, which was not liver-selective, as it was associated with decreased arterial pressure. The potential use of terutroban for portal hypertension requires further investigation.

Síntese e caracterização de novos compostos de coordenação de cobre (II) com ligantes não-simétricos N,O-doadores: contribuições para o sítio ativo da galactose oxidase

The reactions of four new unsymmetrical N,O-donor ligands, {H2BBPETEN= [N-(2-hydroxybenzyl) - N,N' - bis(2 methylpyridyl) -N'-(hydroxyethyl) ethylenodiamine], H3BPETEN=[N,N'- bis(2-hydroxybenzyl) -N- (2-methylpyridyl) -N'- (hydroxyethyl) ethylenodiamine], HTPETEN=[N,N,N'- tris(2-methylpyridyl) -N'- (hydroxyethyl) ethylenodiamine] and H3BIMETEN=[N,N'-(2-hydroxybenzyl)-N-(1-methylimidazol-2-il-methyl)-N'- (hydroxyethyl)ethylenodiamine]}, with Cu(II) salts afforded the following mononuclear compounds: [CuII(HBBPETEN)]ClO4, [CuII(H2BPETEN)]ClO4 , [CuII(HTPETEN)](PF6)2 and [CuII(H2BIMETEN)]ClO4 . All were characterized by EPR, electronic spectroscopy and electrochemistry. The four copper (II) compounds showed interesting electrochemistry properties. All presented an anodic wave that can be attributed to the Cu (I) oxide formation at the electrode surface, or to a Cu0 sediment at the same surface or yet, to Cu(I) -> Cu(II) oxidation process with coupled chemistry reaction, due to their irreversibility. Two of the complexes are described as interesting synthetic models for the active site of the metalloenzyme galactose oxidase.