APOPTOSIS PRODUCIDA POR SALES BILIARES EN EL EPITELIO INTESTINAL. IMPLICANCIAS EN LA ABSORCI��N INTESTINAL DE CALCIO

En altas concentraciones, el deoxicolato de sodio (DXCS, sal biliar) produce da��o hep��tico durante la colestasis y act��a como promotor de c��ncer de colon en animales de experimentaci��n. El estr��s oxidativo que el DXCS desencadena produce alteraciones mitocondriales y del ret��culo endopl��smico, las cuales pueden llevar a la apoptosis. Las dietas occidentales, ricas en grasas y pobres en fibras, y el incremento de las expectativas de vida hacen que el DXCS circule mayor n��mero de veces por el circuito enterohep��tico aumentando, en consecuencia, sus efectos citot��xicos. Dado que el intestino constituye la ��nica puerta de entrada de calcio al organismo y que la absorci��n intestinal del cati��n es sensible al estr��s oxidativo nos planteamos como HIP��TESIS que el DXCS, en concentraciones fisiol��gicas altas, podr��a alterar la absorci��n intestinal de Ca2+, quiz��s por desencadenamiento de estr��s oxidativo que estimular��a los procesos apopt��ticos de las c��lulas epiteliales, resultando en una disminuci��n de la capacidad de transporte del cati��n. Para demostrar esta hip��tesis, se plantearon los siguientes objetivos. OBJETIVO GENERAL: Conocer los mecanismos moleculares que pueden desencadenar altas concentraciones de DXCS en el duodeno y sus implicancias sobre la absorci��n intestinal de calcio. OBJETIVOS ESPEC��FICOS: 1) Analizar la histolog��a del intestino en presencia y ausencia de altas concentraciones de DXCS.2) Determinar el efecto del DXCS sobre la absorci��n intestinal de calcio en funci��n del tiempo de exposici��n y la dosis. 3) Evaluar el efecto del DXCS sobre la expresi��n de genes relacionados con la absorci��n intestinal de calcio. 4) Analizar la expresi��n de prote��nas que participan en la absorci��n intestinal de calcio tales como Ca2+-ATPasa, intercambiador Na+/Ca2+ y CB28k. 5) Estudiar el efecto del DXCS sobre el sistema redox intestinal, a trav��s de la cuantificaci��n del contenido de glutati��n y carbonilos, de la medici��n de radicales libres hidroxilo y de las actividades de las enzimas del sistema antioxidante.6) Determinar la localizaci��n subcelular y la expresi��n de mol��culas proapopt��ticas de la v��a intr��nseca (Bax, citocromo c) y la fragmentaci��n del ADN como indicadores de apoptosis en enterocitos expuestos a altas concentraciones de DXCS. 7) Analizar la expresi��n de mol��culas proapopt��ticas de la v��a extr��nseca (Fas, FasL, etc) en enterocitos tratados con DXCS.8) Interpretar los posibles mecanismos moleculares desencadenados por el DXCS que podr��an afectar el proceso global de la absorci��n intestinal de calcio. Metodolog��a: Se utilizar��n pollos Cobb, los cuales ser��n alimentados con una dieta comercial. Al cabo de cuatro semanas de edad, se dividir��n en dos grupos: a) controles y b) tratados con DXCS en el lumen intestinal a diferentes tiempos y concentraciones (1-100 mM). En ellos se medir�� la absorci��n intestinal de calcio mediante la t��cnica del asa intestinal ligada in situ utilizando 45Ca2+ como trazador .Se medir�� la expresi��n de genes y prote��nas que participan en la v��a transcelular de calcio por RT-PCR y Western blot, respectivamente. Se estudiar��n variables asociadas al estres oxidativo tales como grupos carbonilos, radicales libres hidroxilos, niveles de glutati��n y se medir�� la actividad de enzimas del sistema antioxidante. Se evaluar��n mol��culas de las v��as apopt��ticas extr��nseca e intr��nseca. Para el an��lisis de los datos se utilizar�� ANOVA seguido del test de Bonferroni en la mayor��a de los estudios. El estudio de la absorci��n intestinal de calcio bajo la influencia del DXCS, sal biliar no conjugada que est�� en gran proporci��n en el l��quido fecal, arrojar�� informaci��n no s��lo sobre los factores moleculares que influyen sobre la absorci��n intestinal del Ca2+ sino tambi��n puede brindar elementos que orienten hacia el conocimiento de la etiopatogenia de enfermedades que transcurren con alteraciones en la absorci��n del cati��n como es el caso de la osteoporosis o de otras patolog��as ��seas.

Estudio de la muerte celular por Apoptosis en el ovario de Columba maculosa y Columbina picui (Aves:Columbidae) durante el Ciclo Reproductivo Anual. Study of cell death by apoptosis in the ovary of Columba maculosa and Columbina picui (Aves: Columbidae) during the reproductive annual cycle.

En este trabajo se analizar��n las caracter��sticas estructurales, cuantitativas y el proceso de muerte celular por apoptosis en el ovario de C. maculosa y C.picui con el objetivo de aportar conocimientos b��sicos a la biolog��a reproductiva de estas aves. Cincuenta hembras adultas de cada especie se capturar��n en el Dpto. R��o Primero (Pcia. de Cba), R. Argentina, durante el ciclo reproductivo 2011-2012. Las muestras de ovarios se fijaran en Formalina Neutra pH 7.0, procesar��n con la t��cnica de inclusi��n en parafina y colorearan con Hematoxilina /Eosina y Reacci��n Nuclear de Feulgen. Cinco muestras ser��n utilizadas para la determinaci��n de muerte celular por apoptosis con la t��cnica de TUNEL. Se estudiar��n las caracter��sticas morfohistol��gicas del ovario de C.maculosa y C. picui e identificar��n, categorizar��n y cuantificar��n los fol��culos atr��sicos no bursting (fol��culos atr��sicos previtelog��nicos y vcitelog��nicos peque��os que conservan la integridad de la pared folicular) y bursting (fol��culos vitelog��nicos mayores de 2 mm que liberar el contenido folicular por ruptura de la pared folicular) durante el ciclo reproductivo anual. Mediante la marcaci��n de ADN fragmentado, se revelar�� la muerte celular por apoptosis en las c��lulas granulosas de los fol��culos atr��sicos. Se comparar��n las semejanzas y diferencias estructurales y cuantitativas y el proceso de muerte celular en los fol��culos regresivos entre las dos especies. Los resultados de este trabajo representar��n un importante aporte al conocimiento de la atresia folicular como as�� tambi��n a la muerte celular, un proceso estrechamente asociado a la misma, a��n poco estudiado en el ovario de las aves. In this work we analyse the structural, quantitative and the process of the cell death by apoptosis in the ovary of Columba maculosa and Columbina picui in order of providing basic knowledge of reproductive biology of these birds. Fifty adult female of each species will be caught in the R��o Primero (Pcia. Cba) R.Argentina, during 2011 - 2012. The ovarian samples will be fixed en neutral buffered Formalin (pH 7.0), processed with the technique of inclusion in paraffin and stained with Haematoxylin - Eosin, and nuclear Reaction of Feulgen. Five samples will be used to reveal cell death by apoptosis with the technique of TUNEL. Will be studied the morphohistological characteristics of ovary of both species, and identify, categorize and quantify the atretic follicles over the cycle, the non-bursting (pre-vitellogenic follicles and vitellogenic small < 2 mm, involution an without follicular rupture) and bursting (vitellogenic follicle > 2 mm in the which the yolk falls into the peritoneum due to rupture of with out put of the wall follicular). Will be revealed DNA fragmentation in the granulosa cells of the atretic follicles. Will compared the similaritues and differences in structure and quantitative and the cell death process by apoptosis in the regresive follicles, between the two species. The results of this study represent an important contribution to the knowledge of follicular atresia as well cell death a process closely associated with it and still poorly studied in the ovary of birds.

Expresi��n de las prote��nas relacionadas con la proliferaci��n celular y la apoptosis en lesiones blancas de la mucosa oral, como precursoras de la cancerizaci��n en el epitelio escamoso oral.

El c��ncer de la mucosa oral es una patolog��a muy frecuente que llega en muchos casos a conformar entre el 8 y el 10% de los tumores malignos del hombre. La baja concientizaci��n de la poblaci��n general sobre el tema es un factor importante que hace que estas lesiones sean detectadas en forma tard��a y cuando ya se trata de lesiones avanzadas de muy mal pron��stico. Si bien ha habido grandes avances en el diagn��stico y tratamiento de lesiones blancas de la mucosa oral como el liquen plano oral, estas lesiones siguen siendo entidades con muchos interrogantes para todos los expertos en medicina oral, sobre todo en lo referente a su proceso de aparici��n y a su tratamiento. Es importante la diferenciaci��n correcta de estas lesiones, ya que el carcinoma epidermoide bucal puede aparecer tambi��n como una lesi��n blanca. No hay suficiente conocimiento de lo que ocurre en otras lesiones blancas de la mucosa oral no liquen plano. Para evaluar el verdadero potencial de transformaci��n maligna de leucoplasias, liquen plano oral, reacciones liquenoides y lesiones escamosas intraepiteliales (inclu��das las inducidas por acci��n viral) se evaluar�� la expresi��n de las prote��nas relacionadas con la proliferaci��n celular y la apoptosis en estas lesiones. Se evaluar�� con t��cnicas inmunohistoqu��micas la relaci��n de bcl-2 como marcador apopt��tico en leucoplasias, liquen plano oral, lesiones escamosas intraepiteliales y carcinomas epidermoides orales, as�� como tambi��n en estas mismas lesiones la expresi��n proteica de MIB-1 (ki67), ciclina D1, p16 y p53 para valorar si las alteraciones en la expresi��n proteica de estos marcadores, suceden de forma secuencial a trav��s de las distintas etapas en la cancerizaci��n del campo de cavidad oral, ya que no existe consenso en los resultados y las conclusiones obtenidas en los diferentes estudios efectuados sobre la influencia exclusiva de los marcadores apopt��ticos en el desarrollo de las lesiones de algunas de las lesiones como el LPO.

Function and regulation of death-associated protein kinase1 (DAPK) in colon cancer cells and macrophage-like cell line : identifcation of p38 as a novel DAPK interacting partner during TNF��-mediated apoptosis in colon cancer

DAPK, Apoptosis, p38, macrophages, survival

Dynamics of proximal signaling events after TCR/CD8-mediated induction of proliferation or apoptosis in mature T-cells

Magdeburg, Univ., Fak. f��r Naturwiss., Diss., 2009

CD95-induced apoptosis in health and disease dimers at the DISC and the effect of c-FLIP R on L. Monocytogenes infection

Magdeburg, Univ., Fak. f��r Naturwiss., Diss., 2013

role of Gadd45�� in apoptosis and autophagy

Magdeburg, Univ., Fak. f��r Naturwiss., Diss., 2013

apoptosis-regulator c-FLIP : functional role in urothelial carcinoma and autoimmunity and identification of novel CD95 DISC-interacting proteins

Magdeburg, Univ., Fak. f��r Naturwiss., Diss., 2013

Tumor necrosis factor (TNF) signaling, but not TWEAK (TNF-like weak inducer of apoptosis)-triggered cIAP1 (cellular inhibitor of apoptosis protein 1) degradation, requires cIAP1 RING dimerization and E2 binding.

Cellular inhibitor of apoptosis (cIAP) proteins, cIAP1 and cIAP2, are important regulators of tumor necrosis factor (TNF) superfamily (SF) signaling and are amplified in a number of tumor types. They are targeted by IAP antagonist compounds that are undergoing clinical trials. IAP antagonist compounds trigger cIAP autoubiquitylation and degradation. The TNFSF member TWEAK induces lysosomal degradation of TRAF2 and cIAPs, leading to elevated NIK levels and activation of non-canonical NF-kappaB. To investigate the role of the ubiquitin ligase RING domain of cIAP1 in these pathways, we used cIAP-deleted cells reconstituted with cIAP1 point mutants designed to interfere with the ability of the RING to dimerize or to interact with E2 enzymes. We show that RING dimerization and E2 binding are required for IAP antagonists to induce cIAP1 degradation and protect cells from TNF-induced cell death. The RING functions of cIAP1 are required for full TNF-induced activation of NF-kappaB, however, delayed activation of NF-kappaB still occurs in cIAP1 and -2 double knock-out cells. The RING functions of cIAP1 are also required to prevent constitutive activation of non-canonical NF-kappaB by targeting NIK for proteasomal degradation. However, in cIAP double knock-out cells TWEAK was still able to increase NIK levels demonstrating that NIK can be regulated by cIAP-independent pathways. Finally we show that, unlike IAP antagonists, TWEAK was able to induce degradation of cIAP1 RING mutants. These results emphasize the critical importance of the RING of cIAP1 in many signaling scenarios, but also demonstrate that in some pathways RING functions are not required.

Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors.

Viruses have evolved many distinct strategies to avoid the host's apoptotic response. Here we describe a new family of viral inhibitors (v-FLIPs) which interfere with apoptosis signalled through death receptors and which are present in several gamma-herpesviruses (including Kaposi's-sarcoma-associated human herpesvirus-8), as well as in the tumorigenic human molluscipoxvirus. v-FLIPs contain two death-effector domains which interact with the adaptor protein FADD, and this inhibits the recruitment and activation of the protease FLICE by the CD95 death receptor. Cells expressing v-FLIPs are protected against apoptosis induced by CD95 or by the related death receptors TRAMP and TRAIL-R. The herpesvirus saimiri FLIP is detected late during the lytic viral replication cycle, at a time when host cells are partially protected from CD95-ligand-mediated apoptosis. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses.

Peptabody-EGF: a novel apoptosis inducer targeting ErbB1 receptor overexpressing cancer cells.

The epidermal growth factor receptor (EGFR) plays a central role in cell life by controlling processes such as growth or proliferation. This receptor is commonly overexpressed in a number of epithelial malignancies and its upregulation is often associated with an aggressive phenotype of the tumor. Thus, targeting of EGFR represents a very promising challenge in oncology, and antibodies raised against this receptor have been investigated as potential antitumor agents. Various putative mechanisms of action were proposed for such antibodies, including decreased proliferation, induction of apoptosis, stimulation of the immunological response against targeted cancer cells or combinations thereof. We report here the development of an alternative high affinity molecule that is directed against EGFR. Production of this pentameric protein, named peptabody-EGF, includes expression in a bacterial expression system and subsequent refolding and multimerization of peptabody monomers. The protein complex contains 5 human EGF ligand domains, which confer specific binding towards the extracellular portion of EGFR. Receptor binding of the peptabody-EGF had a strong antiproliferative effect on different cancer cell lines overexpressing EGFR. However, cells expressing constitutive levels of the target receptor were barely affected. Peptabody-EGF treated cancer cells exhibited typical characteristics of apoptosis, which was found to be induced within 30 min after the addition of the peptabody-EGF. In vitro experiments demonstrated a significantly higher binding activity for peptabody-EGF than for the therapeutic monoclonal EGFR antibody Mab-425. Furthermore, the antitumor action provoked by the peptabody-EGF was greatly superior than antibody mediated effects when tested on EGFR overexpressing cancer cell lines. These findings suggest a potential application of this high affinity molecule as a novel tool for anti-EGFR therapy.

Cutting edge: apoptosis of superantigen-activated T cells occurs preferentially after a discrete number of cell divisions in vivo.

Staphylococcal enterotoxins are bacterial products that display superantigen activity in vitro as well as in vivo. For instance, staphylococcal enterotoxin B (SEB) polyclonally activates T cells that bear the Vbeta8 gene segment of the TCR. SEB-activated T cells undergo a burst of proliferation that is followed by apoptosis. Using an in vivo adaptation of a fluorescent cell division monitoring technique, we show here that SEB-activated T cells divide asynchronously, and that apoptosis of superantigen-activated T cells is preferentially restricted to cells which have undergone a discrete number of cell divisions. Collectively, our data suggest that superantigen-activated T cells are programmed to undergo a fixed number of cell divisions before undergoing apoptosis. A delayed death program may provide a mechanistic compromise between effector functions and homeostasis of activated T cells.

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major.

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.

Dominant negative MyD88 proteins inhibit interleukin-1beta /interferon-gamma -mediated induction of nuclear factor kappa B-dependent nitrite production and apoptosis in beta cells.

Insulin-dependent diabetes mellitus is an autoimmune disease in which pancreatic islet beta cells are destroyed by a combination of immunological and inflammatory mechanisms. In particular, cytokine-induced production of nitric oxide has been shown to correlate with beta cell apoptosis and/or inhibition of insulin secretion. In the present study, we investigated whether the interleukin (IL)-1beta intracellular signal transduction pathway could be blocked by overexpression of dominant negative forms of the IL-1 receptor interacting protein MyD88. We show that overexpression of the Toll domain or the lpr mutant of MyD88 in betaTc-Tet cells decreased nuclear factor kappaB (NF-kappaB) activation upon IL-1beta and IL-1beta/interferon (IFN)-gamma stimulation. Inducible nitric oxide synthase mRNA accumulation and nitrite production, which required the simultaneous presence of IL-1beta and IFN-gamma, were also suppressed by approximately 70%, and these cells were more resistant to cytokine-induced apoptosis as compared with parental cells. The decrease in glucose-stimulated insulin secretion induced by IL-1beta and IFN-gamma was however not prevented. This was because these dysfunctions were induced by IFN-gamma alone, which decreased cellular insulin content and stimulated insulin exocytosis. These results demonstrate that IL-1beta is involved in inducible nitric oxide synthase gene expression and induction of apoptosis in mouse beta cells but does not contribute to impaired glucose-stimulated insulin secretion. Furthermore, our data show that IL-1beta cellular actions can be blocked by expression of MyD88 dominant negative proteins and, finally, that cytokine-induced beta cell secretory dysfunctions are due to the action of IFN-gamma.