Characterization of Staphylococcus aureus adhesins important for respiratory tract infection

Staphylococcus aureus is a leading cause of lower respiratory tract infections in both adult and pediatric populations. In the past two decades, reports have described emergent incidence of severe necrotizing pneumonia in previously healthy individuals, frequently caused by antibiotic resistant strains. Additionally, S. aureus remains the most common cause of ventilator-associated pneumonia, contributing morbidity and mortality in intensive care units. As treatment of infection is made more difficult by the resistance to multiple antibiotics including vancomycin, there is a pressing need for novel strategies to prevent and treat S. aureus infections. Targeting essential mechanisms that promote infection such as adhesion, colonization, invasion, evasion of immune system and signaling may lead to inhibition of pathogenic surge. Staphylococcal adhesins of the MSCRAMM family (microbial surface components recognizing adherent matrix molecules) represent viable targets for such investigations. Understanding the molecular mechanism of binding is the first step toward the development of such therapies. Analysis of bacterial strains isolated from patients with staphylococcal pneumonia show increased expression of protein A, SdrD, SdrC and ClfB, cell surface proteins members of the MSCRAMM family. In this study the interaction of these MSCRAMMs with candidate ligands has been examined. We found that SdrD mediates S. aureus adherence to the lung epithelial cell line A549. Consistently, bacteria expressing SdrD have increased persistence in the lungs of infected mice after bronchoalveolar lavage in comparison with bacteria lacking this protein. Inhibition studies revealed that bacterial attachment can be abolished using neutralizing antibodies against SdrD. Using phage display, neurexin β isoforms were identified as SdrC binding partners. Previous reports postulated that MSCRAMMS bind their ligands by a 'dock, lock and latch' mechanism of interaction. Our data suggested that ClfB, an MSCRAMM responsible for nasal colonization, binds cytokeratin 10 by a 'dock and lock' variant of this model, in which the 'latching' event is not necessary. In summary, we have characterized aspects of molecular interaction between several MSCRAMMS and host components. We hope that continued delineation of these interactions will lead to identification of novel therapeutic targets or preventive strategies against S. aureus infections. ^

STIMULATION THROUGH TLR4 INCREASES FVIII INHIBITOR FORMATION IN A MOUSE MODEL OF HEMOPHILIA A

Hemophilia A is a clotting disorder caused by functional factor VIII (FVIII) deficiency. About 25% of patients treated with therapeutic recombinant FVIII develop antibodies (inhibitors) that render subsequent FVIII treatments ineffective. The immune mechanisms of inhibitor formation are not entirely understood, but circumstantial evidence indicates a role for increased inflammatory response, possibly via stimulation of Toll-like receptors (TLRs), at the time of FVIII immunization. I hypothesized that stimulation through TLR4 in conjunction with FVIII treatments would increase the formation of FVIII inhibitors. To test this hypothesis, FVIII K.O. mice were injected with recombinant human FVIII with or without concomitant doses of TLR4 agonist (lipopoysaccharide; LPS). The addition of LPS combined with FVIII significantly increased the rate and the production of anti-FVIII IgG antibodies and neutralizing FVIII inhibitors. In the spleen, repeated in vivo TLR4 stimulation with LPS increased the relative percentage of macrophages and dendritic cells (DCs) over the course of 4 injections. However, repeated in vivo FVIII stimulation significantly increased the density of TLR4 expressed on the surface of all spleen antigen presenting cells (APCs). Culture of splenocytes isolated from mice revealed that the combined stimulation of LPS and FVIII also synergistically increased early secretion of the inflammatory cytokines IL-6, TNF-α, and IL-10, which was not maintained throughout the course of the repeated injections. While cytokine secretion was relatively unchanged in response to FVIII re-stimulation in culture, LPS re-stimulation in culture induced increased and prolonged inflammatory cytokine secretion. Re-stimulation with both LPS and FVIII induced cytokine secretion similar to LPS stimulation alone. Interestingly, long term treatment of mice with LPS alone resulted in splenocytes that showed reduced response to FVIII in culture. Together these results indicated that creating a pro-inflammatory environment through the combined stimulation of chronic, low-dose LPS and FVIII changed not only the populations but also the repertoire of APCs in the spleen, triggering the increased production of FVIII inhibitors. These results suggested an anti-inflammatory regimen should be instituted for all hemophilia A patients to reduce or delay the formation of FVIII inhibitors during replacement therapy.

The effects of IL -10 producing monocytes on T-cell activation in patients with epithelial ovarian cancer

A newly described subset of monocytes has been identified in peritoneal exudate cells (PEC) from the malignant ascites of patients with ovarian cancer. These cells were characterized by the production of IL-10 and TGF-β2, but not IL-12, IL-1α, or TNF-α, and expressed CD14, CD16, and CD54, but not HLA-DR, CD80, CD86, CD11a, CD11b, or CD25 cell surface antigens. Since this subset of monocytes could affect the modulation of tumor immune responses in vivo, studies were undertaken to determine their effect on the activation and proliferation of autologous T-cells from the peritoneal cavity of patients with ovarian carcinoma. Cytokine transcripts, including IL-2, GM-CSF, and IFN-γ were detected in T-cells isolated from patient specimens that also contained the IL-10 producing monocytes, although the IFN-γ and IL-2 proteins could not be detected in T-cells co-incubated with the IL-10 producing monocytes in vitro. Additionally, IL-10 producing monocytes co-cultured with autologous T-cells inhibited the proliferation of the T-cells in response to PHA. T-cell proliferation and cytokine protein production could be restored by the addition of neutralizing antibodies to IL-10R and TGF-β to the co-culture system. These results suggested that this subset of monocytes may modulate antitumor immune responses by inhibiting T-cell proliferation and cytokine protein production. Further studies determined that the precursors to the inhibitory monocytes were tumor-associated and only present in the peripheral blood of patients with ovarian cancer and not present in the peripheral blood of healthy donors. These precursors could be induced to the suppressor phenotype by the addition of IL-2 and GM-CSF, two cytokines detected in the peritoneal cavity of ovarian cancer patients. Lastly, it was shown that the suppressor monocytes from the peritoneal cavity of ovarian cancer patients could be differentiated to a non-inhibitory phenotype by the addition of TNF-α and IFN-γ to the culture system. The differentiated monocytes did not produce IL-10, expressed the activation antigens HLA-DR, CD80, and CD86, and were able to stimulate autologous T-cells in vitro. Since a concomitant reduction in immune function is associated with tumor growth and progression, the effects of these monocytes are of considerable importance in the context of tumor immunotherapy. ^

コンゴ民主共和国における武装勢力掃討は成功するか? -- 対ADF作戦の難しさ

コンゴ民主共和国の安定を図るため、国連は周辺諸国、アフリカ連合などと協力して新たな構想を立ち上げた。その構想「フレームワーク」ではコンゴ軍と協力して、コンゴ東部の武装勢力を一掃する作戦を展開することとなった。ADFと呼ばれる武装勢力もその標的の1つであるが、いまだに武装解除できていない。本稿ではADFの誕生から現在までの歴史をたどり、彼らが地元住民と社会的、経済的に緊密なネットワークを形成して共存していることを示す。またADFが、コンゴ軍やそのほかの武装勢力による暴力から地元住民を保護する役割を果たしていることを示唆し、コンゴ軍よりも支持されている可能性を示す。コンゴを安定させるためには武装勢力への軍事行動だけでなく、コンゴ軍を含む、政治、行政機構の改革にこれまで以上に積極的に取り組む必要がある。

VISIONS Vehicular communication Improvement : solution based on IMS Operational Nodes and Services

Digital services and communications in vehicular scenarios provide the essential assets to improve road transport in several ways like reducing accidents, improving traffic efficiency and optimizing the transport of goods and people. Vehicular communications typically rely on VANET (Vehicular Ad hoc Networks). In these networks vehicles communicate with each other without the need of infrastructure. VANET are mainly oriented to disseminate information to the vehicles in certain geographic area for time critical services like safety warnings but present very challenging requirements that have not been successfully fulfilled nowadays. Some of these challenges are; channel saturation due to simultaneous radio access of many vehicles, routing protocols in topologies that vary rapidly, minimum quality of service assurance and security mechanisms to efficiently detect and neutralize malicious attacks. Vehicular services can be classified in four important groups: Safety, Efficiency, Sustainability and Infotainment. The benefits of these services for the transport sector are clear but many technological and business challenges need to be faced before a real mass market deployment. Service delivery platforms are not prepared for fulfilling the needs of this complex environment with restrictive requirements due to the criticism of some services To overcome this situation, we propose a solution called VISIONS “Vehicular communication Improvement: Solution based on IMS Operational Nodes and Services”. VISIONS leverages on IMS subsystem and NGN enablers, and follows the CALM reference Architecture standardized by ISO. It also avoids the use of Road Side Units (RSUs), reducing complexity and high costs in terms of deployment and maintenance. We demonstrate the benefits in the following areas: 1. VANET networks efficiency. VISIONS provide a mechanism for the vehicles to access valuable information from IMS and its capabilities through a cellular channel. This efficiency improvement will occur in two relevant areas: a. Routing mechanisms. These protocols are responsible of carrying information from a vehicle to another (or a group of vehicles) using multihop mechanisms. We do not propose a new algorithm but the use of VANET topology information provided through our solution to enrich the performance of these protocols. b. Security. Many aspects of security (privacy, key, authentication, access control, revocation mechanisms, etc) are not resolved in vehicular communications. Our solution efficiently disseminates revocation information to neutralize malicious nodes in the VANET. 2. Service delivery platform. It is based on extended enablers, reference architectures, standard protocols and open APIs. By following this approach, we reduce costs and resources for service development, deployment and maintenance. To quantify these benefits in VANET networks, we provide an analytical model of the system and simulate our solution in realistic scenarios. The simulations results demonstrate how VISIONS improves the performance of relevant routing protocols and is more efficient neutralizing security attacks than the widely proposed solutions based on RSUs. Finally, we design an innovative Social Network service based in our platform, explaining how VISIONS facilitate the deployment and usage of complex capabilities. RESUMEN Los servicios digitales y comunicaciones en entornos vehiculares proporcionan herramientas esenciales para mejorar el transporte por carretera; reduciendo el número de accidentes, mejorando la eficiencia del tráfico y optimizando el transporte de mercancías y personas. Las comunicaciones vehiculares generalmente están basadas en redes VANET (Vehicular Ad hoc Networks). En dichas redes, los vehículos se comunican entre sí sin necesidad de infraestructura. Las redes VANET están principalmente orientadas a difundir información (por ejemplo advertencias de seguridad) a los vehículos en determinadas zonas geográficas, pero presentan unos requisitos muy exigentes que no se han resuelto con éxito hasta la fecha. Algunos de estos retos son; saturación del canal de acceso de radio debido al acceso simultáneo de múltiples vehículos, la eficiencia de protocolos de encaminamiento en topologías que varían rápidamente, la calidad de servicio (QoS) y los mecanismos de seguridad para detectar y neutralizar los ataques maliciosos de manera eficiente. Los servicios vehiculares pueden clasificarse en cuatro grupos: Seguridad, Eficiencia del tráfico, Sostenibilidad, e Infotainment (información y entretenimiento). Los beneficios de estos servicios para el sector son claros, pero es necesario resolver muchos desafíos tecnológicos y de negocio antes de una implementación real. Las actuales plataformas de despliegue de servicios no están preparadas para satisfacer las necesidades de este complejo entorno con requisitos muy restrictivos debido a la criticidad de algunas aplicaciones. Con el objetivo de mejorar esta situación, proponemos una solución llamada VISIONS “Vehicular communication Improvement: Solution based on IMS Operational Nodes and Services”. VISIONS se basa en el subsistema IMS, las capacidades NGN y es compatible con la arquitectura de referencia CALM estandarizado por ISO para sistemas de transporte. También evita el uso de elementos en las carreteras, conocidos como Road Side Units (RSU), reduciendo la complejidad y los altos costes de despliegue y mantenimiento. A lo largo de la tesis, demostramos los beneficios en las siguientes áreas: 1. Eficiencia en redes VANET. VISIONS proporciona un mecanismo para que los vehículos accedan a información valiosa proporcionada por IMS y sus capacidades a través de un canal de celular. Dicho mecanismo contribuye a la mejora de dos áreas importantes: a. Mecanismos de encaminamiento. Estos protocolos son responsables de llevar información de un vehículo a otro (o a un grupo de vehículos) utilizando múltiples saltos. No proponemos un nuevo algoritmo de encaminamiento, sino el uso de información topológica de la red VANET a través de nuestra solución para enriquecer el funcionamiento de los protocolos más relevantes. b. Seguridad. Muchos aspectos de la seguridad (privacidad, gestión de claves, autenticación, control de acceso, mecanismos de revocación, etc) no están resueltos en las comunicaciones vehiculares. Nuestra solución difunde de manera eficiente la información de revocación para neutralizar los nodos maliciosos en la red. 2. Plataforma de despliegue de servicios. Está basada en capacidades NGN, arquitecturas de referencia, protocolos estándar y APIs abiertos. Siguiendo este enfoque, reducimos costes y optimizamos procesos para el desarrollo, despliegue y mantenimiento de servicios vehiculares. Para cuantificar estos beneficios en las redes VANET, ofrecemos un modelo de analítico del sistema y simulamos nuestra solución en escenarios realistas. Los resultados de las simulaciones muestran cómo VISIONS mejora el rendimiento de los protocolos de encaminamiento relevantes y neutraliza los ataques a la seguridad de forma más eficientes que las soluciones basadas en RSU. Por último, diseñamos un innovador servicio de red social basado en nuestra plataforma, explicando cómo VISIONS facilita el despliegue y el uso de las capacidades NGN.

El Prado, de territorio a escenario : la construcción del espacio en un lugar tangible y su significado

"Si el hombre es el cuidador de las palabras y sólo de ellas emerge el sentido de las cosas, la arquitectura tiene un cometido preciso: hacer de las condiciones ya dadas de cada lugar palabras que signifiquen las cualidades de la existencia, y que desvelen la riqueza y contenidos que en ellas se contienen potencialmente" Ignasi Solá Morales. Lugar: permanencia o producción, 1992. Esta tesis surge tanto del afán por comprender la identidad de uno de los espacios más representativos de mi ciudad, asumido familiarmente pero que plantea muchas dudas respecto a su caracterización, como de la preocupación personal respecto a la aparente hegemonía del modelo urbano de la "ciudad genérica", crudamente expuesto por Rem Koolhaas a finales del siglo XX, que pone en crisis la ciudad histórica. El territorio, espacio físico concreto, y la memoria asociada a este, obliterados, son considerados como punto de partida para confrontarlos con la proclamación del nuevo modelo de "ciudad genérica", de raíz eminentemente económica y tecnológica. La realidad tangible de un espacio, aparentemente forjado en base a los valores denostados por el nuevo modelo propuesto, se estudia desde las premisas opuestas. La idea del no-lugar, teorizado por Marc Augé y tomado como modelo por Koolhaas, supone éste emancipado tanto de las preexistencias históricas como de su ubicación física concreta, planteando un tipo de espacio de representación al margen del territorio y la memoria. Sin pretender adoptar una postura resistente u opuesta, sino antitética y complementaria, se toman aquí las premisas de Koolhaas para contrastarlas con una porción del territorio a medio camino entre la arquitectura y la ciudad, a fin de desarrollar una reflexión que sirva de complemento y contrapeso al paradigma espacial que la “ciudad genérica” implica y cuya inmediatez y supuesta anomia parecen anular cualquier intención interpretativa al neutralizar los centros históricos y proclamar el agotamiento de la historia. El planteamiento de una teoría dicotómica frente al espacio y las teorías arquitectónicas asociadas a este ya fue formulado por Colin Rowe y Fred Koetter a finales de los años setenta del siglo pasado. Se plantea aquí la idea de una “ciudad tangible” como opuesta a la idea de la "ciudad genérica" enunciada por Koolhaas. Tomando el territorio y la memoria como referencia principal en un lugar concreto y huyendo de la premisa de la inmediatez del instante y el "presente perpetuo" proclamado por Koolhaas, del que según él seríamos prisioneros, se establece una distancia respecto al objeto de análisis que desarrolla el estudio en la dirección opuesta al supuesto origen del mismo, planteando la posibilidad de reactivar una reflexión en torno al territorio y la memoria en el seno del proceso global de habitación para poner de manifiesto determinados mecanismos de configuración de un espacio de representación al margen de la urgencia del presente, reactivando la memoria y su relación con el territorio como punto de partida. Desde de la reconstrucción hipotética del territorio, partiendo de la propia presencia física del mismo, su orografía, la paleo-biología, las analogías etológicas, los restos arqueológicos, la antropología o la historia, se reivindica la reflexión arquitectónica como disciplina diversa y privilegiada en cuanto al análisis espacial, tratando de discernir el proceso mediante el cual el Prado pasó de territorio a escenario. La organización cronológica del estudio y la incorporación de muy diversas fuentes, en su mayoría directas, pretende poner de manifiesto la condición transitiva del espacio de representación y contrastar el pasado remoto del lugar y su construcción con el momento actual, inevitablemente encarnado por el punto de vista desde el cual se desarrolla la tesis. El Prado parece albergar, agazapado en su nombre, la raíz de un origen remoto y olvidado. Si como enunciaba Ignasi Solá-Morales la función de la arquitectura es hacer aflorar los significados inherentes al lugar, esta tesis se plantea como una recuperación de la idea del vínculo entre el territorio y la memoria como fuente fundamental en la definición de un espacio de representación específico. El escrutinio del pasado constituye un acto eminentemente contemporáneo, pues el punto de vista y la distancia, inevitablemente condicionados por el presente, determinan la mirada. El culto contemporáneo a la inmediatez y la proclamación de la superación de los procesos históricos han relegado el pasado, en cierto grado, a depósito de restos o referente a superar, obviando su ineluctable condición de origen o momento anterior condicionante. Partiendo de la reconstrucción del lugar sobre el cual se halla el Prado ubicado y reconsiderando, según las premisas desarrolladas por la moderna historiografía, fundamentalmente desarrolladas por la Escuela francesa de los Annales, la cotidianeidad y lo anónimo como fuente de la que dimanan muchos de los actuales significados de nuestros espacios de representación, tomando como punto de partida un lugar remoto y olvidado, se estudia como se fue consolidando el Prado hasta devenir un lugar insigne de referencia asociado a los poderes fácticos y el espacio áulico de la capital de las Españas en el siglo XVII. El proceso mediante el cual el Prado pasó de territorio a escenario implica la recuperación de la memoria de un espacio agropecuario anónimo y el análisis de cómo, poco a poco, se fue depositando sobre el mismo el acervo de los diversos pobladores de la región que con sus particularidades culturales y sociales fueron condicionando, en mayor o menor grado, un lugar cuyo origen se extiende retrospectivamente hasta hace más de dos mil años, cuando se considera que pudo darse la primera habitación a partir de la cual, de manera ininterrumpida, el Prado ha venido siendo parte de lo que devino, más tarde, Madrid. La llegada de nuevos agentes, vinculados con estructuras de poder y territoriales que trascendían la inmediatez del territorio sobre el que se comenzó a erigir dicho lugar, sirven para repasar los diferentes depósitos ideológicos y culturales que han ido conformando el mismo, reivindicando la diversidad y lo heterogéneo del espacio de representación frente a la idea homogeneizadora que el modelo genérico implica. La constitución del Prado como un espacio de referencia asociado al paganismo arcaico a partir de la praxis espacial cotidiana, su relación con las estructuras defensivas de Al-Andalus y la atalaya Omeya, la apropiación del los primitivos santuarios por parte la iglesia, su relación con un determinado tipo de espiritualidad y las órdenes religiosas más poderosas de la época, la preferencia de Carlos V por Madrid y sus vínculos con la cultura europea del momento, o la definitiva metamorfosis del lugar a partir del siglo XVI y el advenimiento de un nuevo paganismo emblemático y estetizado, culminan con el advenimiento de lo económico como representación del poder en el seno de la corte y la erección del Palacio del Buen Retiro como manifestación tangible de la definitiva exaltación del Prado a espacio de representación áulico. Decía T.S. Elliot que la pugna por el espacio de la memoria constituye el principal rasgo del clasicismo, y el Prado, ciertamente, participa de ese carácter al que está profundamente asociado en la conciencia espacial de los madrileños como lugar de referencia. Acaso la obliteración del territorio y la memoria, propuestas en la “ciudad genérica” también tengan algo que ver con ello. ABSTRACT "If man is the caretaker of words and only they provide the sense of things, the architecture has a precise mission: to make out from the given conditions of each place words that mean the qualities of existence, and which unveil the wealth and content they potentially contain " Ignasi Solá Morales. Place: permanence or production, 1992. This thesis arises from both the desire to understand the identity of one of the most representative spaces of my city, assumed in a familiar way but that raises many doubts about its characterization, and from a personal concern about the apparent hegemony of the urban model of the "generic city " so crudely exposed by Rem Koolhaas in the late twentieth century that puts a strain on the historic city. The obliteration of the territory, specific physical space, and its associated memory, are considered as a starting point to confront them with the proclamation of the new model of "generic city" raised from eminently economic and technological roots. The tangible reality of a space, apparently forged based on the values reviled by the proposed new model, is studied from opposite premises. The idea of non-place, theorized by Marc Augé and modeled by Koolhaas, implies the emancipation from both historical preexistences and physical location, posing a type of space representation outside the territory and memory. Without wishing to establish a confrontational or opposite position, but an antithetical and complementary stance, the premises of Koolhaas are here taken to contrast them with a portion of territory halfway between architecture and the city, to develop a study that will complement and counterbalance the spatial paradigm that the "generic city" means and whose alleged immediacy and anomie appear to nullify any interpretative intention by neutralizing the historic centers and proclaiming the exhaustion of history. The approach of a dichotomous theory versus space and architectural theories associated with this were already formulated by Colin Rowe and Fred Koetter during the late seventies of last century. The idea of a "tangible city" as opposed to the idea of the "generic city" enunciated by Koolhaas arises here. Taking the territory and memory as the main reference in a particular place and trying to avoid the premise of the immediacy of the moment and the "perpetual present" proclaimed by Koolhaas, of which he pleas we would be prisoners, a distance is established from the object of analysis developing the study in the opposite direction to the alleged origin of it, raising the possibility of reactivating a reflection on the territory and memory within the overall process of inhabiting to reveal certain representational space configuration mechanisms outside the urgency of the present, reviving the memory and its relationship with the territory as a starting point. From the hypothetical reconstruction of the territory, starting from its physical presence, geography, paleo-biology, ethological analogies, archaeological remains, anthropology or history, architecture is claimed as a diverse as privileged discipline for spatial analysis, trying to discern the process by which the Prado moved from territory to stage. The chronological organization of the study and incorporating a variety of sources, most direct, aims to highlight the transitive condition of representational space and contrast the remote past of the place and its construction with the current moment, inevitably played by the view point from which the thesis develops. The Prado seems to harbor, in its name, the root of a remote and forgotten origin. If, as Ignasi Sola-Morales said, the aim of architecture is to bring out the meanings inherent in the site, this thesis is presented as a recovery of the idea of the link between the territory and memory as a key source in defining a specific space of representation. The scrutiny of the past is an eminently contemporary act, for the view and distance inevitably conditioned by the present, determine the way we look. The contemporary cult of immediacy and the proclamation of overcoming historical processes have relegated the past, to some extent, to remains deposit or a reference to overcome, ignoring its ineluctable condition as origin or previous constraint. From rebuilding the site on which the Prado is located and reconsidering everyday life and the anonymous as a source of many arising current meanings of our space of representation, according to the premises developed by modern historiography mainly developed by the French school of Annales, trying to recover the remote and forgotten is attempted, the thesis studies how el Prado was consolidated to become the most significant place of Madrid, deeply associated with the power in the capital of Spain during the XVII century. The process by which the Prado evolved from territory to stage involves the recovery of the memory of an anonymous agricultural space and the analysis of how, little by little, the influence of the various inhabitants of the region with their own and how their cultural and social peculiarities was deposited through time on the common ground and how that determined, to a greater or lesser degree, a place whose origin retrospectively extends over more than two thousand years ago, when we can consider the first inhabiting from which, without interruption, the Prado has come to be part of what became, later, Madrid. The arrival of new players, linked to power structures and territorial issues which transcended the immediacy of the territory on which the place begun to be a characteristic space, serve to review the different ideological and cultural deposits that have shaped the place, claiming diversity and heterogeneous space of representation before the homogenizing idea which the generic model implies. The constitution of the Prado as a benchmark associated with the archaic paganism developed from the ancient everyday spatial praxis, its relationship with the defensive structures of Al-Andalus and the Umayyad watchtower, the appropriation of the early sanctuaries by the roman church, its relationship with a certain type of spirituality and the most powerful religious orders of the time, the preference of Carlos V towards Madrid and its links with the European culture of the moment and the final metamorphosis of the place during the sixteenth century, end at the moment on which the advent of the economic as a representation of power within the court and the erection of the Palacio del Buen Retiro, as a tangible manifestation of the ultimate exaltation of courtly Prado space representation, happened in the mid XVII century. T. S. Elliot said that the struggle for memory space is the main feature of classicism, and the Prado certainly shares part of that character deeply associated in the mental spatial structure of the locals as a landmark. Perhaps the obliteration of territory and memory proposed in the "generic city" might also have something to do with that.

A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

P-glycoprotein (MDR-1) is a well-known transporter that mediates efflux of chemotherapeutic agents from the intracellular milieu and thereby contributes to drug resistance. MDR-1 also is expressed by nonmalignant cells, including leukocytes, but physiologic functions for MDR-1 are poorly defined. Using an initial screening assay that included >100 mAbs, we observed that neutralizing mAbs MRK16, UIC2, and 4E3 against MDR-1 specifically and potently blocked basal-to-apical transendothelial migration of mononuclear phagocytes, a process that may mimic their migration into lymphatic vessels. Antagonists of MDR-1 then were used in a model of authentic lymphatic clearance. In this model, antigen-presenting dendritic cells (DC) migrate out of explants of cultured human skin and into the culture medium via dermal lymphatic vessels. DC and T cells derived from skin expressed MDR-1 on their surfaces. Addition of anti-MDR-1 mAbs MRK16, UIC2, or the MDR-1 antagonist verapamil to skin explants at the onset of culture inhibited the appearance of DC, and accompanying T cells, in the culture medium by approximately 70%. Isotype-matched control mAbs against other DC molecules including CD18, CD31, and major histocompatibility complex I did not block. In the presence of MDR-1 antagonists, epidermal DC were retained in the epidermis, in contrast to control conditions. In summary, this work identifies a physiologic function for MDR-1 during the mobilization of DC and begins to elucidate how these critical antigen-presenting cells migrate from the periphery to lymph nodes to initiate T lymphocyte-mediated immunity.

Patterns of brain angiogenesis after vascular endothelial growth factor administration in vitro and in vivo

Vascular endothelial growth factor (VEGF) is a secreted endothelial cell mitogen that has been shown to induce vasculogenesis and angiogenesis in many organ systems and tumors. Considering the importance of VEGF to embryonic vascularization and survival, the effects of administered VEGF on developing or adult cerebrovasculature are unknown: can VEGF alter brain angiogenesis or mature cerebrovascular patterns? To examine these questions we exposed fetal, newborn, and adult rat cortical slice explants to graduated doses of recombinant VEGF. The effects of another known angiogenic factor, basic fibroblast growth factor (bFGF), were evaluated in a comparable manner. In addition, we infused VEGF via minipump into the adult cortex. Significant angiogenic effects were found in all VEGF experiments in a dose-responsive manner that were abolished by the addition of VEGF neutralizing antibody. Fetal and newborn explants had a highly complex network of branched vessels that immunoexpressed the flt-1 VEGF receptor, and flk-1 VEGF receptor expression was determined by reverse transcription–PCR. Adult explants had enlarged, dilated vessels that appeared to be an expansion of the existing network. All bFGF-treated explants had substantially fewer vascular profiles. VEGF infusions produced both a remarkable localized neovascularization and, unexpectedly, the expression of flt-1 on reactive astrocytes but not on endothelial cells. The preponderance of neovascularization in vitro and in vivo, however, lacked the blood–brain barrier (BBB) phenotype marker, GLUT-1, suggesting that in brain the angiogenic role of VEGF may differ from a potential BBB functional role, i.e., transport and permeability. VEGF may serve an important capacity in neovascularization or BBB alterations after brain injury.

Interleukin 18 stimulates HIV type 1 in monocytic cells

The cytokine interleukin (IL) 18 (formerly interferon γ-inducing factor) induces the T helper type 1 response. In the present studies, IL-18 increased HIV type 1 (HIV-1) production from 5- to 30-fold in the chronically infected U1 monocytic cell line. Inhibition of tumor necrosis factor (TNF) activity by the addition of TNF-binding protein reduced IL-18-stimulated HIV-1 production by 48%. In the same cultures, IL-18-induced IL-8 was inhibited by 96%. Also, a neutralizing anti-IL-6 mAb reduced IL-18-induced HIV-1 by 63%. Stimulation of U1 cells with IL-18 resulted in increased production of IL-6, and exogenous IL-6 added to U1 cells increased HIV-1 production 4-fold over control. A specific inhibitor of the p38 mitogen-activated protein kinase reduced IL-18-induced HIV-1 by 73%, and a 50% inhibition was observed at 0.05 μM. In the same cultures, IL-8 was inhibited by 87%. By gel-shift and supershift analyses, increased binding activity of the transcription factor NF-κB was measured in nuclear extracts from U1 cells 1 h after exposure to IL-18. These results demonstrate induction of HIV-1 by IL-18 in a monocyte target associated with an intermediate role for TNF and IL-6, activation of p38 mitogen-activated protein kinase, and nuclear translocation of NF-κB.

Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs

Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA. B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines. Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA. In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance. However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5′ side or a G on the 3′ side. Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo. Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs. Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing. These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination

It is generally thought that an effective vaccine to prevent HIV-1 infection should elicit both strong neutralizing antibody and cytotoxic T lymphocyte responses. We recently demonstrated that potent, boostable, long-lived HIV-1 envelope (Env)-specific cytotoxic T lymphocyte responses can be elicited in rhesus monkeys using plasmid-encoded HIV-1 env DNA as the immunogen. In the present study, we show that the addition of HIV-1 Env protein to this regimen as a boosting immunogen generates a high titer neutralizing antibody response in this nonhuman primate species. Moreover, we demonstrate in a pilot study that immunization with HIV-1 env DNA (multiple doses) followed by a final immunization with HIV-1 env DNA plus HIV-1 Env protein (env gene from HXBc2 clone of HIV IIIB; Env protein from parental HIV IIIB) completely protects monkeys from infection after i.v. challenge with a chimeric virus expressing HIV-1 env (HXBc2) on a simian immmunodeficiency virusmac backbone (SHIV-HXBc2). The potent immunity and protection seen in these pilot experiments suggest that a DNA prime/DNA plus protein boost regimen warrants active investigation as a vaccine strategy to prevent HIV-1 infection.

Strain-specific differences in mouse hepatic wound healing are mediated by divergent T helper cytokine responses

Hepatic fibrosis represents the generalized response of the liver to injury and is characterized by excessive deposition of extracellular matrix. The cellular basis of this process is complex and involves interplay of many factors, of which cytokines are prominent. We have identified divergent fibrosing responses to injury among mouse strains and taken advantage of these differences to examine and contrast T helper (Th)-derived cytokines during fibrogenesis. Liver injury was induced with carbon tetrachloride, fibrosis was quantitated, and Th1/Th2 cytokine mRNAs measured. Liver injury in BALB/c mice resulted in severe fibrosis, whereas C57BL/6 mice developed comparatively minimal fibrosis. Fibrogenesis was significantly modified in T and B cell-deficient BALB/c and C57BL/6 severe combined immunodeficient (SCID) mice compared with wild-type counterparts, suggesting a role of Th subsets. Fibrogenic BALB/c mice exhibited a Th2 response during the wounding response, whereas C57BL/6 mice displayed a Th1 response, suggesting that hepatic fibrosis is influenced by different T helper subsets. Moreover, mice lacking interferon γ, which default to the Th2 cytokine pathway, exhibited more pronounced fibrotic lesions than did wild-type animals. Finally, shifting of the Th2 response toward a Th1 response by treatment with neutralizing anti-interleukin 4 or with interferon γ itself ameliorated fibrosis in BALB/c mice. These data support a role for immune modulation of hepatic fibrosis and suggest that Th cytokine subsets can modulate the fibrotic response to injury.

Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15INK4B blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27kip-1 blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of β transforming growth factor (TGFβ) in serum-free medium decreased levels of p15INK4B and increased colony formation and retroviral-mediated transduction of primary human CD34+ cells. Although TGFβ neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27kip-1 coupled with TGFβ-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFβ, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.

Emergence of a highly pathogenic simian/human immunodeficiency virus in a rhesus macaque treated with anti-CD8 mAb during a primary infection with a nonpathogenic virus

Although simian/human immunodeficiency virus (SHIV) strain DH12 replicates to high titers and causes immunodeficiency in pig-tailed macaques, virus loads measured in SHIVDH12-infected rhesus monkeys are consistently 100-fold lower and none of 22 inoculated animals have developed disease. We previously reported that the administration of anti-human CD8 mAb to rhesus macaques at the time of primary SHIVDH12 infection resulted in marked elevations of virus loads. One of the treated animals experienced rapid and profound depletions of circulating CD4+ T lymphocytes. Although the CD4+ T cell number partially recovered, this monkey subsequently suffered significant weight loss and was euthanized. A tissue culture virus stock derived from this animal, designated SHIVDH12R, induced marked and rapid CD4+ cell loss after i.v. inoculation of rhesus monkeys. Retrospective analyses of clinical specimens, collected during the emergence of SHIVDH12R indicated: (i) the input cloned SHIV remained the predominant virus during the first 5–7 months of infection; (ii) variants bearing only a few of the SHIVDH12R consensus changes first appeared 7 months after the administration of anti-CD8 mAb; (iii) high titers of neutralizing antibody directed against the input SHIV were detected by week 10 and persisted throughout the infection; and (iv) no neutralizing antibody against SHIVDH12R ever developed.

Loss of ovarian function promotes angiogenesis in human ovarian carcinoma

We show here that elevated levels of gonadotropins (luteinizing hormone and follicle stimulating hormone), as found in menopause or after ovariectomy, promote growth of human ovarian carcinoma by induction of tumor angiogenesis. Human epithelial ovarian cancer tumors progressed faster in ovariectomized mice. This induced growth could be attributed to the elevated levels of gonadotropins associated with loss of ovarian function because direct administration of gonadotropins also was effective in promoting tumor progression in vivo. On the other hand, gonadotropins had no direct effect on the proliferation of human ovarian cancer cells in vitro. Using MRI, we demonstrated that ovariectomy significantly (P < 0.02) induces neovascularization of human ovarian carcinoma spheroids implanted in nude mice. Moreover, conditioned medium of gonadotropin-treated human ovarian carcinoma cells showed increased mitogenic activity to bovine endothelial cells, and this activity could be blocked by neutralizing antibodies against luteinizing hormone and against vascular endothelial growth factor. Accordingly, gonadotropin stimulation resulted in a dose-dependent-induced expression of vascular endothelial growth factor in monolayer culture as well as in the outer proliferating cells of human ovarian cancer spheroids. These results demonstrate the significance of the elevated levels of gonadotropins, as found in menopause and in all ovarian cancer patients, on the progression of ovarian cancer and could explain the protective effect of estrogen replacement therapy. Based on these results, we suggest that hormonal therapy aimed at lowering the circulating levels of gonadotropins may possibly prolong remission in ovarian cancer by extending tumor dormancy.