Páncreas artificial basado en un controlador Smith paramétrico

La diabetes mellitus es una enfermedad que se caracteriza por la nula o insuficiente producción de insulina, o la resistencia del organismo a la misma. La insulina es una hormona que ayuda a que la glucosa llegue a los tejidos periféricos y al sistema nervioso para suministrar energía. Actualmente existen dos tipos de terapias aplicada en tejido subcutáneo: mediante inyección múltiple realizada con plumas, y la otra es mediante infusión continua de insulina por bomba (CSII). El mayor problema de esta terapia son los retardos por la absorción, tanto de los carbohidratos como de la insulina, y los retardos introducidos por el sensor subcutáneo de glucosa que mide la glucosa del líquido intersticial, lo deseable es controlar la glucosa en sangre. Para intentar independizar al paciente de su enfermedad se está trabajando en el desarrollo del páncreas endocrino artificial (PEA) que dotaría al paciente de una bomba de insulina, un sensor de glucosa y un controlador, el cual se encargaría de la toma de decisiones de las infusiones de insulina. Este proyecto persigue el diseño de un regulador en modo de funcionamiento en CL, con el objetivo de conseguir una regulación óptima del nivel de glucosa en sangre. El diseño de dicho regulador va a ser acometido utilizando la teoría del control por modelo interno (IMC). Esta teoría se basa en la idea de que es necesario realimentar la respuesta de un modelo aproximado del proceso que se quiere controlar. La salida del modelo, comparada con la del proceso real nos da la incertidumbre del modelo de la planta, frente a la planta real. Dado que según la teoría del modelo interno, estas diferencias se dan en las altas frecuencias, la teoría IMC propone un filtro paso bajo como regulador en serie con la inversa del modelo de la planta para conseguir el comportamiento deseado. Además se pretende implementar un Predictor Smith para minimizar los efectos del retardo de la medida del sensor. En el proyecto para conseguir la viabilidad del PEA se ha adaptado el controlador IMC clásico utilizando las ganancias estáticas de un modelo de glucosa, a partir de la ruta subcutánea de infusión y la vía subcutánea de medida. El modo de funcionamiento del controlador en SCL mejora el rango de normoglucemia, necesitando la intervención del paciente indicando anticipadamente el momento de las ingestas al controlador. El uso de un control SCL con el Predictor de Smith mejora los resultados pues se añade al controlador una variable sobre las ingestas con la participación del paciente. ABSTRACT. Diabetes mellitus is a group of metabolic diseases in which a person has high blood sugar, due to the body does not produce enough insulin, or because cells do not respond to the insulin produced. The insulin is a hormone that helps the glucose to reach to outlying tissues and the nervous system to supply energy. There are currently two types of therapies applied in subcutaneous tissue: the first one consists in using the intensive therapy with an insulin pen, and the other one is by continuous subcutaneous insulin infusion (CSII). The biggest problems of this therapy are the delays caused by the absorption of carbohydrates and insulin, and the delays introduced by the subcutaneous glucose sensor that measures glucose from interstitial fluid, it is suitable to control glucose blood. To try to improve these patients quality of life, work is being done on the development of an artificial endocrine pancreas (PEA) consisting of a subcutaneous insulin pump, a subcutaneous glucose sensor and an algorithm of glucose control, which would calculate the bolus that the pump would infuse to patient. This project aims to design a controller for closed-loop therapy, with the objective of obtain an optimal regulation of blood glucose level. The design of this controller will be formed using the theory of internal model control (IMC). This theory is based on the uncertainties given by a model to feedback the system control. Output model, in comparison with the actual process gives the uncertainty of the plant model, compared to the real plant. Since the theory of the internal model, these differences occur at high frequencies, the theory proposes IMC as a low pass filter regulator in series with the inverse model of the plant to get the required behavior. In addition, it will implement a Smith Predictor to minimize the effects of the delay measurement sensor. The project for the viability of PEA has adapted the classic IMC controller using the gains static of glucose model from the subcutaneous infusion and subcutaneous measuring. In simulation the SemiClosed-Loop controller get on the normoglycemia range, requiring patient intervention announce the bolus priming connected to intakes. Using an SCL control with the Smith Predictor improves the outcome because a variable about intakes is added to the controller through patient intervention.

Railway Rapid Transit timetables with variable and elastic demand.

This paper focuses on the design of railway timetables considering a variable elastic demand profile along a whole design day. Timetabling is the third stage in the classical hierarchical railway planning process. Most of previous works on this topic consider a uniform demand behavior for short planning intervals. In this paper, we propose a MINLP model for designing non-periodic timetables on a railway corridor where demand is dependent on waiting times. In the elastic demand case, long waiting times lead to a loss of passengers, who may select an alternative transportation mode. The mode choice is modeled using two alternative methods. The first one is based on a sigmoid function and can be used in case of absence of information for competitor modes. In the second one, the mode choice probability is obtained using a Logit model that explicitly considers the existence of a main alternative mode. With the purpose of obtaining optimal departure times, in both cases, a minimization of the loss of passengers is used as objective function. Finally, as illustration, the timetabling MINLP model with both mode choice methods is applied to a real case and computational results are shown.

Perfil antropométrico y respuesta psico-fisiológica en escalada deportiva en roca: Diferencias entre modalidades

En la literatura se ha descrito el perfil antropométrico y la respuesta psicofisiológica en escalada deportiva en roca, pero hasta la fecha, no se habían analizado las diferencias existentes entre sus principales modalidades. El objetivo de la presente tesis fue describir las características antropométricas del escalador de competición y comprobar la existencia de diferencias entre los participantes de distintas modalidades, así como analizar la respuesta psico-fisiológica durante la ejecución de un búlder y una vía, además de evaluar las diferencias entre su realización a vista o tras un ensayo. Para ello, efectuamos dos estudios diferentes: en el primero participaron voluntariamente 61 hombres y 18 mujeres, participantes en cuatro pruebas del circuito nacional de competición de escalada durante el año 2009, tres de ellas de la modalidad de dificultad a vista y una de búlder. Se realizaron mediciones antropométricas, prueba de fuerza de prensión manual antes y después de competir, y se cumplimentó un cuestionario donde se evaluaba la percepción del esfuerzo y la experiencia deportiva. En el segundo estudio, 23 escaladores, 15 hombres y 8 mujeres, divididos en tres grupos en función de su nivel de rendimiento, realizaron de manera voluntaria distintas pruebas durante tres días separados entre sí al menos 48 horas. El primer día rellenaron un cuestionario sobre su experiencia deportiva y nivel de rendimiento, fueron pesados, tallados y sometidos a un escáner de cuerpo completo en densitómetro con objeto de medir la composición corporal. El segundo día realizaron previo calentamiento, un búlder a vista y, tras un descanso de 15 minutos, escalaron una vía a vista acorde con su nivel. El tercer día, después de calentar y disponer de 20 minutos para ensayarlo, repitieron la escalada del búlder. Tras un descanso de 15 minutos y 20 minutos de ensayo, realizaron un segundo intento a la vía. Se registraron los valores en la respuesta cardiorrespiratoria, se obtuvieron muestras de lactato en sangre del lóbulo de la oreja y se realizaron pruebas de fuerza de prensión manual antes y después de la escalada. También se pasó un cuestionario para medir la ansiedad y autoconfianza así como el esfuerzo percibido. Los resultados no mostraron diferencias antropométricas significativas entre los participantes en competiciones de búlder y los que participaron en competiciones de escalada de dificultad a vista. Se dieron diferencias en la pérdida de fuerza antes y después de escalar entre dichos participantes. Las mujeres obtuvieron menor fuerza de prensión manual que los hombres pero la misma pérdida de fuerza entre el instante antes de competir y el posterior. La respuesta fisiológica durante la ejecución del búlder fue menor que la obtenida durante la ejecución de la vía. Hubo pérdida de fuerza de prensión manual entre el instante anterior y el posterior a ejecutar la vía, pero no al hacer el búlder. Sin embargo, no se dieron diferencias en la ansiedad y la autoestima provocada por ambas modalidades, por lo que deducimos que la ejecución de un búlder y una vía presentan una respuesta fisiológica distinta. Proponemos que la respuesta está relacionada, sobre todo, con las variables de ejecución, de tal manera que a mayor distancia y/o tiempo recorrido en la escalada, mayor será la contribución anaeróbica al esfuerzo y la fatiga manifestada como pérdida de fuerza que, en el caso del búlder, fue mínima o inexistente. En el segundo intento, tras un ensayo de 20 minutos en el búlder, se consiguió mejorar el rendimiento respecto al primer intento, que se manifestó con un aumento en la distancia recorrida. Sin embargo, en la vía no se dieron diferencias entre ambos intentos, ni en la ejecución, ni en la respuesta fisiológica, ni en la ansiedad, ni siquiera en la fuerza de prensión manual. ABSTRACT It has been described in the literature the anthropometric profile and psychophysiological response in rock climbing, but so far not been analyzed differences between its main modalities. The aim of this thesis was to describe the anthropometric characteristics of the climber competitor and check for differences between participants of different modalities and to analyze the psycho-physiological response during the execution of a boulder and a route also to assess differences between on sight and redpoint attempts. We made two different studies: in the first 61 men and 18 women who attended four competitions of national climbing circuit in 2009, three of them on-sight difficulty competitions and a boulder competition participated voluntarily. Anthropometric measurements, a hand grip strength test before and after competing were registered for each climber, and a questionnaire which assessed perception of effort and the climbing experience was fulfilled. In the second study, various tests were conducted on 23 volunteer climbers, 15 men and 8 women, during three days separated for, at least, 48 hours of resting, divided into three groups according to their performance. The first day, climbers completed a questionnaire on their experience and performance level. It was recorded weight, height and they underwent a full body scan densitometer in order to measure body composition. The second day, after previous warming-up, they climbed a boulder on sight and, after a break of 15 minutes, climbed a route on-sight according to their level. The third day, after warming-up and have 20 minutes to try it, they repeated the bouldering climbing. After a break of 15 minutes and 20 minutes of essaying, they made a second attempt at the route. Values in the cardiorespiratory response were recorded, blood lactate samples were obtained from earlobe, and hand grip strength was tested before and after the climb. They also filled a questionnaire to measure anxiety and self-confidence and perceived exertion. The results showed no significant anthropometric differences between participants in bouldering competitions and participants in competitions on-sight difficulty climbing. There were found differences in strength loss before and after climbing between those participants. Women had less hand grip strength than men but the same loss of strength between the records carried out before and after competing. The physiological response recorded for boulder climbing was lower than the obtained for the route. There was loss of hand grip strength between the time before and after running the route but not for bouldering. However, there were no differences in anxiety and self-esteem caused by both modalities, so we conclude that the implementation of a boulder and a route have different physiological responses. We think that this response is mainly related to performance variables, as a greater distance and/or travel time on the climb, the higher the anaerobic contribution to the effort and fatigue as manifested by loss of strength in the case of the boulder was minimal or nonexistent. In the second attempt after 20 minutes in the boulder better performance was achieved on the first attempt, which was manifested by an increment of climbing distance. However, there were the differences in the route between the two attempts, either in execution or in the physiological response, or anxiety, or even in hand grip strength.

DA TERCEIRA REVOLUÇÃO INDUSTRIAL À PRODUÇÃO ENXUTA SEU IMPACTO NA INDÚSTRIA DE AUTOPEÇAS CASO: MAHLE METAL LEVE S/A

A presente dissertação teve como objetivo verificar as razões pelas quais a empresa Metal Leve cedeu o controle acionário a uma empresa concorrente de porte internacional e comparar a situação existente à época da sua desnacionalização e o estágio em que se encontra a nova controladora Mahle Metal Leve S. A. em termos de produção e gestão, após a reorganização produtiva. Esse processo de internacionalização produtiva acarretou modificações na empresa ensejando uma reestruturação da produção e da gestão e um novo círculo vicioso, constituindo as bases de um novo crescimento econômico, com um projeto estratégico de longo prazo, associado ao seu poder econômico, a sua capacidade gerencial e a sua tradição. O estudo está fundamentado em um conjunto de informações sobre os dois momentos, focalizando os resultados financeiros, aspectos gerenciais, liderança, capacidade competitiva e a evolução ao longo desses dois momentos comparados. Concluindo que não apenas o fenômeno da globalização da economia internacional tornou inevitável a cessão do controle acionário. Porém, também faltou visão estratégica para perceber as mudanças que inevitavelmente ocorriam a sua volta e que a nova controladora, a Mahler Metal Leve, trouxe uma competência gerencial que resultou em ganhos de produtividade e melhorou sua competitividade

DA TERCEIRA REVOLUÇÃO INDUSTRIAL À PRODUÇÃO ENXUTA SEU IMPACTO NA INDÚSTRIA DE AUTOPEÇAS CASO: MAHLE METAL LEVE S/A

A presente dissertação teve como objetivo verificar as razões pelas quais a empresa Metal Leve cedeu o controle acionário a uma empresa concorrente de porte internacional e comparar a situação existente à época da sua desnacionalização e o estágio em que se encontra a nova controladora Mahle Metal Leve S. A. em termos de produção e gestão, após a reorganização produtiva. Esse processo de internacionalização produtiva acarretou modificações na empresa ensejando uma reestruturação da produção e da gestão e um novo círculo vicioso, constituindo as bases de um novo crescimento econômico, com um projeto estratégico de longo prazo, associado ao seu poder econômico, a sua capacidade gerencial e a sua tradição. O estudo está fundamentado em um conjunto de informações sobre os dois momentos, focalizando os resultados financeiros, aspectos gerenciais, liderança, capacidade competitiva e a evolução ao longo desses dois momentos comparados. Concluindo que não apenas o fenômeno da globalização da economia internacional tornou inevitável a cessão do controle acionário. Porém, também faltou visão estratégica para perceber as mudanças que inevitavelmente ocorriam a sua volta e que a nova controladora, a Mahler Metal Leve, trouxe uma competência gerencial que resultou em ganhos de produtividade e melhorou sua competitividade

Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine

To enhance the efficacy of DNA malaria vaccines, we evaluated the effect on protection of immunizing with various combinations of DNA, recombinant vaccinia virus, and a synthetic peptide. Immunization of BALB/c mice with a plasmid expressing Plasmodium yoelii (Py) circumsporozoite protein (CSP) induces H-2Kd-restricted CD8+ cytotoxic T lymphocyte (CTL) responses and CD8+ T cell- and interferon (IFN)-γ-dependent protection of mice against challenge with Py sporozoites. Immunization with a multiple antigenic peptide, including the only reported H-2Kd-restricted CD8+ T cell epitope on the PyCSP (PyCSP CTL multiple antigenic peptide) and immunization with recombinant vaccinia expressing the PyCSP induced CTL but only modest to minimal protection. Mice were immunized with PyCSP DNA, PyCSP CTL multiple antigenic peptide, or recombinant vaccinia expressing PyCSP, were boosted 9 wk later with the same immunogen or one of the others, and were challenged. Only mice immunized with DNA and boosted with vaccinia PyCSP (D-V) (11/16: 69%) or DNA (D-D) (7/16: 44%) had greater protection (P < 0.0007) than controls. D-V mice had significantly higher individual levels of antibodies and class I-restricted CTL activity than did D-D mice; IFN-γ production by ELIspot also was higher in D-V than in D-D mice. In a second experiment, three different groups of D-V mice each had higher levels of protection than did D-D mice, and IFN-γ production was significantly greater in D-V than in D-D mice. The observation that priming with PyCSP DNA and boosting with vaccinia-PyCSP is more immunogenic and protective than immunizing with PyCSP DNA alone supports consideration of a similar sequential immunization approach in humans.

Major histocompatibility complex (MHC) class I KbDb −/− deficient mice possess functional CD8+ T cells and natural killer cells

We obtained mice deficient for major histocompatibility complex (MHC) molecules encoded by the H-2K and H-2D genes. H-2 KbDb −/− mice express no detectable classical MHC class I-region associated (Ia) heavy chains, although β2-microglobulin and the nonclassical class Ib proteins examined are expressed normally. KbDb −/− mice have greatly reduced numbers of mature CD8+ T cells, indicating that selection of the vast majority (>90%) of CD8+ T cells cannot be compensated for by β2-microglobulin-associated molecules other than classical H-2K and D locus products. In accord with the greatly reduced number of CD8+ T cells, spleen cells from KbDb −/− mice do not generate cytotoxic responses in primary mixed-lymphocyte cultures against MHC-disparate (allogeneic) cells. However, in vivo priming of KbDb −/− mice with allogeneic cells resulted in strong CD8+ MHC class Ia-specific allogeneic responses. Thus, a minor population of functionally competent peripheral CD8+ T cells capable of strong cytotoxic activity arises in the complete absence of classical MHC class Ia molecules. KbDb −/− animals also have natural killer cells that retain their cytotoxic potential.

BAC-VAC, a novel generation of (DNA) vaccines: A bacterial artificial chromosome (BAC) containing a replication-competent, packaging-defective virus genome induces protective immunity against herpes simplex virus 1

This study aimed to exploit bacterial artificial chromosomes (BAC) as large antigen-capacity DNA vaccines (BAC-VAC) against complex pathogens, such as herpes simplex virus 1 (HSV-1). The 152-kbp HSV-1 genome recently has been cloned as an F-plasmid-based BAC in Escherichia coli (fHSV), which can efficiently produce infectious virus progeny upon transfection into mammalian cells. A safe modification of fHSV, fHSVΔpac, does not give rise to progeny virus because the signals necessary to package DNA into virions have been excluded. However, in mammalian cells fHSVΔpac DNA can still replicate, express the HSV-1 genes, cause cytotoxic effects, and produce virus-like particles. Because these functions mimic the lytic cycle of the HSV-1 infection, fHSVΔpac was expected to stimulate the immune system as efficiently as a modified live virus vaccine. To test this hypothesis, mice were immunized with fHSVΔpac DNA applied intradermally by gold-particle bombardment, and the immune responses were compared with those induced by infection with disabled infectious single cycle HSV-1. Immunization with either fHSVΔpac or disabled infectious single cycle HSV-1 induced the priming of HSV-1-specific cytotoxic T cells and the production of virus-specific antibodies and conferred protection against intracerebral injection of wild-type HSV-1 at a dose of 200 LD50. Protection probably was cell-mediated, as transfer of serum from immunized mice did not protect naive animals. We conclude that BAC-VACs per se, or in combination with genetic elements that support replicative amplification of the DNA in the cell nucleus, represent a useful new generation of DNA-based vaccination strategies for many viral and nonviral antigens.

Temporal variation, dormancy, and coexistence: A field test of the storage effect

Theoretical models suggest that overlapping generations, in combination with a temporally fluctuating environment, may allow the persistence of competitors that otherwise would not coexist. Despite extensive theoretical development, this “storage effect” hypothesis has received little empirical attention. Herein I present the first explicit mathematical analysis of the contribution of the storage effect to the dynamics of competing natural populations. In Oneida Lake, NY, data collected over the past 30 years show a striking negative correlation between the water-column densities of two species of suspension-feeding zooplankton, Daphnia galeata mendotae and Daphnia pulicaria. I have demonstrated competition between these two species and have shown that both possess long-lived eggs that establish overlapping generations. Moreover, recruitment to this long-lived stage varies annually, so that both daphnids have years in which they are favored (for recruitment) relative to their competitor. When the long-term population growth rates are calculated both with and without the effects of a variable environment, I show that D. galeata mendotae clearly cannot persist without the environmental variation and prolonged dormancy (i.e., storage effect) whereas D. pulicaria persists through consistently high per capita recruitment to the long-lived stage.

DNA immunization circumvents deficient induction of T helper type 1 and cytotoxic T lymphocyte responses in neonates and during early life

The relative deficiency of T helper type 1 (Th1) and cytotoxic T lymphocyte (CTL) responses in early life is associated with an increased susceptibility to infections by intracellular microorganisms. This is likely to reflect a preferential polarization of immature CD4 T cells toward a Th2 rather than a Th1 pattern upon immunization with conventional vaccines. In this report, it is shown that a single immunization within the first week of life with DNA plasmids encoding viral (measles virus hemagglutinin, Sendai virus nucleoprotein) or bacterial (C fragment of tetanus toxin) vaccine antigens can induce adult-like Th1 or mixed Th1/Th2 responses indicated by production of IgG2a vaccine-specific antibodies and preferential secretion of interferon-γ (IFN-γ) compared with interleukin (IL)-5 by antigen-specific T cells, as well as significant CTL responses. However, in spite of this potent Th1-driving capacity, subsequent DNA immunization was not capable of reverting the Th2-biased responses induced after early priming with a recombinant measles canarypox vector. Thus, DNA vaccination represents a novel strategy capable of inducing Th1 or mixed Th1/Th2 and CTL responses in neonates and early life, providing it is performed prior to exposure to Th2-driving conventional vaccine antigens.

Inhibition of interferon γ induced interleukin 12 production: A potential mechanism for the anti-inflammatory activities of tumor necrosis factor

Inflammation is associated with production of cytokines and chemokines that recruit and activate inflammatory cells. Interleukin (IL) 12 produced by macrophages in response to various stimuli is a potent inducer of interferon (IFN) γ production. IFN-γ, in turn, markedly enhances IL-12 production. Although the immune response is typically self-limiting, the mechanisms involved are unclear. We demonstrate that IFN-γ inhibits production of chemokines (macrophage inflammatory proteins MIP-1α and MIP-1β). Furthermore, pre-exposure to tumor necrosis factor (TNF) inhibited IFN-γ priming for production of high levels of IL-12 by macrophages in vitro. Inhibition of IL-12 by TNF can be mediated by both IL-10-dependent and IL-10-independent mechanisms. To determine whether TNF inhibition of IFN-γ-induced IL-12 production contributed to the resolution of an inflammatory response in vivo, the response of TNF+/+ and TNF−/− mice injected with Corynebacterium parvum were compared. TNF−/− mice developed a delayed, but vigorous, inflammatory response leading to death, whereas TNF+/+ mice exhibited a prompt response that resolved. Serum IL-12 levels were elevated 3-fold in C. parvum-treated TNF−/− mice compared with TNF+/+ mice. Treatment with a neutralizing anti-IL-12 antibody led to resolution of the response to C. parvum in TNF−/− mice. We conclude that the role of TNF in limiting the extent and duration of inflammatory responses in vivo involves its capacity to regulate macrophage IL-12 production. IFN-γ inhibition of chemokine production and inhibition of IFN-γ-induced IL-12 production by TNF provide potential mechanisms by which these cytokines can exert anti-inflammatory/repair function(s).

ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

Biparental Inheritance of γ-Tubulin during Human Fertilization: Molecular Reconstitution of Functional Zygotic Centrosomes in Inseminated Human Oocytes and in Cell-free Extracts Nucleated by Human Sperm

Human sperm centrosome reconstitution and the parental contributions to the zygotic centrosome are examined in mammalian zygotes and after exposure of spermatozoa to Xenopus laevis cell-free extracts. The presence and inheritance of the conserved centrosomal constituents γ-tubulin, centrin, and MPM-2 (which detects phosphorylated epitopes) are traced, as is the sperm microtubule-nucleating capability on reconstituted centrosomes. γ-Tubulin is biparentally inherited in humans (maternal >> than paternal): Western blots detect the presence of paternal γ-tubulin. Recruitment of maternal γ-tubulin to the sperm centrosome occurs after sperm incorporation in vivo or exposure to cell-free extract, especially after sperm “priming” induced by disulfide bond reduction. Centrin is found in the proximal sperm centrosomal region, demonstrates expected calcium sensitivity, but appears absent from the zygotic centrosome after sperm incorporation or exposure to extracts. Sperm centrosome phosphorylation is detected after exposure of primed sperm to egg extracts as well as during the early stages of sperm incorporation after fertilization. Finally, centrosome reconstitution in cell-free extracts permits sperm aster microtubule assembly in vitro. Collectively, these results support a model of a blended zygotic centrosome composed of maternal constituents attracted to an introduced paternal template after insemination.

Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide

A cytotoxic T lymphocyte (CTL) clone generated in vitro from the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specific in vitro priming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.

Efficient IgG-mediated suppression of primary antibody responses in Fcγ receptor-deficient mice

IgG antibodies can suppress more than 99% of the antibody response against the antigen to which they bind. This is used clinically to prevent rhesus-negative (Rh−) women from becoming immunized against Rh+ erythrocytes from their fetuses. The suppressive mechanism is poorly understood, but it has been proposed that IgG/erythrocyte complexes bind to the inhibitory Fc receptor for IgG (FcγRIIB) on the B cell surface, thereby triggering negative signals that turn off the B cell. We show that IgG induces the same degree of suppression of the response to sheep erythrocytes in animals lacking the known IgG-binding receptors FcγRIIB, FcγRI + III, FcγRI + IIB + III, and FcRn (the neonatal Fc receptor) as in wild-type animals. Reinvestigation of the ability of F(ab′)2 fragments to suppress antibody responses demonstrated that they were nearly as efficient as intact IgG. In addition, monoclonal IgE also was shown to be suppressive. These findings suggest that IgG inhibits antibody responses through Fc-independent mechanisms, most likely by masking of antigenic epitopes, thereby preventing B cells from binding and responding to antigen. In agreement with this, we show that T cell priming is not abolished by passively administered IgG. The results have implications for the understanding of in vivo regulation of antibody responses and Rh prophylaxis.