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CONFERENCIA
MEDICINA (Buenos Aires) 2006; 66 (Supl. II): 27-33 ELIZABETH JARES-ERIJMAN
Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Argentina El desarrollo de las nanopartículas semiconductoras trol sobre las propiedades ópticas de estas nanopartículas, conocidas como quantum dots ha evolucionado en las dos con un énfasis en las aplicaciones en biología celular y últimas décadas del área de la ciencia de los materiales a animal, así como nuevos desarrollos para estudios clíni- las aplicaciones biológicas y clínicas. Los quantum dots cos. Ejemplos recientes incluyen la observación de los están emergiendo como una nueva clase de marcadores primeros pasos en la cascada de eventos iniciada por el luminiscentes con especial luminosidad, resistencia a la EGF por su interacción con la familia de receptores tirosina fotodestrucción y emisión multicolor. Propiedades ópticas kinasa y la identificación de nódulos centinela en anima- especiales de estas nanopartículas son su emisión en for- les vivos. Las nuevas generaciones de quantum dots tie- ma de líneas angostas y su muy ancho espectro de exci- nen un gran potencial para el estudio de procesos tación, que permite la observación de un elevado número intracelulares a nivel de moléculas individuales, para las de marcadores diferentes en forma simultanea. La carac- observaciones por tiempos prolongados in vivo de even- terística única y fundamental cuando se realizan observa- tos de tráfico celular, la detección de tumores tempranos ciones por tiempos prolongados es su alta fotoestabilidad.
y para el desarrollo de nuevos métodos de diagnóstico.
Esta propiedad ha permitido el rastreo de eventos biológi- Por otra parte se discutirá un rol adicional de estas cos a nivel celular imposibles de realizar con marcadores nanopartículas, la posibilidad de actuar como iniciadores fluorescentes usuales. Se discutirán nuevos métodos que de procesos biológicos, ejemplificando con estudios so- permiten la funcionalización, marcaje específico, y el con- bre la agregación de proteínas amiloides.
CONFERENCIA LEONARDO SATZ
LECTINAS EN INMUNIDAD INNATA: LA PRIMERA TRINCHERA GERARDO R. VASTA
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Las lectinas solubles y asociadas a las membranas opsónicos, líticos o tóxicos. En los mamíferos, incluyen- de células fagocíticas constituyen componentes clave do el hombre, las lectinas no solo participan como pro- de las respuestas de inmunidad innata en invertebrados teínas de fase aguda en el reconocimiento de antígenos y vertebrados. Sus funciones biológicas incluyen no sólo glicosilados, sino que cumplen un papel fundamental en el reconocimiento de patógenos y parásitos microbianos, la regulación de respuestas inmunes adaptativas. Sin sino también funciones efectoras, tales como aglutina- embargo, estudios estructurales y funcionales de lectinas ción, inmobilización, y activación de las cascadas de en modelos animales pertenecientes a niveles evoluti- complemento y melanización, cuyos productos son vos que carecen de inmunidad adaptativa (invertebra- MEDICINA - Volumen 66 - (Supl. II), 2006 dos tales como Drosophila sp., C. elegans), o poseen (isolectinas) llevan a un sistema con alta capacidad de mecanismos inmunes adaptativos menos complejos que reconocimiento, que incluye a la mayoría de los glicanos en los mamíferos (por ejemplo, Danio rerio), sugiere que de superficie comunes a patógenos microbianos y pará- los repertorios de lectinas en estos taxones también es- sitos. Así, a través de sus funciones de reconocimiento tán altamente diversificados, estructural y funcionalmente.
immune que a través de mecanismos efectores llevan a Estos incluyen no sólo representantes de las familias de la destrucción del patógeno, las lectinas contribuyen lectinas y vías efectoras caracterizadas en los mamífe- significativamente a formar una barrera defensiva tem- ros, sino familias de lectinas tales como las de tipo F, prana contra el desafío infeccioso. Sin embargo, algu- descriptas recientemente en modelos animales no con- nos microorganismos (por ejemplo, Perkinsus marinus), vencionales. Si bien la capacidad de reconocimiento in- no sólo se han adaptado para evadir o bloquear los me- mune de las lectinas no se incrementa por mecanismos canismos de defensa immune del hospedador, sino que de recombinación genética como los observados en las aparentemente subvierten sus mecanismos de recono- inmunoglobulinas o los VLRs, la diversidad individual de cimiento immune para facilitar su entrada en ciertos ti- los repertorios de lectinas basados en la expresión de pos celulares. (Estudios financiados a través de subsi- múltiples miembros de cada familia (lectinas tipo C, F, dios de National Science Foundation, Maryland Sea ficolinas, pentraxinas, etc) y de sus respectivas isoformas Grant, NOAA, and NIGMH, National Institutes of Health) CONFERENCIA ALBERTO TAQUINI
KEVIN J. CATT
Endocrinology and Reproduction Research Branch, NICHD, National Institutes of Health, Bethesda, MD, USA Activation of the AT receptor (AT R) by angiotensin in which agonist activation of either species leads to II (Ang II) initiates diverse signaling responses that me- complex formation and transphosphorylation between diate its physiological regulation of blood pressure and the two types of receptors. Such interactions are de- electrolyte balance, as well as its several pathogenic pendent on co-localization of the receptors in caveolin- rich domains of the plasma membrane, which have an PLCb, InsP /Ca2+ signaling and activation of PKC essential scaffolding role during agonist-induced isoforms, MAP kinases, cytoplasmic tyrosine kinases transactivation and reciprocal signaling between the two (Pyk2, Src, Tyk2, FAK), receptor tyrosine kinases (RTKs) such as the EGF-R, reactive oxygen species (ROS) and Most of the deleterious actions of AT R activation are the NFkB pathway. Ang II also stimulates G protein-in- caused by locally generated Ang II, and in the heart are dependent signaling pathways, including β-arrestin- often associated with the harmful effects of aldosterone.
mediated MAP kinases and the Jak/STAT pathway. The AT R-mediated overproduction of ROS causes growth- ability of certain GPCRs to utilize the growth-promoting promoting, proinflammatory and profibrotic responses in actions of RTKs is exemplified by the manner in which vascular cells, leucocytes and monocytes. Activation of the Ang II-activated AT R increases HB-EGF formation the AT R also accelerates the development of diabetes and other pathways to transactivate the EGFR and stimu- and contributes to tumor progression and metastasis late ERK phosphorylation. In addition to its regulation of through its growth-promoting and proangiogenic activi- cell growth, survival, and proliferation, such AT R acti- ties. The increasing recognition of Ang II-induced disor- vation of the EGFR is relevant to its pathogenic actions ders has led to novel clinical applications of angiotensin in the cardiovascular system, and in renal disease, dia- converting enzyme inhibitors and AT R blockers, in addi- betes and cancer. Recent studies have revealed recip- tion to their established therapeutic actions in essential rocal signaling from RTKs to GPCRs such as the AT R, CONFERENCIA
PROLACTIN RECEPTORS: INHIBITORY SHORT FORMS IN CANCER CELLS AND ESTROGEN MARIA L. DUFAU
Section on Molecular Endocrinology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD, USA. Prolactin (PRL) exerts diverse functions in target (PIII/hE1 ). In breast cancer cells, E increases PRLR hE1 tissues through its membrane receptors, and is a potent transcripts directed by hPIII which lack an ERE. This mitogen in normal and neoplastic breast cells. PRL acts promoter contains functional Sp1 and C/EBP sites that through the long form of the receptor (LF) to cause bind Sp1/Sp3 and CEBPβ, respectively. Abolition of the differentiation of mammary epithelium and to initiate and E effect by mutation of either element indicated the maintain lactation through activation of the Jak2/Stat5 cooperation of these transfactors in E -induced trans- pathway and subsequent transcriptional events. We have cription of the hPRLR. E activated-estrogen receptorα identified two alternatively spliced short forms (SF) of (ERα) through interaction with Sp1/Sp3 and C/EBPβ the human PRL receptor (hPRLR) with abbreviated bound to DNA caused transcriptional activation of the cytoplasmic domains that inhibit the activation by PRL promoter and of hPRLR expression in cancer cells. The of the LF. A significant decrease in the ratio of SFs/LF is ligand binding domain of ERα was essential for its physical observed in the breast tumor tissue and cancer cell lines.
interaction with C/EBPβ and E promoted this association, The relatively lower expression of SFs in cancer could and its DNA binding domain was required for transac- cause unopposed PRL-mediated LF stimulatory function, tivation of PIII. Other studies revealed tethering of C/EBPβ and contribute to breast tumor development/progression.
to Sp1 by the E -activated ERα, favouring interaction with Inhibition by SFs may result from hormone-independent its cognate element, and recruitment of coactivators to heterodimer formation. Although SF homodimers and the complex, with consequent region-specific changes in their heterodimers with LF bind hormone and mediate histone acetylation. These hormone/receptor-induced JAK2 activation, the SF heterodimer partner lacks associations and chromatin changes favored TFIIB and sequences essential for activation of the STAT5 pathway RNA Pol II recruitment and the activation of PIII directed and prevents the LF from mediating activation of PRL- hPRLR transcription. Stromal and adipose tissue, which are major sources of estrogen in post-menopausal The hPRLR gene has six alternative non-coding women, could exert paracrine control of PRL and PRLR exons 1 driven by individual promoters. These include expression in adjacent mammary epithelial cells and the preferentially utilized generic promoterIII/ exon1 , CONFERENCIA
SELECTIVE STEROID SIGNALING IN THE NERVOUS SYSTEM: MICHAEL SCHUMACHER, COSIMA FONTE, AMALIA TROUSSON,
JULIEN GRENIER, CHARBEL MASSAAD
UMR 788 Inserm - University Paris 11, 80 rue du Général Leclerc, 94276 Kremlin-Bicêtre, France Work over the past 10 years has revealed that the coactivators and corepressors. Coactivators include mol- transcriptional efficiency of liganded steroid receptors is ecules that facilitate the access of the basal transcrip- determined by nuclear coregulator proteins, comprising tional machinery to the promoter. Among the histone MEDICINA - Volumen 66 - (Supl. II), 2006 acetyltransferase (HAT) coactivators, the best character- tion of the p160s, which was dependent on the duration ized groups are the p160 (the Steroid Receptor of the hormonal induction. Overexpression and siRNA Coactivators SRC-1a, SRC-1e, SRC-2 and SRC-3) and knock-down experiments allowed us to show that de- the CREB Binding Protein (CBP)/p300 families. However, pending on the glial cell type, the GR differentially re- only few studies have so far examined the functional re- cruits p160 family members. Moreover, the interaction lationship between steroid receptors and their between SRC-1a and GR is unusual in glial cells: the C- coregulators in the nervous system. Nevertheless, the terminal nuclear receptor interacting domain of SRC-1a still fragmentary findings show that coactivators of the participates in its exclusion from the GR complex in p160 family are critically involved in the amplification of astrocytes, while in Schwann cells, SRC-1a interacts with nuclear receptor actions within the brain.
GR via its two nuclear receptor binding domains. As a We have studied the interactions between the gluco- consequence of this atypical interaction between SRC- corticoid receptor (GR) and coactivators of the p160 and 1a and GR in Schwann cells, the actions of CBP and CBP/p300 families in two types of glial cells: astrocytes p300 are modified. CBP is not implicated in the GR com- and Schwann cells. First, we showed that the recruit- plex, and p300 unexpectedly repressed GR tran- ment of the p160s by the GR is dependent on the pro- sactivation. Functional and pull-down assays showed moter context. We also demonstrated by immunolocali- that β-catenin is the coactivator replacing CBP in zation experiments a cell-specific intracellular distribu- CONFERENCIA
HUMAN GENETIC OF PREMATURE OVARIAN FAILURE: M. FELLOUS1; S. COPELLI2
1Cochin Institute, Paris, France, 2CAECE University, Buenos Aires, Argentina Premature ovarian failure (POF) is a disease affecting standing of the genetics and physiopathology of POF.
1 to 3% of women before the age of 40 years, and 0,1 % We have collected a large panel of familials and sporadic of women aged of 30 years. Patients may present a pri- mary or secondary amenorhea, depending on the age of Mapping approach: Genome-wide scanning in famil- occurrence. POF may result from (i) a decrease in the ial cases : We collected 11 families with familial cases of primordial follicle pool; (ii) an increased or accelerated follicular atresia; (iii) an alteration of the recruitment of With the help of the French Genome Center, the ge- the dominant follicle; and (iv) an interruption of the matu- nome of the member of the families was scanned by ration of the follicle. Infertility is usually definitive and can genotyping 412 microsatellite markers at the Center Na- be currently treated only by ovum donation.
Several arguments are in favor of a genetic aetiology Evidence for significant linkage was detected on the of POF. The first one is the existence of familial cases.
long arm of human chromosome 7q22-1 with a peak lod- Even if familial cases are uncommon, they have already score of 3.85. The region of interest is presently 10 Mb allowed the identification of disease loci such as the FSH- long and contains interesting candidate genes.
R. Other genetic alterations or mutations have been de- Candidate genes approach on sporadic POF: FOXL2, scribed as responsible for POF. The most frequent anomalies are chromosome X alterations, such as The candidates genes involved in meiosis and follicular monosomy associated with Turner syndrome or deletions maturation were selected based on the fact that they are responsible of similar phenotypes in “knock out” mice.
Unfortunately, in more 90% of POF patients, the aeti- Three hundred sporadic POF cases were sequenced ology of the disease is still unknown. For this reason, we for these genes, and genetic variants were found for have set up an international network gathering clinical DMC1, GDF9 and BMP15 genes. These variants where and research teams, in order to obtain a better under- ORAL TOLERANCE ASSOCIATED WITH TOLEROGENIC ADJUVANTS TO TREAT ALLERGY AND AUTOIMMUNE DISORDERS PIERRE L. MASSON
Université Catholique de Louvain. Belgium The feeding of antigens leads typically to a state of Regarding autoimmune diseases, oral administration of unresponsiveness, known as oral tolerance. Two types autoantigens in animal models produced very promising of cells are playing a key role in oral tolerance, i.e. mu- results, but the clinical trials were disappointing. A tolerogenic cosal dendritic cells, which send processes probing the adjuvant could however enhance oral tolerance. So far the content of the gut lumen, and regulatory T cells, which main tolerogenic adjuvants are bacterial heat shock pro- secrete immunomodulating cytokines such as IL-10 and teins (HSPs), which are molecular “chaperones”. They are TGF-β. The oral intake of an antigen can induce a by- involved in the clearance of proteins that are improperly stander suppression; when oral tolerance has been in- folded, for example under conditions of cellular stress. When duced toward antigen X, the injection of antigen Y to- bound to ATP, a hydrophobic pocket in HSPs opens and gether with X in the same body site fails to induce an binds certain peptides. The hydrolysis of ATP into ADP en- immune response against both antigens Y and X. Oral closes the peptide in the pocket, which can be reopened by administration of allergens significantly improves aller- ATP. In animal models, either injection or oral administra- gic rhinitis and induces much less adverse effects than tion of a microbial HSP leads to the production of T cells allergen injections. Increased efficacy can still be obtained that cross-react with self-HSPs. These T cells produce regu- by using antigen peptides instead of the whole antigen.
latory cytokines, and have disease-suppressive activity. If Peptides are apparently more tolerogenic and reduce the peptides from either allergens or autoantigens are combined risk of adverse effects because they cannot trigger the to HSP, one can expect that the HSP tolerogenic effect will CONFERENCIA
EDUARDO M. SOTOMAYOR
Division of Immunology and Division of Malignant Hematology. H. Lee Moffitt Cancer Center & Research Institute. Tampa, Florida, USA The spontaneous interaction between tumor cells and lineage-specific tissue differentiation antigens also ex- the immune system results in reciprocal changes lead- pressed in normal tissues5, 6. These observations raised ing to a less immunogenic tumor and immune cells less the concern that the same mechanisms that normally pre- capable to mount an effective response against a grow- vent attack against self-antigens may also blunt the abil- ing malignancy1, 2. Although several mechanisms have ity of the immune system to recognize and respond to been proposed to explain how tumors might influence antigens expressed by tumors7. This different view of tu- the function of tumor-antigen specific T cells, one mecha- mor immunity profoundly changed our approach to can- nism that has gained particular attention relates to the cer immunotherapy since the greatest obstacle to the ability of malignant cells to induce antigen-specific T-cell development of successful immunotherapeutic ap- tolerance3, 4. The immune tolerance hypothesis was first proaches is the immune system itself, and more specifi- evoked following the surprising findings that most of the cally, its complex mechanisms for tolerance induction.
identified tumor antigens were not necessarily neoanti- Thus, over the past decade, a significant amount of effort gens uniquely expressed by cancer cells, but rather were has been devoted towards answering those questions MEDICINA - Volumen 66 - (Supl. II), 2006 pertaining to how tolerance towards tumor-antigens is specific T cell function during tumor progression. J Exp established and maintained, and what steps can be taken 3. Bogen, B. Peripheral T cell tolerance as a tumor escape to break this state of unresponsiveness in a controllable mechanism: deletion of CD4+ T cells specific for a monoclonal immunoglobulin idiotype secreted by a During our presentation we will discuss therefore the plasmacytoma. Eur J Immunol 1996; 26: 2671-9.
experimental and clinical evidence that help identify the 4. Staveley-O’ Carroll K, Sotomayor E, Montgomery J, Borrello I, Hwang L, Fein S, Pardoll D, and Levitsky H.
remarkable barrier that tolerance to tumor antigens has Induction of antigen-specific T cell anergy: An early event imposed to our efforts to effectively harness the immune in the course of tumor progression. Proc Natl Acad Sci system against malignancies. In particular, we will dis- U S A, 1998; 95: 1178-83.
cuss the central role of bone marrow-derived antigen-pre- 5. Rosenberg SA. The Development of New Cancer Therapies Based on the Molecular Identification of senting cells (APCs) in the induction of this state of T-cell Cancer Regression Antigens. Cancer J Sci Am 1995; 1: unresponsiveness and the role of the tumor microenvi- ronment in determining the tolerogenic properties of these 6. Boon T. and van der Bruggen, P. Human tumor antigens APCs8, 9. Finally, we provide information on receptor- recognized by T lymphocytes. J Exp Med 1996, 183:725-9.
ligands and intracellular pathways (CD40, STAT3, c-kit, 7. Sotomayor EM, Borrello, I., and Levitsky, HI. Tolerance Histone deacetylases) that given their role in influencing and cancer: a critical issue in tumor immunology. Crit Rev the inflammatory properties of APCs are being exploited Oncog 1996; 7: 433-56.
as targets to revert mechanisms of T-cell unresponsive- 8. Sotomayor EM, Borrello, I, Rattis, FM., Cuenca, AG., Abrams, J, Staveley-O’Carroll, K, and Levitsky, H. I.
Cross-presentation of tumor antigens by bone marrow-derived antigen-presenting cells is the dominantmechanism in the induction of T-cell tolerance during B- References
cell lymphoma progression. Blood 2001; 98: 1070-17.
9. Horna P, Cuenca, A, Cheng F, Brayer J, Wang HW, 1. Pure E, Allison JP, and Schreiber, RD. Breaking down Borrello I, Levitsky H, and Sotomayor, E M. In vivo the barriers to cancer immunotherapy. Nat Immunol, disruption of tolerogenic cross-presentation mechanisms uncovers an effective T-cell activation by B-cell lym- 2. Zhou G, Lu Z, McCadden JD, Levitsky HI, and Marson, phomas leading to antitumor immunity. Blood 2006; 107: AL. Reciprocal changes in tumor antigenicity and antigen- CONFERENCIA
THE AMPK SYSTEM - AN ENERGY SENSOR AT THE CELLULAR D. GRAHAME HARDIE
Division of Molecular Physiology, University of Dundee, Scotland, UK The AMP-activated protein kinase (AMPK) is the down- catabolic pathways that generate ATP such as the up- stream component of a kinase cascade that acts as a take and oxidation of glucose and fatty acids, while at the sensor of cellular energy charge. Stresses that inhibit same time conserving ATP by switching off ATP consum- ATP production (e.g. hypoxia, hypoglycemia, ischemia) ing processes, including lipid, polysaccharide and pro- or that accelerate ATP consumption (e.g. muscle con- tein synthesis. The ability of AMPK to inhibit cell growth traction) cause an increase in ADP:ATP that is amplified and proliferation may explain why the upstream kinase by adenylate kinase into a much larger rise in the LKB1 was first identified as a tumor suppressor.
AMP:ATP ratio. Binding of AMP to the gamma subunit of As well as acting as an energy sensor at the level of AMPK triggers phosphorylation and activation of the cata- the individual cell, it is now clear that AMPK also regu- lytic alpha subunit by the upstream kinase LKB1. Once lates energy balance at the whole body level, making it a activated, AMPK has numerous rapid effects due to di- prime target for drugs aimed at treatment of obesity, Type rect phosphorylation of target proteins, as well as longer- 2 diabetes and the metabolic syndrome. AMPK is acti- term effects on gene expression. It switches on alternate vated by leptin and adiponectin in skeletal muscle, stimulating glucose and fat oxidation, and hence energy the respiratory chain, and the thiaziolidinediones also expenditure. In the hypothalamus it stimulates food in- act indirectly by stimulating adiponectin release. Re- take and is regulated by hormones known to affect ap- cent work from our laboratory suggests that in the ca- petite, such as leptin, ghrelin and cannabinoids. It is a rotid body and pulmonary smooth muscle the AMPK target for two major classes of anti-diabetic drugs, i.e.
system acts as a sensor involved in the physiological the biguanides (e.g. metformin) and the thiazoli- responses to hypoxia that control breathing and blood dinediones. These drugs activate AMPK by inhibiting CONFERENCIA
SIGNAL TRANSDUCTION BY DIACYLGLYCEROL IN CANCER: MARCELO G. KAZANIETZ
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. Email: marcelo@spirit.gcrc.upenn.edu Diacylglycerol (DAG) is a key lipid second messenger 2004). RNAi depletion of chimaerins led to a prolonged generated upon activation of phospholipase C (PLC) Rac activation in response to EGF, suggesting that they isozymes by tyrosine kinase and G-protein-coupled re- negatively modulate Rac signaling. There are several ceptors. Work from many laboratories, particularly com- important implications for these findings. First, we can prehensive biochemical studies from Alexandra Newton’s unambiguously conclude that PKCs are not the only DAG lab at UCSD, established a crucial role for DAG as a pro- receptors and that divergence in DAG signaling via re- tein kinase C (PKC) activator. Our studies revealed that ceptor activation exists. Second, we established for the other intracellular receptors for DAG exist in addition to first time that tyrosine kinase receptors trigger a mecha- PKC isozymes that play important roles in cancer. We nism that self-limits Rac activation via the PLC/DAG found that α- and β-chimaerins, “non-kinase” DAG recep- branch. Third, our recent demonstration that chimaerins tors with GAP activity for the small G-protein Rac, are are down-regulated in some types of cancers and inhibit effectors of the epidermal growth factor receptor (EGFR) cell proliferation and migration in response to growth fac- (EMBO J. 25: 2062-2074, 2006). Upon stimulation, EGFR tors suggests a role for chimaerins as tumor suppressors activates Rac to promote a proliferative and motile re- (JBC 280:24363-24370, 2005; Mol. Cell. Biol. 26:831-842, sponse. Experiments using co-IP and Fluorescence Reso- 2006). Lastly, as we have recently established using a nance Energy Transfer (FRET) revealed that EGF causes knock-down approach in zebrafish that α-chimaerin regu- the translocation of chimaerins to the plasma membrane lates gastrulation (PNAS 103:5373-5378, 2006), DAG sig- in a DAG-dependent manner, where they associate with nals must play a crucial role in early development. Our active Rac (Rac-GTP) to promote its inactivation, as pre- challenge now is to dissect the functional relevance of these dicted from our structural studies (Cell 119:407-418, findings using knockout and knockin mouse models.

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