Optimization of organotypic cultures of mouse spleen for staining and functional assays

By preserving cell viability and three-dimensional localization, organotypic culture stands out among the newest frontiers of cell culture. It has been successfully employed for the study of diseases among which neoplasias, where tumoral cells take advantage of the surrounding stroma to promote their own proliferation and survival. Organotypic culture acquires major importance in the context of the immune system, whose cells cross-talk in a complex and dynamic fashion to elicit productive responses. However, organotypic culture has been as yet poorly developed for and applied to primary and secondary lymphoid organs. Here we describe in detail the development of a protocol suitable for the efficient cutting of mouse spleen, which overcomes technical difficulties related to the peculiar organ texture, and for optimized organotypic culture of spleen slices. Moreover, we used microscopy, immunofluorescence, flow cytometry and qRT-PCR to demonstrate that the majority of cells residing in spleen slices remain alive and maintain their original location in the organ architecture for several days after cutting. The development of this protocol represents a significant technical improvement in the study of the lymphoid microenvironment in both physiological and pathological conditions involving the immune system

The small GTPase Rab29 is a common regulator of immune synapse assembly and ciliogenesis

Acknowledgements We wish to thank Jorge Galán, Gregory Pazour, Derek Toomre, Giuliano Callaini, Joel Rosenbaum, Alessandra Boletta and Francesco Blasi for generously providing reagents and for productive discussions, and Sonia Grassini for technical assistance. The work was carried out with the financial support of Telethon (GGP11021) and AIRC.Peer reviewedPostprin

Optimización de métodos para el estudio del riesgo asociado a la contaminación fecal del agua y alimentos: cuantificación de patógenos y del riesgo de infecciones virales y análisis metagenómico de virus asociados a hepatitis

El agua es esencial para el sostenimiento de la vida en la tierra, por lo tanto, la calidad debe ser controlada para garantizar su uso de forma segura. Los objetivos planteados en esta tesis están orientados a la optimización de métodos para el estudio del riesgo asociado a la contaminación fecal del agua y alimentos. Este trabajo se dividen en tres secciones: La primera parte busca mejorar las herramientas para cuantificar de una manera más precisa los patógenos y el riesgo microbiológico que tiene el consumo de agua contaminada. En la segunda parte se estima el riesgo microbiológico de la utilización de agua regenerada para el riego de vegetales. En la última parte estudiamos pacientes con hepatitis aguda de etiología desconocida para la valoración de virus emergentes o virus nuevos de posible transmisión fecal-oral como posibles causantes de hepatitis. Los objetivos desarrollados en la presente tesis doctoral han dado lugar a una serie de resultados publicados o en proceso de publicación, las principales conclusiones de estos trabajos fueron: (a) El método SMF pudo ser utilizado eficientemente para concentrar simultáneamente los virus, bacterias y protozoos y se desarrolló un método para estimar la concentración real usando la cuantificaciones obtenidas por q(RT)PCR y la predicción de intervalos al 95% mediante “bootstrap” de las recuperaciones; (b) ninguna de los dos planta evaluadas fueron capaces de superar el umbral de <1.00E-6 DALYs pppy considerado como aceptable por la WHO para irrigar vegetales con agua regenerada y (c) la técnica de secuenciación masiva aplicada en esta tesis permitió detectar una gran variedad de secuencias de especies virales en pacientes con hepatitis y las principales familias identificadas fueron Anelloviridae, Caliciviridae y Astroviridae, todas estas relacionadas con transmisión fecal-oral

Thymic development beyond β-selection requires phosphatidylinositol 3-kinase activation by CXCR4

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110δ, and class IB, p110γ catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110δ, but not p110γ. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein–coupled receptor signaling in β-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of β-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond β-selection

Quantitative Phosphoproteomics of CXCL12 (SDF-1) Signaling

CXCL12 (SDF-1) is a chemokine that binds to and signals through the seven transmembrane receptor CXCR4. The CXCL12/CXCR4 signaling axis has been implicated in both cancer metastases and human immunodeficiency virus type 1 (HIV-1) infection and a more complete understanding of CXCL12/CXCR4 signaling pathways may support efforts to develop therapeutics for these diseases. Mass spectrometry-based phosphoproteomics has emerged as an important tool in studying signaling networks in an unbiased fashion. We employed stable isotope labeling with amino acids in cell culture (SILAC) quantitative phosphoproteomics to examine the CXCL12/CXCR4 signaling axis in the human lymphoblastic CEM cell line. We quantified 4,074 unique SILAC pairs from 1,673 proteins and 89 phosphopeptides were deemed CXCL12-responsive in biological replicates. Several well established CXCL12-responsive phosphosites such as AKT (pS473) and ERK2 (pY204) were confirmed in our study. We also validated two novel CXCL12-responsive phosphosites, stathmin (pS16) and AKT1S1 (pT246) by Western blot. Pathway analysis and comparisons with other phosphoproteomic datasets revealed that genes from CXCL12-responsive phosphosites are enriched for cellular pathways such as T cell activation, epidermal growth factor and mammalian target of rapamycin (mTOR) signaling, pathways which have previously been linked to CXCL12/CXCR4 signaling. Several of the novel CXCL12-responsive phosphoproteins from our study have also been implicated with cellular migration and HIV-1 infection, thus providing an attractive list of potential targets for the development of cancer metastasis and HIV-1 therapeutics and for furthering our understanding of chemokine signaling regulation by reversible phosphorylation

P66shc: A pleiotropic regulator of B cell trafficking and a gatekeeper in chronic lymphocytic leukemia

Neoplastic B cells from chronic lymphocytic leukemia patients (CLL) have a profound deficiency in the expression of p66Shc, an adaptor protein with pro-apoptotic and pro-oxidant activities. This defect results in leukemic B cell resistance to apoptosis and additionally impinges on the balance between chemokine receptors that control B cell homing to secondary lymphoid organs and the sphingosine phosphate receptor S1PR1 that controls their egress therefrom, thereby favoring leukemic B cell accumulation in the pro-survival lymphoid niche. Ablation of the gene encoding p66Shc in the Eµ-TCL1 mouse model of human CLL enhances leukemogenesis and promotes leukemic cell invasiveness in both nodal and extranodal organs, providing in vivo evidence of the pathogenic role of the p66Shc defect in CLL pathogenesis. Here we present an overview of the functions of p66Shc in B lymphocytes, with a specific focus on the multiple mechanisms exploited by p66Shc to control B cell trafficking and the abnormalities in this process caused by p66Shc deficiency in CLL

Dysfunctional immune synapses in T cell immunodeficiencies

Adaptive immune responses take place in the T cell area of secondary lymphoid organs, where, on encounter of antigen-presenting cells (APCs) bearing specific MHC-associated antigen, T lymphocytes assemble a highly organized intercellular junction, referred to as the immune synapse, on which T cell activation, proliferation, and differentiation crucially depend. Immune synapse assembly and function are impaired in a subgroup of human immunodeficiencies, characterized by altered adhesion or defective cross talk between the two synaptic partners. In this chapter, we will provide an overview of the immune synapse and describe how dysfunctional immune synapses can impinge on the pathogenesis of T cell immunodeficiencies, highlighting the reciprocal contribution of alterations at either side of the IS

p66Shc-dependent apoptosis requires Lck and CaMKII activity

p66Shc, an adaptor molecule which enhances reactive oxygen species (ROS) production by mitochondria, promotes T-cell apoptosis by inducing mitochondrial dysfunction and impairing Ca(2+) homeostasis. We have addressed the potential role of Lck, a kinase which has been implicated in T-cell apoptosis induced by a number of stimuli, in the proapoptotic activity of p66Shc. Lck deficiency in Jurkat T cells overexpressing p66Shc leads to impaired apoptotic responses to supraphysiological increases in [Ca(2+)](c). This defect could be rescued by reconstitution of Lck expression, indicating that Lck is required for p66Shc-dependent apoptosis. Furthermore, p66Shc phosphorylation on serine 36 (S36), an event on which the proapoptotic function of p66Shc depends, requires Lck. p66Shc-dependent mitochondrial dysfunction, altered Ca(2+) homeostasis and S36 phosphorylation require moreover the activity of CaMKII, a Ca(2+)/calmodulin-dependent kinase known to be implicated in the proapoptotic activity of Lck in T cells. The results suggest that increases in [Ca(2+)](c) lead to CaMKII activation and subsequent Lck-dependent p66Shc phosphorylation on S36. This event causes both mitochondrial dysfunction and impaired Ca(2+) homeostasis, which synergize in promoting Jurkat T-cell apoptosis