Descripción: Two populations of human natural killer (NK) cells can be identified in peripheral blood. The majority are CD3-CD56dim cells while the minority exhibits a CD3-CD56bright phenotype. In vitro evidence indicates that CD56bright cells are precursors of CD56dim cells, but in vivo evidence is lacking. Here, we studied NK cells from a patient that suffered from a melanoma and opportunistic fungal infection during childhood. The patient exhibited a stable phenotype characterized by a reduction in the frequency of peripheral blood CD3-CD56dim NK cells, accompanied by an overt increase in the frequency and absolute number of CD3-CD56bright cells. These NK cells exhibited similar expression of perforin, CD57 and CD158, the major activating receptors CD16, NKp46, NKG2D, DNAM-1, and 2B4, as well as the inhibitory receptor CD94/NKG2A, on both CD56bright and CD56dim NK cells as healthy controls. Also, both NK cell subpopulations produced IFN-γ upon stimulation with cytokines, and CD3-CD56dim NK cells degranulated in response to cytokines or K562 cells. However, upon stimulation with cytokines, a substantial fraction of CD56dim cells failed to up-regulate CD57 and CD158, showed a reduction in the percentage of CD16+ cells, and CD56bright cells did not down-regulate CD62L, suggesting that CD56dim cells could not acquire a terminally differentiated phenotype and that CD56bright cells exhibit a maturation defect that might result in a potential altered migration pattern. These observations, support the notion that NK cells of this patient display a maturation/activation defect that precludes the generation of mature NK cells at a normal rate accompanied by CD56dim NK cells that cannot completely acquire a terminally differentiated phenotype. Thus, our results provide evidence that support the concept that in vivo CD56bright NK cells differentiate into CD56dim NK cells, and contribute to further understand human NK cell ontogeny. © 2012 Domaica et al.

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: If irradiated tumor cells could be rendered immunogenic, they would provide a safe, broad, and patient-specific array of antigens for immunotherapies. Prior approaches have emphasized genetic transduction of live tumor cells to express cytokines, costimulators, and surrogate foreign antigens. We asked if immunity could be achieved by delivering irradiated, major histocompatibility complex-negative plasmacytoma cells to maturing mouse dendritic cells (DCs) within lymphoid organs. Tumor cells injected intravenously (i.v.) were captured by splenic DCs, whereas subcutaneous (s.c.) injection led only to weak uptake in lymph node or spleen. The natural killer T (NKT) cells mobilizing glycolipid α-galactosyl ceramide, used to mature splenic DCs, served as an effective adjuvant to induce protective immunity. This adjuvant function was mimicked by a combination of poly IC and agonistic αCD40 antibody. The adjuvant glycolipid had to be coadministered with tumor cells i.v. rather than s.c. Specific resistance was generated both to a plasmacytoma and lymphoma. The resistance afforded by a single vaccination lasted >2 mo and required both CD4+ and CD8+ T cells. Mature tumor capturing DCs stimulated the differentiation of P1A tumor antigen-specific, CD8+ T cells and uniquely transferred tumor resistance to naive mice. Therefore, the access of dying tumor cells to DCs that are maturing to activated NKT cells efficiently induces long-lived adaptive resistance. JEM © The Rockefeller University Press.

Descripción: Type 1 diabetes (T1D) is a disease caused by the destruction of the β cells of the pancreas by activated T cells. Dendritic cells (BC) are the APC that initiate the T cell response that triggers T1D. However, DC also participate in T cell tolerance, and genetic engineering of DC to modulate T cell immunity is an area of active research. Galectin-1 (gal-1) is an endogenous lectin with regulatory effects on activated T cells including induction of apoptosis and down-regulation of the Th1 response, characteristics that make gal-1 an ideal transgene to transduce DC to treat T1D. We engineered bone marrow-derived DC to synthesize transgenic gal-1 (gal-1-DC) and tested their potential to prevent T1D through their regulatory effects on activated T cells. NOD-derived gal-1-DC triggered rapid apoptosis of diabetogenic BDC2.5 TCR-transgenic CD4+ T cells by TCR-dependent and -independent mechanisms. Intravenously administered gal-1-DC trafficked to pancreatic lymph nodes and spleen and delayed onset of diabetes and insulitis in the NODrag1 -/- lymphocyte adoptive transfer model. The therapeutic effect of gal-1-DC was accompanied by increased percentage of apoptotic T cells and reduced number of IFN-γ-secreting CD4+ T cells in pancreatic lymph nodes. Treatment with gal-1-DC inhibited proliferation and secretion of IFN-γ of T cells in response to β cell Ag. Unlike other DC-based approaches to modulate T cell immunity, the use of the regulatory properties of gal-1-DC on activated T cells might help to delete β cell-reactive T cells at early stages of the disease when the diabetogenic T cells are already activated. Copyright © 2005 by The American Association of Immunologists, Inc.

Descripción: The sense of olfaction in armadillos plays an important role, suggested by the great development of the nasal structures, olfactory bulbs, and related brain regions. The mammalian olfactory mucosa is a privileged site of neuronal death and regeneration during the whole life span. A detailed knowledge of its ultrastructure is convenient for gaining insight into the factors controlling those phenomena. We performed this work in species not previously studied in order to provide a firm basis for further research on those factors. No information is available on the histology and ultrastructure of the olfactory mucosa in the order Xenarthra to which armadillos belong. Samples from the endoturbinals of the armadillo Chaetophractus villosus were prepared for light and electron microscopic examination by the usual conventional means. The olfactory epithelium of Chaetophractus villosus shows the classical three types of cells: supporting cells, olfactory receptor neurons, and basal cells. The olfactory neurons and the basal cells were similar to that described in other species. Two different types of supporting cells are described. An outstanding characteristic of the supporting cells is the normal presence of abundant phagosomes, apical secretory granules, apocrine-like protrusions, and highly developed smooth endoplasmic reticulum. Apoptotic bodies are frequently found in the infranuclear cytoplasm of supporting cells. The ductular epithelium of Bowman's glands reveals secretory activity. The lamina propria shows mixed Bowman's glands. Great development of smooth endoplasmic reticulum is observed in the mucous acinar cells. Evidence for merocrine and apocrine mechanisms in the Bowman's glands is presented. The presence of apoptotic bodies and phagosomes in supporting cells suggests a participation in the cellular events induced by cell death and proliferation of the olfactory epithelium. The variety of characteristics exhibited by the supporting cells of the olfactory mucosa may contribute to a deeper understanding of their scarcely known functions.

Descripción: Oligonucleotides (ODNs) of the PyNTTTTGT class directly stimulate B lymphocytes and plasmacytoid dendritic cells of the immune system of primates. Here we investigated the ability of the PyNTTTTGT ODN prototype IMT504 to regulate the expression of surface molecules and apoptosis in human B-chronic lymphocytic leukemia (CLL) cells. The surface molecules CD25, CD40, CD80 and CD86 were up-regulated upon incubation of the B-CLL cells with IMT504. Co-stimulation with IL-2 resulted in further up-regulation. IMT504-activated B-CLL cells were also good stimulators of T cells in allogeneic mixed lymphocyte reactions and co-stimulation with IL-2 improved this stimulation capacity. Apoptosis of the B-CLL cells in vitro was also stimulated by incubation with IMT504. In this case, co-stimulation with IL-2 was not significant. Furthermore, B-CLL cells of all the patients studied developed an immunogenic phenotype and entered stimulated apoptosis upon in vitro incubation with IMT504 independently of the mutational status of their 1gVH genes, becoming a good marker for tumor progression.

Descripción: Fn containing an extra type III domain (EIIIA in the rat, ED1 or EDA in humans) is commonly termed "fetal" fibronectin, but it is prominent during the injury response of adult tissues and mediates important early events in the response. This form is particularly apparent in acute liver injury, where it has been shown that sinusoidal endothelial cells produce EIIIA-fibronectin. This fibronectin isoform arises by alternative splicing of the primary transcript. In the present experiments, we have studied the regulation of fibronectin splicing in primary sinusoidal endothelial cells by transfecting a minigene containing the EIIIA exon and its flanking introns, driven by various promoters. The results indicate that fibronectin splicing in endothelial cells from normal liver is in part promoter-dependent. However, in cells from injured liver in which expression of both total and EIIIA-fibronectin is strikingly increased, promoter effects disappear. Because fibronectin splicing is known to be regulated in part by TGFβ, we also examined the effect of a soluble inhibitor of the TGFβ type 2 receptor. This agent had no effect on splicing by normal endothelial cells. By contrast, for endothelial cells from the injured liver, the splicing pattern reverted to that of normal cells, i.e., it became promoter-dependent. We conclude that, in the setting of injury in vivo, TGFβ overrides the promoter dependence of fibronectin splicing in normal cells. The data suggest that TGFβ modifies the spliceosome, if not through its known signaling intermediates, then through the products of genes regulated by this cytokine.

Descripción: Dendritic cells (DCs) are potent APCs and attractive vectors for cancer immunotherapy. Using the B16 melanoma, a poorly immunogenic experimental tumor that expresses low levels of MHC class I products, we investigated whether DCs loaded ex vivo with apoptotic tumor cells could elicit combined CD4+ and CD8+ T cell dependent, long term immunity following injection into mice. The bone marrow-derived DCs underwent maturation during overnight coculture with apoptotic melanoma cells. Following injection, DCs migrated to the draining lymph nodes comparably to control DCs at a level corresponding to ∼0.5% of the injected inoculum. Mice vaccinated with tumor-loaded DCs were protected against an intracutaneous challenge with B16, with 80% of the mice remaining tumor-free 12 wk after challenge. CD4+ and CD8+ T cells were efficiently primed in vaccinated animals, as evidenced by IFN-γ secretion after in vitro stimulation with DCs loaded with apoptotic B16 or DCs pulsed with the naturally expressed melanoma Ag, tyrosinase-related protein 2. In addition, B16 melanoma cells were recognized by immune CD8 + T cells in vitro, and cytolytic activity against tyrosinase-related protein 2180-188-pulsed target cells was observed in vivo. When either CD4+ or CD8+ T cells were depleted at the time of challenge, the protection was completely abrogated. Mice receiving a tumor challenge 10 wk after vaccination were also protected, consistent with the induction of tumor-specific memory. Therefore, DCs loaded with cells undergoing apoptotic death can prime melanoma-specific helper and CTLs and provide long term protection against a poorly immunogenic tumor in mice.

Descripción: In this work we investigate experimentally the dynamics of two coupled optical excitable cells, namely, two semiconductor lasers with optical feedback. We analyze the dynamics observed in terms of the statistical properties of the time series and in terms of the phase space reconstruction from the data. We build a model based on a simple set of deterministic equations (on a two torus) plus noise in order to capture the essential features of the dynamics observed. We discuss the validity of our theoretical results in terms of families of excitable systems and coupling terms. © 2002 The American Physical Society.

Descripción: Biopsies from human metastatic melanomas were analyzed in order to elucidate the relationship between MCP-1/CCL-2 (monocyte chemoattractant protein-1) chemokine expression by tumor cells, angiogenesis and aggressiveness in tumor development. The chemokine was expressed in 100% of the cases, with heterogeneity in the percentage of positive cells within the tumor mass. Tumors presented an important infiltration of macrophages, particularly associated to areas of active angiogenesis. Microvascular development, assessed by immunohistochemistry, correlated with the high percentage of cells expressing MCP-1/CCL-2. There was also significant correlation with vascularization and mitotic index. These results suggest that vascularization could be predictive of more aggressive melanoma metastasis, where the MCP-1/CCL-2 expression would be closely associated to vessel development through macrophages recruitment.

Descripción: Stress granules (SGs) are ephemeral cytoplasmic aggregates containing stalled translation preinitiation complexes involved in mRNA storage and triage during the cellular stress response. SG formation is triggered by the phosphorylation of the alpha subunit of eIF2 (eIF2α), which provokes a dramatic blockage of protein translation. Our results demonstrate that acute infection of Vero cells with the arenavirus Juni{dotless} ́n (JUNV), aetiological agent of Argentine haemorrhagic fever, does not induce the formation of SGs. Moreover, JUNV negatively modulates SG formation in infected cells stressed with arsenite, and this inhibition correlates with low levels of eIF2α phosphorylation. Transient expression of JUNV nucleoprotein (N) or the glycoprotein precursor (GPC), but not of the matrix protein (Z), inhibits SG formation in a similar manner, comparable to infectious virus. Expression of N and GPC also impaired eIF2α phosphorylation triggered by arsenite. A moderate inhibition of SG formation was also observed when DTT and thapsigargin were employed as stress inducers. In contrast, no inhibition was observed when infected cells were treated with hippuristanol, a translational inhibitor and inducer of SGs that bypasses the requirement for eIF2α phosphorylation. Finally, we analysed SG formation in persistently JUNV-infected cells, where N and GPC are virtually absent and truncated N products are expressed abundantly.Wefound that persistently infected cells show a quite normal response to arsenite, with SG formation comparable to that of uninfected cells. This suggests that the presence of GPC and/or N is crucial to control the stress response upon JUNV infection of Vero cells © 2011 SGM.

Descripción: Bacterial lipopolysaccharide (LPS) activates the immune system and induces increases in peripheral cytokines, which, in turn, affect the endocrine system. In particular, LPS-induced cytokines stimulate the hypothalamic-pituitary-adrenal axis to increase levels of antiinflammatory-acting glucocorticoids. In the present work, we show that LPS directly stimulates interleukin (IL)-6 release by mouse pituitary folliculostellate (FS) TtT/GF tumor cells and FS cells of mouse pituitary cell cultures. The stimulatory effect of LPS was strongly enhanced in the presence of serum, suggesting that LPS is only fully active as a complex with LPS-binding protein (LBP). Both TtT/GF cells and mouse pituitaries expressed CD14, which binds the LPS/LBP complex, and Toll-like receptor type 4, which induces LPS signals. LPS increased phospoinositol turnover in TtT/GF cells and induced phosphorylation of p38α mitogen-activated protein kinase and the inhibitor (IκB) of nuclear factor-κ B. Nuclear factor-κ B was activated by LPS in TtT/GF cells. Functional studies demonstrated that My4 (an antibody blocking the interaction between LPS/LBP and CD14), SB203580, (a specific inhibitor of p38α mitogen-activated protein kinase phosphorylation), dexamethasone, and the messenger RNA translation inhibitor cycloheximide all inhibited LPS-induced IL-6 production by TtT/GF cells and mouse pituitary FS cells. LPS-induced intrapituitary IL-6 may modulate the function of anterior pituitary cells during bacterial infection/inflammation.

Descripción: As for any solid tumour, pituitary adenoma expansion is dependent on neovascularization through angiogenesis. In this process, vascular endothelial growth factor (VEGF) and its receptors VEGFR-1, VEGFR-2 and neuropilin-1 (NRP-1) may play an outstanding role. The intention of this work was to study the expression/localization and possible function of VEGF receptors in pituitary adenomas. VEGF receptor mRNA and protein expression was studied by in situ hybridization, immunohistochemistry and RT-PCR in 6 normal human pituitaries, 39 human pituitary adenomas and 4 rodent pituitary adenoma cell lines. VEGFR-1 expressing somatotroph MtT-S cells were used as a model to study the role of VEGF on cell proliferation and to elucidate the underlying mechanism of action. In normal pituitaries, VEGFR-1 was detected in endocrine cells, whereas VEGFR-2 and NRP-1 were exclusively expressed in endothelial cells. In pituitary tumours, a heterogeneous VEGFR expression pattern was observed by IHC. VEGFR-1, VEGFR-2 and NRP-1 were detected in 24, 18 and 17 adenomas respectively. In the adenomas, VEGFR-1 was expressed in epithelial tumour cells and VEGFR-2/NRP-1 in vessel endothelial cells. Functional studies in VEGFR-1-positive MtT-S cells showed that the ligands of VIEGFR-1 significantly stimulated cell proliferation. This effect was mediated through the phosphatidylinositol-3-kinase-signalling pathway and involves induction of cyclin D1 and Bcl-2. Based on our results, we speculate that the ligands of VEGF receptors, such as VEGF-A and placenta growth factor, not only play a role in angiogenesis in pituitary adenomas, but also affect the growth of pituitary tumour cells through VEGFR-1. © 2006 Society for Endocrinology.

Descripción: The endothelium is an organ that is involved in several physiological processes, mainly blood fluid preservation. Non-activated endothelial cells express an anticoagulant, antiadhesive and vasodilative phenotype, whereas activated endothelial cells express procoagulant, proadhesive and vasoconstrictive properties. The structure and function are regulated in space and time and this fact originates a specific vascular bed haemostasis . The objective of this work is to review the new concepts in endothelial cell heterogeneity and their influence in haemostasis regulation. © 2007 Federación Bioquímica de la Provincia de Buenos Aires.

Descripción: Endothelial cells, at the cell-cell borders, express PECAM-1, and have been implicated in vascular functions. The monoclonal antibody MEC 13.3 recognizes PECAM-1 molecule from mouse vessels and allows to analyze the ontogeny of mouse endothelium. At the present, little is known about the molecular basis of differentiation pathways of endothelial cells, that enables its morphological heterogeneity. The purpose of this study was to analyze the pattern of PECAM-1 expression, employing monoclonal antibody MEC 13.3, in cellular suspensions obtained from different mouse organs at pre and postnatal stages. Fluorescence activated cell sorter analysis showed a different profile of the glycoprotein expression in a cell population with size and granularity selected by 1G11 endothelial cell line. The expression differs from prenatal to postnatal developmental stages in a given organ, and among the organs studied. Another cell population, with a size and granularity higher than 1G11 endothelial cell line, coexists in cellular suspensions obtained from liver, gut and brain. These cells could be related to those detected by means of immunoenzyme methods which showed a non-differentiated morphology. The different PECAM-1 pattern expression could reflect potential organ-specific differentiation pathways during development and according to organs environment. The existence of another cell population with a size and granularity higher than 1G11 endothelial cell line required a phenotypic characterization.

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: The Euterpe edulis embryo consists of a prominent single cotyledon, a very short radicle-hypocotyl axis and an epicotyl. The epicotyl is obliquely angled with respect to the cotyledon; consequently it corresponds to one of the two categories recognized for palm seeds by DeMason (1988). Parenchyma, protoderm and procambium can be distinguished on the basis of position and shape of their cells, which are highly vacuolated with one central vacuole and the cytoplasm restricted to a thin parietal layer. Initial cells from both apical meristems are also vacuolated but they have small vacuoles distributed around the nuclei. Silica occurs in cell walls of some protodermal cells. Raphides, silica bodies and tannins all occur occasionally in vacuoles, especially in the basal cotyledon region. Most embryo cells lack storage reserves and exhibit an active state, with numerous mitochondria, RER cisternae and Golgi apparatus, indicating a strategy of continuous development without the interposition, at maturity, of a dry state. The endosperm consists of living cells with very large nuclei and thickened cell walls. Similar to the endosperm of other studied palm species, their cells exhibit a quiescent appearance with lipid, protein, minerals (in the cytoplasm) and mannans (in the cell walls) as the insoluble storage reserves. © 2004 The Linnean Society of London.

Descripción: Although homomeric channels assembled from the α9 nicotinic acetylcholine receptor (nAChR) subunit are functional in vitro, electrophysiological, anatomical, and molecular data suggest that native cholinergic olivocochlear function is mediated via heteromeric nAChRs composed of both α9 and α10 subunits. To gain insight into α10 subunit function in vivo, we examined olivocochlear innervation and function in α10 null-mutant mice. Electrophysiological recordings from postnatal (P) days P8-9 inner hair cells revealed ACh-gated currents in α10 +/+ and α10+/- mice, with no detectable responses to ACh in α10+/+ mice. In contrast, a proportion of α10-/- outer hair cells showed small ACh-evoked currents. In α10-/- mutant mice, olivocochlear fiber stimulation failed to suppress distortion products, suggesting that the residual α9 homomeric nAChRs expressed by outer hair cells are unable to transduce efferent signals in vivo. Finally, α10-/- mice exhibit both an abnormal olivocochlear morphology and innervation to outer hair cells and a highly disorganized efferent innervation to the inner hair cell region. Our results demonstrate that α9-/- and α10-/- mice have overlapping but nonidentical phenotypes. Moreover, α10 nAChR subunits are required for normal olivocochlear activity because α9 homomeric nAChRs do not support maintenance of normal olivocochlear innervation or function in α10-/- mutant mice. © 2007 by The National Academy of Sciences of the USA.

Descripción: The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the α9α10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the α9α10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 μM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 μM ryanodine. This facilitatory effect on current through the AChR could enhance brief (∼1 s) activation of associated calcium-dependent K+ (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate α9α10- mediated responses and for potential inner ear therapeutics based on this interaction. © 2007 Association for Research in Otolaryngology.