Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Distribución de las células presentadoras de antígenos en las glándulas salivales

Las células presentadoras de antígenos profesionales como las células de Langerhans o las células dendríticas intersticiales del tejido conectivo, son las células capturadoras de antígenos más importantes del sistema inmune. En trabajos previos describimos variaciones numéricas y estructurales de las células dendríticas; con especial referencia a las células de Langerhans, en diversos epitelios del tracto digestivo. El propósito de este trabajo fue estudiar la distribución, en forma comparativa, de las células dendríticas en glándulas salivales mayores y menores de la mucosa oral. Material y métodos: para esta investigación se utilizaron muestras de glándulas salivales mayores y menores tomadas de ratas Wistar. Las muestras fueron procesadas utilizando técnicas de microscopía óptica convencional, electrónica e inmunohistoquímica. Los resultados muestran numerosas células dendríticas en los conductos salivares excretores (con epitelio estratificado) así como en el parénquima glandular. Aunque no detectamos diferencias estadísticas significativas entre los dos componentes glandulares analizados, nuestros hallazgos sugieren que, debido a su localización, estas células cumplen un rol fundamental en las defensas inmunes de la mucosa oral.

Oscillatory genetic circuits: new designs and mathematical models

Introduction and motivation: A wide variety of organisms have developed in-ternal biomolecular clocks in order to adapt to cyclic changes of the environment. Clock operation involves genetic networks. These genetic networks have to be mod¬eled in order to understand the underlying mechanism of oscillations and to design new synthetic cellular clocks. This doctoral thesis has resulted in two contributions to the fields of genetic clocks and systems and synthetic biology, generally. The first contribution is a new genetic circuit model that exhibits an oscillatory behav¬ior through catalytic RNA molecules. The second and major contribution is a new genetic circuit model demonstrating that a repressor molecule acting on the positive feedback of a self-activating gene produces reliable oscillations. First contribution: A new model of a synthetic genetic oscillator based on a typical two-gene motif with one positive and one negative feedback loop is pre¬sented. The originality is that the repressor is a catalytic RNA molecule rather than a protein or a non-catalytic RNA molecule. This catalytic RNA is a ribozyme that acts post-transcriptionally by binding to and cleaving target mRNA molecules. This genetic clock involves just two genes, a mRNA and an activator protein, apart from the ribozyme. Parameter values that produce a circadian period in both determin¬istic and stochastic simulations have been chosen as an example of clock operation. The effects of the stochastic fluctuations are quantified by a period histogram and autocorrelation function. The conclusion is that catalytic RNA molecules can act as repressor proteins and simplify the design of genetic oscillators. Second and major contribution: It is demonstrated that a self-activating gene in conjunction with a simple negative interaction can easily produce robust matically validated. This model is comprised of two clearly distinct parts. The first is a positive feedback created by a protein that binds to the promoter of its own gene and activates the transcription. The second is a negative interaction in which a repressor molecule prevents this protein from binding to its promoter. A stochastic study shows that the system is robust to noise. A deterministic study identifies that the oscillator dynamics are mainly driven by two types of biomolecules: the protein, and the complex formed by the repressor and this protein. The main conclusion of this study is that a simple and usual negative interaction, such as degradation, se¬questration or inhibition, acting on the positive transcriptional feedback of a single gene is a sufficient condition to produce reliable oscillations. One gene is enough and the positive transcriptional feedback signal does not need to activate a second repressor gene. At the genetic level, this means that an explicit negative feedback loop is not necessary. Unlike many genetic oscillators, this model needs neither cooperative binding reactions nor the formation of protein multimers. Applications and future research directions: Recently, RNA molecules have been found to play many new catalytic roles. The first oscillatory genetic model proposed in this thesis uses ribozymes as repressor molecules. This could provide new synthetic biology design principles and a better understanding of cel¬lular clocks regulated by RNA molecules. The second genetic model proposed here involves only a repression acting on a self-activating gene and produces robust oscil¬lations. Unlike current two-gene oscillators, this model surprisingly does not require a second repressor gene. This result could help to clarify the design principles of cellular clocks and constitute a new efficient tool for engineering synthetic genetic oscillators. Possible follow-on research directions are: validate models in vivo and in vitro, research the potential of second model as a genetic memory, investigate new genetic oscillators regulated by non-coding RNAs and design a biosensor of positive feedbacks in genetic networks based on the operation of the second model Resumen Introduccion y motivacion: Una amplia variedad de organismos han desarro-llado relojes biomoleculares internos con el fin de adaptarse a los cambios ciclicos del entorno. El funcionamiento de estos relojes involucra redes geneticas. El mo delado de estas redes geneticas es esencial tanto para entender los mecanismos que producen las oscilaciones como para diseiiar nuevos circuitos sinteticos en celulas. Esta tesis doctoral ha dado lugar a dos contribuciones dentro de los campos de los circuitos geneticos en particular, y biologia de sistemas y sintetica en general. La primera contribucion es un nuevo modelo de circuito genetico que muestra un comportamiento oscilatorio usando moleculas de ARN cataliticas. La segunda y principal contribucion es un nuevo modelo de circuito genetico que demuestra que una molecula represora actuando sobre el lazo de un gen auto-activado produce oscilaciones robustas. Primera contribucion: Es un nuevo modelo de oscilador genetico sintetico basado en una tipica red genetica compuesta por dos genes con dos lazos de retroa-limentacion, uno positivo y otro negativo. La novedad de este modelo es que el represor es una molecula de ARN catalftica, en lugar de una protefna o una molecula de ARN no-catalitica. Este ARN catalitico es una ribozima que actua despues de la transcription genetica uniendose y cortando moleculas de ARN mensajero (ARNm). Este reloj genetico involucra solo dos genes, un ARNm y una proteina activadora, aparte de la ribozima. Como ejemplo de funcionamiento, se han escogido valores de los parametros que producen oscilaciones con periodo circadiano (24 horas) tanto en simulaciones deterministas como estocasticas. El efecto de las fluctuaciones es-tocasticas ha sido cuantificado mediante un histograma del periodo y la función de auto-correlacion. La conclusion es que las moleculas de ARN con propiedades cataliticas pueden jugar el misnio papel que las protemas represoras, y por lo tanto, simplificar el diseno de los osciladores geneticos. Segunda y principal contribucion: Es un nuevo modelo de oscilador genetico que demuestra que un gen auto-activado junto con una simple interaction negativa puede producir oscilaciones robustas. Este modelo ha sido estudiado y validado matematicamente. El modelo esta compuesto de dos partes bien diferenciadas. La primera parte es un lazo de retroalimentacion positiva creado por una proteina que se une al promotor de su propio gen activando la transcription. La segunda parte es una interaction negativa en la que una molecula represora evita la union de la proteina con el promotor. Un estudio estocastico muestra que el sistema es robusto al ruido. Un estudio determinista muestra que la dinamica del sistema es debida principalmente a dos tipos de biomoleculas: la proteina, y el complejo formado por el represor y esta proteina. La conclusion principal de este estudio es que una simple y usual interaction negativa, tal como una degradation, un secuestro o una inhibition, actuando sobre el lazo de retroalimentacion positiva de un solo gen es una condition suficiente para producir oscilaciones robustas. Un gen es suficiente y el lazo de retroalimentacion positiva no necesita activar a un segundo gen represor, tal y como ocurre en los relojes actuales con dos genes. Esto significa que a nivel genetico un lazo de retroalimentacion negativa no es necesario de forma explicita. Ademas, este modelo no necesita reacciones cooperativas ni la formation de multimeros proteicos, al contrario que en muchos osciladores geneticos. Aplicaciones y futuras lineas de investigacion: En los liltimos anos, se han descubierto muchas moleculas de ARN con capacidad catalitica. El primer modelo de oscilador genetico propuesto en esta tesis usa ribozimas como moleculas repre¬soras. Esto podria proporcionar nuevos principios de diseno en biologia sintetica y una mejor comprension de los relojes celulares regulados por moleculas de ARN. El segundo modelo de oscilador genetico propuesto aqui involucra solo una represion actuando sobre un gen auto-activado y produce oscilaciones robustas. Sorprendente-mente, un segundo gen represor no es necesario al contrario que en los bien conocidos osciladores con dos genes. Este resultado podria ayudar a clarificar los principios de diseno de los relojes celulares naturales y constituir una nueva y eficiente he-rramienta para crear osciladores geneticos sinteticos. Algunas de las futuras lineas de investigation abiertas tras esta tesis son: (1) la validation in vivo e in vitro de ambos modelos, (2) el estudio del potential del segundo modelo como circuito base para la construction de una memoria genetica, (3) el estudio de nuevos osciladores geneticos regulados por ARN no codificante y, por ultimo, (4) el rediseno del se¬gundo modelo de oscilador genetico para su uso como biosensor capaz de detectar genes auto-activados en redes geneticas.

Nepfix: on-demand cloud elastic simulation of networks of evolutionary processors

Una Red de Procesadores Evolutivos o NEP (por sus siglas en ingles), es un modelo computacional inspirado por el modelo evolutivo de las celulas, específicamente por las reglas de multiplicación de las mismas. Esta inspiración hace que el modelo sea una abstracción sintactica de la manipulation de information de las celulas. En particu¬lar, una NEP define una maquina de cómputo teorica capaz de resolver problemas NP completos de manera eficiente en tóerminos de tiempo. En la praóctica, se espera que las NEP simuladas en móaquinas computacionales convencionales puedan resolver prob¬lemas reales complejos (que requieran ser altamente escalables) a cambio de una alta complejidad espacial. En el modelo NEP, las cóelulas estóan representadas por palabras que codifican sus secuencias de ADN. Informalmente, en cualquier momento de cómputo del sistema, su estado evolutivo se describe como un coleccion de palabras, donde cada una de ellas representa una celula. Estos momentos fijos de evolucion se denominan configuraciones. De manera similar al modelo biologico, las palabras (celulas) mutan y se dividen en base a bio-operaciones sencillas, pero solo aquellas palabras aptas (como ocurre de forma parecida en proceso de selection natural) seran conservadas para la siguiente configuracióon. Una NEP como herramienta de computation, define una arquitectura paralela y distribuida de procesamiento simbolico, en otras palabras, una red de procesadores de lenguajes. Desde el momento en que el modelo fue propuesto a la comunidad científica en el año 2001, múltiples variantes se han desarrollado y sus propiedades respecto a la completitud computacional, eficiencia y universalidad han sido ampliamente estudiadas y demostradas. En la actualidad, por tanto, podemos considerar que el modelo teórico NEP se encuentra en el estadio de la madurez. La motivación principal de este Proyecto de Fin de Grado, es proponer una aproxi-mación práctica que permita dar un salto del modelo teórico NEP a una implantación real que permita su ejecucion en plataformas computacionales de alto rendimiento, con el fin de solucionar problemas complejos que demanda la sociedad actual. Hasta el momento, las herramientas desarrolladas para la simulation del modelo NEP, si bien correctas y con resultados satisfactorios, normalmente estón atadas a su entorno de ejecucion, ya sea el uso de hardware específico o implementaciones particulares de un problema. En este contexto, el propósito fundamental de este trabajo es el desarrollo de Nepfix, una herramienta generica y extensible para la ejecucion de cualquier algo¬ritmo de un modelo NEP (o alguna de sus variantes), ya sea de forma local, como una aplicación tradicional, o distribuida utilizando los servicios de la nube. Nepfix es una aplicacion software desarrollada durante 7 meses y que actualmente se encuentra en su segunda iteration, una vez abandonada la fase de prototipo. Nepfix ha sido disenada como una aplicacion modular escrita en Java 8 y autocontenida, es decir, no requiere de un entorno de ejecucion específico (cualquier maquina virtual de Java es un contenedor vólido). Nepfix contiene dos componentes o móodulos. El primer móodulo corresponde a la ejecución de una NEP y es por lo tanto, el simulador. Para su desarrollo, se ha tenido en cuenta el estado actual del modelo, es decir, las definiciones de los procesadores y filtros mas comunes que conforman la familia del modelo NEP. Adicionalmente, este componente ofrece flexibilidad en la ejecucion, pudiendo ampliar las capacidades del simulador sin modificar Nepfix, usando para ello un lenguaje de scripting. Dentro del desarrollo de este componente, tambióen se ha definido un estóandar de representacióon del modelo NEP basado en el formato JSON y se propone una forma de representation y codificación de las palabras, necesaria para la comunicación entre servidores. Adicional-mente, una característica importante de este componente, es que se puede considerar una aplicacion aislada y por tanto, la estrategia de distribution y ejecución son total-mente independientes. El segundo moódulo, corresponde a la distribucióon de Nepfix en la nube. Este de-sarrollo es el resultado de un proceso de i+D, que tiene una componente científica considerable. Vale la pena resaltar el desarrollo de este modulo no solo por los resul-tados prócticos esperados, sino por el proceso de investigation que se se debe abordar con esta nueva perspectiva para la ejecución de sistemas de computación natural. La principal característica de las aplicaciones que se ejecutan en la nube es que son gestionadas por la plataforma y normalmente se encapsulan en un contenedor. En el caso de Nepfix, este contenedor es una aplicacion Spring que utiliza el protocolo HTTP o AMQP para comunicarse con el resto de instancias. Como valor añadido, Nepfix aborda dos perspectivas de implementation distintas (que han sido desarrolladas en dos iteraciones diferentes) del modelo de distribution y ejecucion, que tienen un impacto muy significativo en las capacidades y restricciones del simulador. En concreto, la primera iteration utiliza un modelo de ejecucion asincrono. En esta perspectiva asincrona, los componentes de la red NEP (procesadores y filtros) son considerados como elementos reactivos a la necesidad de procesar una palabra. Esta implementation es una optimization de una topologia comun en el modelo NEP que permite utilizar herramientas de la nube para lograr un escalado transparente (en lo ref¬erente al balance de carga entre procesadores) pero produce efectos no deseados como indeterminacion en el orden de los resultados o imposibilidad de distribuir eficiente-mente redes fuertemente interconectadas. Por otro lado, la segunda iteration corresponde al modelo de ejecucion sincrono. Los elementos de una red NEP siguen un ciclo inicio-computo-sincronizacion hasta que el problema se ha resuelto. Esta perspectiva sincrona representa fielmente al modelo teórico NEP pero el proceso de sincronizacion es costoso y requiere de infraestructura adicional. En concreto, se requiere un servidor de colas de mensajes RabbitMQ. Sin embargo, en esta perspectiva los beneficios para problemas suficientemente grandes superan a los inconvenientes, ya que la distribuciín es inmediata (no hay restricciones), aunque el proceso de escalado no es trivial. En definitiva, el concepto de Nepfix como marco computacional se puede considerar satisfactorio: la tecnología es viable y los primeros resultados confirman que las carac-terísticas que se buscaban originalmente se han conseguido. Muchos frentes quedan abiertos para futuras investigaciones. En este documento se proponen algunas aproxi-maciones a la solucion de los problemas identificados como la recuperacion de errores y la division dinamica de una NEP en diferentes subdominios. Por otra parte, otros prob-lemas, lejos del alcance de este proyecto, quedan abiertos a un futuro desarrollo como por ejemplo, la estandarización de la representación de las palabras y optimizaciones en la ejecucion del modelo síncrono. Finalmente, algunos resultados preliminares de este Proyecto de Fin de Grado han sido presentados recientemente en formato de artículo científico en la "International Work-Conference on Artificial Neural Networks (IWANN)-2015" y publicados en "Ad-vances in Computational Intelligence" volumen 9094 de "Lecture Notes in Computer Science" de Springer International Publishing. Lo anterior, es una confirmation de que este trabajo mas que un Proyecto de Fin de Grado, es solo el inicio de un trabajo que puede tener mayor repercusion en la comunidad científica. Abstract Network of Evolutionary Processors -NEP is a computational model inspired by the evolution of cell populations, which might model some properties of evolving cell communities at the syntactical level. NEP defines theoretical computing devices able to solve NP complete problems in an efficient manner. In this model, cells are represented by words which encode their DNA sequences. Informally, at any moment of time, the evolutionary system is described by a collection of words, where each word represents one cell. Cells belong to species and their community evolves according to mutations and division which are defined by operations on words. Only those cells are accepted as surviving (correct) ones which are represented by a word in a given set of words, called the genotype space of the species. This feature is analogous with the natural process of evolution. Formally, NEP is based on an architecture for parallel and distributed processing, in other words, a network of language processors. Since the date when NEP was pro¬posed, several extensions and variants have appeared engendering a new set of models named Networks of Bio-inspired Processors (NBP). During this time, several works have proved the computational power of NBP. Specifically, their efficiency, universality, and computational completeness have been thoroughly investigated. Therefore, we can say that the NEP model has reached its maturity. The main motivation for this End of Grade project (EOG project in short) is to propose a practical approximation that allows to close the gap between theoretical NEP model and a practical implementation in high performing computational platforms in order to solve some of high the high complexity problems society requires today. Up until now tools developed to simulate NEPs, while correct and successful, are usu¬ally tightly coupled to the execution environment, using specific software frameworks (Hadoop) or direct hardware usage (GPUs). Within this context the main purpose of this work is the development of Nepfix, a generic and extensible tool that aims to execute algorithms based on NEP model and compatible variants in a local way, similar to a traditional application or in a distributed cloud environment. Nepfix as an application was developed during a 7 month cycle and is undergoing its second iteration once the prototype period was abandoned. Nepfix is designed as a modular self-contained application written in Java 8, that is, no additional external dependencies are required and it does not rely on an specific execution environment, any JVM is a valid container. Nepfix is made of two components or modules. The first module corresponds to the NEP execution and therefore simulation. During the development the current state of the theoretical model was used as a reference including most common filters and processors. Additionally extensibility is provided by the use of Python as a scripting language to run custom logic. Along with the simulation a definition language for NEP has been defined based on JSON as well as a mechanisms to represent words and their possible manipulations. NEP simulator is isolated from distribution and as mentioned before different applications that include it as a dependency are possible, the distribution of NEPs is an example of this. The second module corresponds to executing Nepfix in the cloud. The development carried a heavy R&D process since this front was not explored by other research groups until now. It's important to point out that the development of this module is not focused on results at this point in time, instead we focus on feasibility and discovery of this new perspective to execute natural computing systems and NEPs specifically. The main properties of cloud applications is that they are managed by the platform and are encapsulated in a container. For Nepfix a Spring application becomes the container and the HTTP or AMQP protocols are used for communication with the rest of the instances. Different execution perspectives were studied, namely asynchronous and synchronous models were developed for solving different kind of problems using NEPs. Different limitations and restrictions manifest in both models and are explored in detail in the respective chapters. In conclusion we can consider that Nepfix as a computational framework is suc-cessful: Cloud technology is ready for the challenge and the first results reassure that the properties Nepfix project pursued were met. Many investigation branches are left open for future investigations. In this EOG implementation guidelines are proposed for some of them like error recovery or dynamic NEP splitting. On the other hand other interesting problems that were not in the scope of this project were identified during development like word representation standardization or NEP model optimizations. As a confirmation that the results of this work can be useful to the scientific com-munity a preliminary version of this project was published in The International Work- Conference on Artificial Neural Networks (IWANN) in May 2015. Development has not stopped since that point and while Nepfix in it's current state can not be consid¬ered a final product the most relevant ideas, possible problems and solutions that were produced during the seven months development cycle are worthy to be gathered and presented giving a meaning to this EOG work.

Dendritic cell ontogeny: A human dendritic cell lineage of myeloid origin

Dendritic cells (DC) have been thought to represent a family of closely related cells with similar functions and developmental pathways. The best-characterized precursors are the epidermal Langerhans cells, which migrate to lymphoid organs and become activated DC in response to inflammatory stimuli. Here, we demonstrate that a large subset of DC in the T cell-dependent areas of human lymphoid organs are nonactivated cells and belong to a separate lineage that can be identified by high levels of the interleukin 3 receptor α chain (IL-3Rαhi). The CD34+IL-3Rαhi DC progenitors are of myeloid origin and are distinct from those that give rise to Langerhans cells in vitro. The IL-3Rαhi DC furthermore appear to migrate to lymphoid organs independently of inflammatory stimuli or foreign antigens. Thus, DC are heterogeneous with regard to function and ontogeny.

neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas

In the mammalian pancreas, the endocrine cell types of the islets of Langerhans, including the α-, β-, δ-, and pancreatic polypeptide cells as well as the exocrine cells, derive from foregut endodermal progenitors. Recent genetic studies have identified a network of transcription factors, including Pdx1, Isl1, Pax4, Pax6, NeuroD, Nkx2.2, and Hlxb9, regulating the development of islet cells at different stages, but the molecular mechanisms controlling the specification of pancreatic endocrine precursors remain unknown. neurogenin3 (ngn3) is a member of a family of basic helix–loop–helix transcription factors that is involved in the determination of neural precursor cells in the neuroectoderm. ngn3 is expressed in discrete regions of the nervous system and in scattered cells in the embryonic pancreas. We show herein that ngn3-positive cells coexpress neither insulin nor glucagon, suggesting that ngn3 marks early precursors of pancreatic endocrine cells. Mice lacking ngn3 function fail to generate any pancreatic endocrine cells and die postnatally from diabetes. Expression of Isl1, Pax4, Pax6, and NeuroD is lost, and endocrine precursors are lacking in the mutant pancreatic epithelium. Thus, ngn3 is required for the specification of a common precursor for the four pancreatic endocrine cell types.

Percutaneous peptide immunization via corneum barrier-disrupted murine skin for experimental tumor immunoprophylaxis

H-2Kb-restricted tumor epitope peptides, including tyrosinase-related protein 2 residues 181–188 (TRP-2) and connexin 37 residues 52–59 (MUT1), were applied to permeability barrier-disrupted C57BL/6 (B6) mouse skin from which the stratum corneum of the epidermis had been removed by tape-stripping. This procedure primed tumor-specific cytotoxic T lymphocytes (CTLs) in the lymph nodes and spleen, protected mice against subsequent challenge with corresponding tumor cells, and suppressed the growth of established tumors. Preventive and therapeutic effectiveness was correlated with the frequency of tumor-specific CTL precursors. MHC class II Iab+ cells separated from tape-stripped skin, compared with those from intact skin, exhibited a strong antigen-presenting capacity for CTL, suggesting that CTL expansion after peptide application is primarily mediated by epidermal Langerhans cells. Thus, percutaneous peptide immunization via barrier-disrupted skin provides a simple and noninvasive means of inducing potent anti-tumor immunity which may be exploited for cancer immunotherapy.

CD1c molecules broadly survey the endocytic system

The ability of antigen-presenting cells to sample distinct intracellular compartments is crucial for microbe detection. Major histocompatibility complex class I and class II molecules sample the cytosol or the late endocytic compartment, allowing detection of microbial peptide antigens that arise in distinct intracellular compartments. In contrast, CD1a and CD1b molecules mediate the presentation of lipid and glycolipid antigens and differentially sample early recycling endosomes or late endocytic compartments, respectively, that contain distinct sets of lipid antigens. Here, we show that, unlike the other CD1 isoforms or major histocompatibility complex molecules that each sample restricted only intracellular compartments, CD1c is remarkable in that it distributes broadly throughout the endocytic system and is expressed in both recycling endosomes and late endocytic compartments. Further, in contrast to CD1b, which requires an acidic environment to function, antigen presentation by CD1c was able to overcome dependence on vesicular acidification. Because CD1c is expressed on essential antigen-presenting cells, such as epidermal Langerhans cells (in the absence of CD1b), or on B cells (without CD1a or -b), we suggest that CD1c molecules allow a comprehensive survey for lipid antigens throughout the endocytic system even in the absence of other CD1 isoforms.

Induction of insulin and islet amyloid polypeptide production in pancreatic islet glucagonoma cells by insulin promoter factor 1.

Insulin promoter factor 1 (IPF1), a member of the homeodomain protein family, serves an early role in pancreas formation, as evidenced by the lack of pancreas formation in mice carrying a targeted disruption of the IPF1 gene [Jonsson, J., Carlsson, L., Edlund, T. & Edlund, H. (1994) Nature (London) 371, 606-609]. In adults, IPF1 expression is restricted to the beta-cells in the islets of Langerhans. We report here that IPF1 induces expression of a subset of beta-cell-specific genes (insulin and islet amyloid polypeptide) when ectopically expressed in clones of transformed pancreatic islet alpha-cells. In contrast, expression of IPF1 in rat embryo fibroblasts factor failed to induce insulin and islet amyloid polypeptide expression. This is most likely due to the lack of at least one other essential insulin gene transcription factor, the basic helix-loop-helix protein Beta 2/NeuroD, which is expressed in both alpha- and beta-cells. We conclude that IPF1 is a potent transcriptional activator of endogenous insulin genes in non-beta islet cells, which suggests an important role of IPF1 in beta-cell maturation.

Expression of a candidate cadherin in T lymphocytes.

Cadherins are homotypic adhesion molecules that classically mediate interactions between cells of the same type in solid tissues. In addition, E-cadherin is able to support homotypic adhesion of epidermal Langerhans cells to keratinocytes (Tang, A., Amagai, M., Granger, L. G., Stanley, J. R. & Udey, M. C. (1993) Nature (London) 361, 82-85) and heterotypic adhesion of mucosal epithelial cells to E-cadherin-negative intestinal intraepithelial T lymphocytes. Thus, we hypothesized that cadherins may play a wider role in cell-to-cell adhesion events involving T lymphocytes. We searched for a cadherin or cadherins in T lymphocytes with a pan-cadherin antiserum and antisera against alpha- or beta-catenin, molecules known to associate with the cytoplasmic domain of cadherins. The anti-beta-catenin antisera coimmunoprecipitated a radiolabeled species in T-lymphocyte lines that had a molecular mass of 129 kDa and was specifically immunoblotted with the pan-cadherin antiserum. Also, the pan-cadherin antiserum directly immunoprecipitated a 129-kDa radiolabeled species from an 125I surface-labeled Jurkat human T-cell leukemic cell line. After V8 protease digestion, the peptide map of this pan-cadherin-immunoprecipitated, 129-kDa species exactly matched that of the 129-kDa species coimmunoprecipitated with the beta-catenin antiserum. These results demonstrate that T lymphocytes express a catenin-associated protein that appears to be a member of the cadherin superfamily and may contribute to T cell-mediated immune surveillance.

The cloned rat pancreatic polypeptide receptor exhibits profound differences to the orthologous receptor.

Pancreatic polypeptide (PP) is produced in the islets of Langerhans and released in response to meals. It belongs to a family of peptides that also includes neuropeptide Y and peptide YY. In the present communication, we describe a rat receptor with high affinity for PP, therefore named PP1. Clones for the PP1 receptor were obtained by PCR using sequence information for the neuropeptide Y receptor Y1 from several species. The PP1 receptor has 46% overall amino acid sequence identity to the rat Y1 receptor and 56% identity in the transmembrane regions. The PP1 receptor displays a pharmacological profile that is distinct from previously described neuropeptide Y-family receptors. In competition with iodinated bovine PP, it binds rat PP with an affinity (K(i)) of 0.017 nM, while the affinities for peptide YY and neuropeptide Y are substantially lower with K(i) values of 162 and 192 nM, respectively. In stably transfected CHO cells, the PP1 receptor inhibits forskolin-stimulated cAMP synthesis. Northern blot hybridizations to a panel of mRNAs detected transcripts in testis and lung. A faint band was seen in colon and total brain. In contrast, the human receptor is expressed primarily in colon and small intestine. Whereas rat and human PP1 bind PP with the same affinity, the rat receptor has much lower affinity than its human ortholog for peptide YY and neuropeptide Y. Interestingly, the amino acid sequence identity between rat and human PP1 is only 75%. Thus, the sequence, the tissue distribution, and the binding profile of the PP1 receptor differ considerably between rat and human.

Participación de las células supresoras mieloides (CSM) y T regulatorias (Treg) en el control de la infección con Trypanosoma cruzi y el desarrollo de la patología inflamatoria asociada a la infeccion

La infección de mamíferos con el T. cruzi resulta en diferentes alteraciones inmunológicas que permiten la persistencia crónica del parásito y destrucción inflamatoria progresiva del tejido cardiaco, nervioso y hepático. Los mecanismos responsables de la patología de la enfermedad de Chagas han sido materia de intensa investigación habiéndose propuesto que el daño producido en esta enfermedad puede ser consecuencia de la respuesta inflamatoria del individuo infectado y/o de una acción directa del parásito sobre los tejidos del hospedador. El propósito del presente proyecto es estudiar comparativamente, en dos cepas de ratones con diferente susceptibilidad a la infección y desarrollo de patología, la participación y los mecanismos efectores de las células supresoras mieloides (CSM) y las celulas T regulatorias inducidas por la infección experimental con Trypanosoma cruzi en el control de la infección con este protozoario y en el desarrollo de la patología hepática siendo los objetivos especificos desarrolar: - Investigar la generación y/o reclutamiento de células de CSM en bazo e hígado de ratones infectados con Trypanosoma cruzi y su contribución a la desigual susceptibilidad a la infección y respuesta inmune desarrollada en las cepas de ratones BALB/c y C57BL/6; - Investigar la capacidad de las CSM inducidas por la infección con T. cruzi en bazo e hígado de ratones de ambas cepas para suprimir la respuesta de células T in vitro e indagar sobre los mecanismos de supresión utilizados; - Investigar la generación y/o reclutamiento de células Treg durante la infección experimental con Trypanosoma cruzi, su participación en la desigual susceptibilidad a la infección y respuesta inmune desarrollada en ambas cepas de ratones y los mecanismos de supresión utilizados. - Analizar en tejido hepático o leucocitos infiltrantes la presencia de COX2, PGE2, MMP2 y 9, IL1b, IL6, IDO, IL10 y GM-CSF capaces de inducir la expansión de las CSM; - Dilucidar si la administración del ligando para TLR2 (Pam3CyS) previo a la infección de ratones C57BL/6 (en los cuales se detecta un menor número de CSM) es capaz de modular la respuesta inflamatoria y el daño hepático a través de la inducción de CSM y/o T reg en hígado y bazo. La comprension de los eventos celulares y moleculares que regulan la producción de citoquinas pro- y anti-inflamatorias y otros mediadores, así como el papel de los receptores de la inmunidad innata durante la infección con T. cruzi contribuirá a responder interrogantes que son claves para el diseño de nuevas estrategias de intervención inmune tendientes a preservar los mecanismos de defensa del huésped.

Angiotensinogen localization and secretion in the rat pancreas

Renin and angiotensinogen have been previously found in the rat pancreas, and angiotensin receptors have been located in the apical domain of duct cells. To evaluate the possibility that angiotensin II could be generated within the duct system, we decided to determine whether angiotensinogen is present in rat pancreatic juice and the angiotensinogen-immunoreactive pancreatic cell types that could be responsible for its production. Angiotensinogen was detected in significant amounts by Western blotting in pancreatic juice collected from several individual rats. Different isoforms between plasma and pancreatic juice angiotensinogens were demonstrated by isoelectric focusing. Immunocytochemical experiments revealed angiotensinogen-immunoreactive cells at the periphery of the islets of Langerhans, and confocal microscopy demonstrated that most angiotensinogen-immunoreactive cells were glucagon-secreting cells. Secretion of angiotensinogen did not follow the regulated secretory pathway since it was absent from the glucagon-containing granules. This was confirmed by electron microscopy immunocytochemistry. Duct and acinar cells did not express angiotensinogen at an immunocytochemical detectable level. The present findings indicated an exocrine secretion of angiotensinogen by glucagon-secreting cells and suggest that one of the final targets of the local pancreatic renin-angiotensin system may be the duct epithelium.

A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse

The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fms transcripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5' flanking sequence and the down-stream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms-positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location Was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (the Fms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function. (C) 2003 by The American Society of Hematology.

Activation of dendritic cells by human papillomavirus-like particles through TLR4 and NF-B-mediated signalling, moderated by TGF-beta

Human papillomavirus-like particles (HPV-VLP) are a candidate vaccine for prevention of HPV infection, and also are a candidate for an immunogenic delivery system for incorporated antigen. VLP activate in vitro generated dendritic cells (DC) but not Langerhans cells (LC); however, the mechanism of this activation is unknown. We have shown that uptake and activation of DC by VLP involves proteoglycan receptors and can be inhibited by heparin. Heparin has been shown to activate DC by signalling through Toll-like receptor 4 (TLR4) and nuclear factor (NF)-kappaB. The pathway of DC activation by VLP was further investigated in the present study. Exposure to VLP induced costimulatory molecule expression, RelB translocation and IL-10 production by DC but not by LC. The lack of LC activation was reversible when TGF-beta was removed from the LC medium. VLP-induced induction of costimulatory molecule expression, RelB activation and cytokine secretion by DC was blocked by inhibition of NF-kappaB activation, heparin or TLR4 mAb. The data provide evidence that HPV-VLP signal DC through a pathway involving proteoglycan receptors, TLR4 and NF-kappaB, and shed light on the mechanism by which VLP stimulate immunity in the absence of adjuvants in vivo. LC may resist activation in normal epithelium abundant in TGF-beta, but not in situations in which TGF-beta concentrations are reduced.