Descripción: Dendritic cell (DC) and natural killer (NK) cell interactions are important for the regulation of innate and adaptive immunity, but their relevance during early pregnancy remains elusive. Using two different strategies to manipulate the frequency of NK cells and DC during gestation, we investigated their relative impact on the decidualization process and on angiogenic responses that characterize murine implantation. Manipulation of the frequency of NK cells, DC or both lead to a defective decidual response characterized by decreased proliferation and differentiation of stromal cells. Whereas no detrimental effects were evident upon expansion of DC, NK cell ablation in such expanded DC mice severely compromised decidual development and led to early pregnancy loss. Pregnancy failure in these mice was associated with an unbalanced production of anti-angiogenic signals and most notably, with increased expression of genes related to inflammation and immunogenic activation of DC. Thus, NK cells appear to play an important role counteracting potential anomalies raised by DC expansion and overactivity in the decidua, becoming critical for normal pregnancy progression. © 2012 González et al.

Descripción: Background: The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity. Methodology/Principal Findings: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GRDNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different GR-ligand complexes. No correlation was found between GR intranuclear distribution, cofactor recruitment and the homodimerization process. Finally, Molecular determinants that support the observed experimental GR LBD-ligand/TIF2 interaction were found by Molecular Dynamics simulation. Conclusions/Significance: The data presented here sustain the idea that in vivo GR homodimerization inside the nucleus can be achieved in a DNA-independent fashion, without ruling out a dependent pathway as well. Moreover, since at least one GR-ligand complex is able to induce homodimer formation while preventing TIF2 coactivator interaction, results suggest that these two events might be independent from each other. Finally, 21-hydroxy-6,19-epoxyprogesterone arises as a selective glucocorticoid with potential pharmacological interest. Taking into account that GR homodimerization and cofactor recruitment are considered essential steps in the receptor activation pathway, results presented here contribute to understand how specific ligands influence GR behavior. © 2010 Presman et al.

Descripción: Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium- binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus. © 2013, American Society for Microbiology.

Descripción: Curcumin (diferuloylmethane) is the active ingredient of the spice plant Curcuma longa and has been shown to act anti-tumorigenic in different types of tumours. Therefore, we have studied its effect in pituitary tumour cell lines and adenomas. Proliferation of lactosomatotroph GH3 and somatotroph MtT/S rat pituitary cells as well as of corticotroph AtT20 mouse pituitary cells was inhibited by curcumin in monolayer cell culture and in colony formation assay in soft agar. Fluorescence-activated cell sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G2/M. Analysis of cell cycle proteins by immunoblotting showed reduction in cyclin D1, cyclin-dependent kinase 4 and no change in p27kip. FACS analysis with Annexin V-FITC/7-aminoactinomycin D staining demonstrated curcumin-induced early apoptosis after 3, 6, 12 and 24 h treatment and nearly no necrosis. Induction of DNA fragmentation, reduction of Bcl-2 and enhancement of cleaved caspase-3 further confirmed induction of apoptosis by curcumin. Growth of GH3 tumours in athymic nude mice was suppressed by curcumin in vivo. In endocrine pituitary tumour cell lines, GH, ACTH and prolactin production were inhibited by curcumin. Studies in 25 human pituitary adenoma cell cultures have confirmed the antitumorigenic and hormone-suppressive effects of curcumin. Altogether, the results described in this report suggest this natural compound as a good candidate for therapeutic use on pituitary tumours. © 2009 Society for Endocrinology Printed in Great Britain.

Descripción: Dimethyl sulfoxide (DMSO) has been known to enhance cell membrane permeability of drugs or DNA. Molecular dynamics (MD) simulations with single-component lipid bilayers predicted the existence of three regimes of action of DMSO: membrane loosening, pore formation and bilayer collapse. We show here that these modes of action are also reproduced in the presence of cholesterol in the bilayer, and we provide a description at the atomic detail of the DMSO-mediated process of pore formation in cholesterol-containing lipid membranes. We also successfully explore the applicability of DMSO to promote plasma membrane permeability to water, calcium ions (Ca2+) and Yo-Pro-1 iodide (Yo-Pro-1) in living cell membranes. The experimental results on cells in culture can be easily explained according to the three expected regimes: in the presence of low doses of DMSO, the membrane of the cells exhibits undulations but no permeability increase can be detected, while at intermediate DMSO concentrations cells are permeabilized to water and calcium but not to larger molecules as Yo-Pro-1. These two behaviors can be associated to the MD-predicted consequences of the effects of the DMSO at low and intermediate DMSO concentrations. At larger DMSO concentrations, permeabilization is larger, as even Yo-Pro-1 can enter the cells as predicted by the DMSO-induced membrane-destructuring effects described in the MD simulations. © 2012 de Ménorval et al.

Descripción: Since apoptosis appeared to be related to neurodegenerative processes, neuroprotection has been involved in investigation of therapeutic approaches focused upon pharmacological agents to prevent neuronal programmed cell death. In this regard, erythropoietin (Epo) seems to play a critical role. The present work was focused on the study of the Epo protective effect upon human neuroblastoma SH-SY5Y cells subjected to differentiation by staurosporine. Under this condition, profuse neurite outgrowth was accompanied by programmed cell death (35% of apoptotic cells by Hoechst assay, showing characteristic DNA ladder pattern). A previous treatment with recombinant human Epo (rHuEpo) increased the expression of the specific receptor for Epo while prevented apoptosis. Simultaneously, morphological changes in neurite elongation and interconnection induced by staurosporine were blocked by Epo. These Epo effects proved to be associated to the induction of Bcl-xL at the mRNA and protein levels (RT-PCR and Western blot after immunoprecipitation) and were mediated by activation of pathways inhibited by wortmannin. In conclusion, the fact that both events induced by staurosporine, cell apoptosis and differentiation, were prevented in SH-SY5Y cells previously exposed to rHuEpo suggests interrelated signaling pathways triggered by the Epo/EpoR interaction. © 2005 Elsevier B.V. All rights reserved.

Descripción: The antiviral activity against dengue virus-2 (DENV-2) of carrageenans reported here has shown a differential susceptibility of C6/36 HT and Vero cells, taken as models of mosquito and mammalian cells, depending on the structural class of polysaccharides: all polysaccharides blocked DENV-2 infection in monkey Vero cells, but only iota-carrageenans were virus inhibitors in mosquito cells. However, iota-carrageenans were less effective in mosquito cells in comparison with mammalian cells with effective concentration 50% (EC50) values in C6/36 HT cells 4.9-17.5-fold higher than in Vero cells, as determined by virus yield reduction assay. The mode of action of iota-carrageenan in both cell types was strikingly different: in Vero cells the inhibitory activity was exerted only at the initiation of the cycle, affecting virion binding, whereas in mosquito cells DENV-2 adsorption was not affected and comparable levels of inhibition were obtained if the compound was added to cells together with the virus, after 8 h of infection or by cell pretreatment before infection. Furthermore, iota-carrageenans induced a subtle alteration in mosquito cells, detected by cell proliferation and protein synthesis analyses, suggesting that a probable cellular target may be responsible for the refractory state of mosquito cells to DENV-2 infection produced by this class of polysulfates. The failure of iota-carrageenan to block DENV-2 adsorption to mosquito cells appeared to be related to the low presence of adequate heparin sulfate (HS) in C6/36 HT cell surface and is indicative of a differential participation of HS residues for DENV-2 entry in both types of cells. © 2011 SGM.

Descripción: The objective of this work was to evaluate the effects of chlorpyrifos on earthworms and on soil functional parameters. An integrated laboratory-field study was performed in a wheat fi eld in Argentina, sprayed with chlorpyrifos at two recommended application rates (240 or 960 g ha -1 a.i.). Laboratory tests included neutral red retention time, comet assay (single cell gel electrophoresis), and avoidance behavior, each using the earthworm Eisenia andrei exposed in soil collected 1 or 14 days after pesticide application, and the bait-lamina test. Field tests assessed organic matter breakdown using the litterbag and bait-lamina assays. Earthworm populations in the field were assessed using formalin application and hand-sorting. The neutral red retention time and comet assays were sensitive biomarkers to the effects of chlorpyrifos on the earthworm E. andrei; however, the earthworm avoidance test was not sufficiently robust to assess these effects. Feeding activity of soil biota, assessed by the bait lamina test, was significantly inhibited by chlorpyrifos after 97 days, but recovered by the 118th day of the test. Litterbag test showed no significant differences in comparison to controls. Earthworm abundance in the fi eld was too low to adequately test the sensitivity of this assessment endpoint.

Descripción: The retrograde transport of nerve growth factor (NGF) in neurite-like processes of living differentiated PC12 cells was studied using streptavidin-quantum dots (QDs) coupled to monobiotin-NGF. These reagents were active in differentiation, binding, internalization, and transport. Ten-35% of the QD-NGF-receptor complexes were mobile. Quantitative single particle tracking revealed a bidirectional step-like motion, requiring intact microtubules, with a net retrograde velocity of 0.054 ± 0.020 μm/s. Individual runs had a mean velocity of ∼0.15 μm/s at room temperature, and the run times were exponentially distributed. The photostability and brightness of QDs permit extended real-time analysis of individual QDbNGF- receptor complexes trafficking within neurites. © 2007 Federation of European Biochemical Societies.

Descripción: The entry of DENV into the host cell appears to be a very complex process which has been started to be studied in detail. In this report, the route of functional intracellular trafficking after endocytic uptake of dengue virus serotype 1 (DENV-1) strain HW, DENV-2 strain NGC and DENV-2 strain 16681 into Vero cells was studied by using a susceptibility to ammonium chloride assay, dominant negative mutants of several members of the family of cellular Rab GTPases that participate in regulation of transport through endosome vesicles and immunofluorescence colocalization. Together, the results presented demonstrate that in spite of the different internalization route among viral serotypes in Vero cells and regardless of the viral strain, DENV particles are first transported to early endosomes in a Rab5-dependent manner. Then a Rab7-dependent pathway guides DENV-2 16681 to late endosomes, whereas a yet unknown sorting event controls the transport of DENV-2 NGC, and most probably DENV-1 HW, to the perinuclear recycling compartments where fusion membrane would take place releasing nucleocapsid into the cytoplasm. Besides the demonstration of a different intracellular trafficking for two DENV-2 strains that shared the initial clathrin-independent internalization route, these studies proved for the first time the involvement of the slow recycling pathway for DENV-2 productive infection. © 2012 Acosta et al.

Descripción: Although the negative regulation of gene expression by insulin has been widely studied, the transcription factors responsible for the insulin effect are still unknown. The purpose of this work was to explore the molecular mechanisms involved in the insulin repression of the 5-aminolevulinate synthase (ALAS) gene. Deletion analysis of the 5′-regulatory region allowed us to identify an insulin-responsive region located at -459 to -354 bp. This fragment contains a highly homologous insulin-responsive (IRE) sequence. By transient transfection assays, we determined that hepatic nuclear factor 3 (HNF3) and nuclear factor 1 (NF1) are necessary for an appropriate expression of the ALAS gene. Insulin overrides the HNF3β or HNF3β plus NF1-mediated stimulation of ALAS transcriptional activity. Electrophoretic mobility shift assay and Southwestern blotting indicate that HNF3 binds to the ALAS promoter. Mutational analysis of this region revealed that IRE disruption abrogates insulin action, whereas mutation of the HNF3 element maintains hormone responsiveness. This dissociation between HNF3 binding and insulin action suggests that HNF3β is not the sole physiologic mediator of insulin-induced transcriptional repression. Furthermore, Southwestern blotting assay shows that at least two polypeptides other than HNF3β can bind to ALAS promoter and that this binding is dependent on the integrity of the IRE. We propose a model in which insulin exerts its negative effect through the disturbance of HNF3β binding or transactivation potential, probably due to specific phosphorylation of this transcription factor by Akt. In this regard, results obtained from transfection experiments using kinase inhibitors support this hypothesis. Due to this event, NF1 would lose accessibility to the promoter. The posttranslational modification of HNF3 would allow the binding of a protein complex that recognizes the core IRE. These results provide a potential mechanism for the insulin-mediated repression of IRE-containing promoters.

Descripción: Background/ Aims: Since the reversible phosphorylation of tyrosyl residues is a critical event in cellular signaling pathways activated by erythropoietin (Epo), attention has been focused on protein tyrosine phosphatases (PTPs) and their coordinated action with protein tyrosine kinases. The prototypic member of the PTP family is PTP1B, a widely expressed non-receptor PTP located both in cytosol and intracellular membranes via its hydrophobic C-terminal targeting sequence. PTP1B has been implicated in the regulation of signaling pathways involving tyrosine phosphorylation induced by growth factors, cytokines, and hormones, such as the downregulation of erythropoietin and insulin receptors. However, little is known about which factor modulates the activity of this enzyme. Methods: The effect of Epo on PTP1B expression was studied in the UT-7 Epo-dependent cell line. PTP1B expression was analyzed under different conditions by Real-Time PCR and Western blot, while PTP1B phosphatase activity was determined by a p-nitrophenylphosphate hydrolysis assay. Results: Epo rapidly induced an increased expression of PTP1B which was associated with higher PTP1B tyrosine phosphorylation and phosphatase activity. The action of Epo on PTP1B induction involved Janus Kinase 2 (JAK2) and Phosphatidylinositol-3 kinase (PI3K). Conclusion: The results allow us to suggest for the first time that, besides modulating Epo/Epo receptor signaling, PTP1B undergoes feedback regulation by Epo. Copyright © 2007 S. Karger AG.

Descripción: The ASR (for ABA/water stress/ripening) protein family, first described in tomato as nuclear and involved in adaptation to dry climates, is widespread in the plant kingdom, including crops of high agronomic relevance. We show both nuclear and cytosolic localization for ASR1 (the most studied member of the family) in histological plant samples by immunodetection, typically found in small proteins readily diffusing through nuclear pores. Indeed, a nuclear localization was expected based on sorting prediction software, which also highlight a monopartite nuclear localization signal (NLS) in the primary sequence. However, here we prove that such an "NLS" of ASR1 from tomato is dispensable and non-functional, being the transport of the protein to the nucleus due to simple diffusion across nuclear pores. We attribute such a targeting deficiency to the misplacing in that cryptic NLS of two conserved contiguous lysine residues. Based on previous in vitro experiments regarding quaternary structure, we also carried out live cell imaging assays through confocal microscopy to explore dimer formation in planta. We found homodimers in both the cytosol and the nucleus and demonstrated that assembly of both subunits together can occur in the cytosol, giving rise to translocation of preformed dimers. The presence of dimers was further corroborated by means of in vivo crosslinking of nuclei followed by SDS-PAGE. © 2012 Ricardi et al.

Descripción: Type IV secretion systems (T4SS) are multiprotein structures that direct the translocation of specific molecules across the bacterial cell envelope. As in other bacteria, pathogenicity of the genus Brucella essentially depends on the integrity of the T4SS-encoding virB operon, whose expression is regulated by multiple transcription factors belonging to different families. Previously, we identified IHF and HutC, two direct regulators of the virB genes that were isolated from total protein extracts of Brucella. Here, we report the identification of MdrA, a third regulatory element that was isolated using the same screening procedure. This transcription factor, which belongs to the MarR-family of transcriptional regulators, binds at two different sites of the virB promoter and regulates expression in a growth phase-dependent manner. Like other members of the MarR family, specific ligands were able to dissociate MdrA from DNA in vitro. Determination of the MdrA-binding sites by DNase I footprinting and analyses of protein-DNA complexes by electrophoresis mobility shift assays (EMSAs) showed that MdrA competes with IHF and HutC for the binding to the promoter because their target DNA sequences overlap. Unlike IHF, both MdrA and HutC bound to the promoter without inducing bending of DNA. Moreover, the two latter transcription factors activated virB expression to similar extents, and in doing so, they are functionally redundant. Taken together, our results show that MdrA is a regulatory element that directly modulates the activity of the virB promoter and is probably involved in coordinating gene expression in response to specific environmental signals. © 2012, American Society for Microbiology.

Descripción: β-Lapachone, an o-naphthoquinone, induces a novel caspase- and p53-independent apoptotic pathway dependent on NAD (P) H:quinone oxidoreductase 1 (NQO1). NQO1 reduces β-lapachone to an unstable hydroquinone that rapidly undergoes a two-step oxidation back to the parent compound, perpetuating a futile redox cycle. A deficiency or inhibition of NQO1 rendered cells resistant to beta;-lapachone. Thus, β-lapachone has great potential for the treatment of specific cancers with elevated NQO1 levels (e.g., breast, non - small cell lung, pancreatic, colon, and prostate cancers). We report the development of mono(arylimino) derivatives of β-lapachone as potential prodrugs. These derivatives are relatively nontoxic and not substrates for NQO1 when initially diluted in water. In solution, however, they undergo hydrolytic conversion to β-lapachone at rates dependent on the electron-withdrawing strength of their substituent groups and pH of the diluent. NQO1 enzyme assays, UV-visible spectrophotometry, high-performance liquid chromatography-electrospray ionization-mass spectrometry, and nuclear magnetic resonance analyses confirmed and monitored conversion of each derivative to β-lapachone. Once converted, β-lapachone derivatives caused NQO1-dependent, μ-calpain-mediated cell death in human cancer cells identical to that caused by β-lapachone. Interestingly, coadministration of N-acetyt-L-cysteine prevented derivative-induced cytotoxicity but did not affect β-lapachone lethality. Nuclear magnetic resonance analyses indicated that prevention of β-lapachone derivative cytotoxicity was the result of direct modification of these derivatives by N-acetyl-L-cysteine, preventing their conversion to β-lapachone. The use of β-lapachone mono(arylimino) prodrug derivatives, or more specifically a derivative converted in a tumor-specific manner (i.e., in the acidic local environment of the tumor tissue), should reduce normal tissue toxicity while eliciting tumor-selective cell killing by NQO1 bioactivation. © 2005 American Association for Cancer Research.

Descripción: EGS (external guide sequence) technology is a promising approach to designing new antibiotics. EGSs are short antisense oligoribonucleotides that induce RNase P-mediated cleavage of a target RNA by forming a precursor tRNA-like complex. The ftsZ mRNA secondary structure was modeled and EGSs complementary to two regions with high probability of being suitable targets were designed. In vitro reactions showed that EGSs targeting these regions bound ftsZ mRNA and elicited RNase P-mediated cleavage of ftsZ mRNA. A recombinant plasmid, pEGSb1, coding for an EGS that targets region "b" under the control of the T7 promoter was generated. Upon introduction of this plasmid into Escherichia coli BL21(DE3)(pLysS) the transformant strain formed filaments when expression of the EGS was induced. Concomitantly, E. coli harboring pEGSb1 showed a modest but significant inhibition of growth when synthesis of the EGSb1 was induced. Our results indicate that EGS technology could be a viable strategy to generate new antimicrobials targeting ftsZ. © 2012 Sala et al.

Descripción: The three related 160-kDa proteins, SRC-1, TIF-2 and RAC-3, were initially identified as factors interacting with nuclear receptors. They have also been reported to potentiate the activity of other transcription factors such as AP-1 or NF-κB. The aim of this work was to identify whether SRC-1 interferes with the TGF-β/Smad signaling pathway, and if so, to identify its underlying mechanisms of action. Using transient cell transfection experiments performed in human dermal fibroblasts with the Smad3/4-specific (SBE) 4-lux reporter construct, as well as the human PAI-1 promoter, we determined that SRC-1 enhances TGF-β-induced, Smad-mediated, transcription. Likewise, SRC-1 overexpression potentiated TGF-β-induced upregulation of PAI-1 steady-state mRNA levels. Using a mammalian two-hybrid system, we demonstrated that SRC-1 interacts with the transcriptional co-activators p300/CBP, but not with Smad3. Overexpression of the adenovirus E1A oncoprotein, an inhibitor of CBP/p300 activity, prevented the enhancing effect of SRC-1 on Smad3/4-mediated transcription, indicating that p300/CBP may be required for SRC-1 effect. Such hypothesis was validated, as expression of a mutant form of SRC-1 lacking the CBP/p300-binding site failed to upregulate Smad3/4-dependent transcription, while full-length SRC-1 potentiated p300-Smad3 interactions. These results identify SRC-1 as a novel Smad3/4 transcriptional partner, facilitating the functional link between Smad3 and p300/CBP. © 2005 Nature Publishing Group All rights reserved.

Descripción: The bcl-X gene plays a critical role in apoptosis. Six different isoforms generated by tissue-specific promoter usage and alternative splicing were described. Some of them exert opposite effects on cell death. In mammary epithelial cells glucocorticoids induce bcl-X expression and increase the ratio bcl-XL (antiapoptotic)/bcl-XS (apoptotic) by activating P4 promoter, which contains two hormone response elements. Here we show that, on mouse thymocytes and T lymphocyte derivative S49 cells, glucocorticoids inhibited transcription from P4 and decreased the ratio bcl-X L/bcl-XS favoring apoptosis. Upon hormonal treatment, glucocorticoid receptor (GR), steroid receptor coactivator-1, and RNA polymerase II were transiently recruited to P4 promoter, whereas STAT5B was also recruited but remained bound. Concomitant with the release of GR, silencing mediator for retinoic acid receptor and thyroid hormone receptor and histone deacetylase 3 were recruited, histone H3 was deacetylated, and RNA polymerase II left the promoter. Inhibition of STAT5 activity reverted glucocorticoid repression to activation of transcription and was accompanied by stable recruitment of GR and RNA polymerase II to P4. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Descripción: Background: Sixteen melanoma patients (1 stage IIC, 8 stage III, and 7 stage IV) were treated in a Phase I study with a vaccine (DC/Apo-Nec) composed of autologous dendritic cells (DCs) loaded with a mixture of apoptotic/necrotic allogeneic melanoma cell lines (Apo-Nec), to evaluate toxicity and immune responses. Also, IL-10 1082 genotype was analyzed in an effort to predict disease progression. Methods: PBMC were obtained after leukapheresis and DCs were generated from monocytes cultured in the presence of GM-CSF and IL-4 in serum-free medium. Immature DCs were loaded with gamma-irradiated Apo-Nec cells and injected id without adjuvant. Cohorts of four patients were given four vaccines each with 5, 10, 15, or 20 × 106 DC/Apo-Nec cell per vaccine, two weeks apart. Immune responses were measured by ELISpot and tetramer analysis. Il-10 genotype was measured by PCR and corroborated by IL-10 production by stimulated PBMC. Results: Immature DCs efficiently phagocytosed melanoma Apo-Nec cells and matured after phagocytosis as evidenced by increased expression of CD83, CD80, CD86, HLA class I and II, and 75.2 ± 16% reduction in Dextran-FITC endocytosis. CCR7 was also up-regulated upon Apo-Nec uptake in DCs from all patients, and accordingly DC/Apo-Nec cells were able to migrate in vitro toward MIP-3 beta. The vaccine was well tolerated in all patients. The DTH score increased significantly in all patients after the first vaccination (Mann-Whitney Test, p < 0.05). The presence of CD8+T lymphocytes specific to gp100 and Melan A/ MART-1 Ags was determined by ELISpot and tetramer analysis in five HLA-A*0201 patients before and after vaccination; one patient had stable elevated levels before and after vaccination; two increased their CD8 + levels, one had stable moderate and one had negligible levels. The analysis of IL-10 promoter -1082 polymorphism in the sixteen patients showed a positive correlation between AA genotype, accompanied by lower in vitro IL-10 production by stimulated PBMC, and faster melanoma progression after lymph nodes surgery (p = 0.04). With a mean follow-up of 49.5 months post-surgery, one stage IIC patient and 7/8 stage III patients remain NED but 7/7 stage IV patients have progressed. Conclusion: We conclude that DC/Apo-Nec vaccine is safe, well tolerated and it may induce specific immunity against melanoma Ags. Patients with a low-producing IL-10 polymorphism appear to have a worst prognosis. © 2008 von Euw et al; licensee BioMed Central Ltd.