From pathogen defence to food allergy; The multiple facets of the mucosal immune system

Résumé destiné à un large public Le système immunitaire associé aux muqueuses gastro-intestinales doit être capable de protéger notre organisme contre l'invasion de pathogènes. Parallèlement, il doit identifier en Cant que tels, des composés inoffensifs comme la nourriture ou les milliards de bactéries qui résident dans notre intestin. Le travail présenté ici aborde ces deux aspects essentiels au bon fonctionnement de notre muqueuse intestinale. Dans une première partie, la protéine nommée pièce sécrétoire a été étudiée pour ses propriétés protectrices contre le pathogène viral rotavirus. Le rôle de la pièce sécrétoire est de transporter les anticorps que nous produisons vers la surface des muqueuses. En dehors de cette fonction bien connue, il se peut que cette protéine soit également capable de protéger notre organisme contre certains virus. L'hypothèse de travail était donc que la pièce sécrétoire se lie directement au virus, l'empêchant ainsi d'infecter des cellules épithéliales de l'intestin. En utilisant différentes techniques biochimiques, cette hypothèse s'est révélée fausse car aucune interaction entre la pièce sécrétoire et le virus n'a pu être observée, et logiquement, aucune protection n'a pu prendre place. En revanche, la pièce sécrétoire se lie à d'autres structures pathogéniques et permet ainsi de neutraliser leurs effets néfastes. La pièce sécrétoire participe donc activement à la protection de nos muqueuses, en plus de son rôle de transporteur. La deuxième partie de ce travail avait pour sujet les réactions inappropriées que le système immunitaire induit parfois contre un aliment, ou, autrement dit, les allergies alimentaires. Un modèle d'allergie alimentaire à donc été développé chez la souris et a permis de mesurer plusieurs symptômes et facteurs liés à l'allergie. Puis, ce modèle a été utilisé afin de tester les effets bénéfiques d'une bactérie lactique, dite probiotique, sur le développement de l'allergie. Il a été observé que, sous certaines circonstances, l'administration de la bactérie lactique protégeait entièrement les souris contre les réactions allergiques. L'effet bénéfique dépend donc du probiotique mais également d'autres facteurs encore inconnus â ce jour. Cette étude ouvre la voie sur la compréhension des mécanismes liés aux allergies alimentaires et sur l'impact que peuvent avoir les bactéries probiotiques sur cette maladie. Résumé Le système immunitaire associé aux muqueuses intestinales doit être capable de différencier les antigènes inoffensifs tels que 1a nourriture ou les bactéries commensales des microorganismes potentiellement dangereux. Cet aspect est essentiel pour le maintien de l'homéostase intestinale et fait l'objet du travail présenté ici. Dans un premier projet, les propriétés protectrices de la protéine appelée pièce sécrétoire (SC) ont été étudiées. SC est une protéine connue pour le transport des immunoglobulines à la surface des muqueuses. Cette protéine est fortement glycosylée paz des sucres complexes, ce qui nous a mené à postuler que SC puisse interagir avec le pathogène rotavirus. Cette hypothèse était soutenue par le fait que ce virus adhère aux cellules épithéliales par des résidus glycosylés. Des analyses biochimiques et biologiques ont démontré qu'aucune interaction entre SC et le virus ne prenait place, et que par conséquent SC n'offrait aucune protection contre ce pathogène. En revanche, SC interagit avec d'autres structures pathogéniques, comme la toxine A de Clostridium difficile, et la molécule d'adhésion intimine de la bactérie entéropathogène Escherichia coli. La liaison se fait par l'intermédiaire des sucres et confère ainsi une protection contre ces pathogènes. Ainsi, SC a été identifié comme agent neutralisant au niveau de l'intestin. La deuxième partie de ce travail abordait le sujet des allergies alimentaires, et avait pour but de tester les effets bénéfiques potentiels d'une bactérie probiotique, Lactobacillus paracasei NCC2461, contre les réactions allergiques. Un modèle marin d'allergie alimentaire a été mis au point, permettant de mesurer des immunoglobulines E, des symptômes allergiques, et la dégranulation de mastocytes. Lorsque le probiotique a été administré aux souris, celles-ci ont été complètement protégées des réactions allergiques dans une première expérience. Cependant, cette protection n'a pas été reproduite et suggère que des facteurs environnementaux encore inconnus sont critiques pour que le probiotique agisse positivement. Ce travail a permis de mettre en évidence la complexité de l'approche des traitements liés aux probiotiques et ouvre la voie sur la compréhension des mécanismes liés à l'allergie. Abstract The mucosal immune system associated to the gastrointestinal mucosa must efficiently distinguish between innocuous antigens, such as food proteins and commensal bacteria and potentially infectious agents. The work presented here deals with these two essential aspects guaranteeing intestinal homeostasis. In the first part of this work, the protective properties of secretory component (SC) toward the pathogen rotavirus were investigated. SC, which allows the transport of polymeric immunoglobulins (Ig) to mucosal surfaces, is highly glycosylated with complex glycan structures. The abundance and the nature of these carbohydrates led us to speculate that SC might interact with rotavirus, which is known to bind target cells with glycan receptors. Using various biological and biochemical techniques, we demonstrated that SC did not interact with rotaviruses, nor protected epithelial cells from infection. However, SC was shown to bind to Clostridium difficile toxin A and to the enteropathogenic Echerischia coli adhesion molecule intimin in a glycan-dependent fashion. These interactions allow in vitro protection of epithelial cells using physiological concentrations of SC. These data identify SC as a microbial scavenger at mucosal surfaces, and in the context of secretory IgA, further enhance the neutralising properties of the complex. The second project was inscribed in the domain of food allergy and aimed to test the modulatory functions of a probiotic strain of Lactobacillus paracasei toward allergic reactions. A model of food-mediated allergy was developed in the mouse using mucosal sensitisation. Several parameters associated to allergy were quantified after allergen challenge, and included allergen-specific IgE, allergic signs like diarrhea and temperature drop, and degranulation of mast cells. Administration of the probiotic strain was shown to completely protect mice from allergic reactions. However, these data were not reproduced, suggesting that unknown environmental factors are required so that protection mediated by the probiotic strain occurs. This study paves the way to the understanding of the mechanisms associated to allergy, and highlights the tremendous complexity that probiotic treatments will have to face.

Study of the physiological role of RasGAP cleavage

Résume Les caspases sont un groupe de protéases à cystéine qui s?activent lors de l'apoptose. Leur activation induit le clivage de nombreuses cibles intracellulaires, conduisant à l'activation de voies pro-apoptotiques et finalement au démantèlement des cellules. Cependant, des caspases ont été décrites dans de nombreux autres processus indépendants de l'apoptose, notamment dans la physiologie des cellules hématopoïétiques, des cellules musculaires, des cellules de la peau et des neurones. Comment est-ce que les cellules réconcilient-elles ces deux fonctions distinctes? Une partie de la réponse réside dans la nature des substrats qu'elles clivent. Certains substrats, une fois clivées, deviennent anti-apoptotiques. RasGAP est une cible des caspases et contient deux sites spécifiques de clivage par les caspases. Lorsque le niveau d?activité des caspases est faible le clivage de RasGAP produit un fragment N-terminal qui active un signal antiapoptotique, relayé par la voie de Ras/PI3K/Akt. Lorsque le niveau d?activité des caspases est plus élevé le fragment RasGAP N-terminal est à nouveau clivé, perdant de ce fait ses propriétés anti-apoptotiques. Dans cette étude, nous avons mis en évidence que l'activation de la voie Ras/PI3K/Akt induite par le fragment RasGAP N-terminal dépend de RasGAP lui-même. Par ailleurs, dans le but d?étudier l?importance du clivage de RasGAP dans un contexte physiologique, nous avons développé un modèle animal exprimant une gêne mutée de RasGAP de sorte que la protéine est devenu insensible a l?action de caspases. Les données préliminaires obtenues montrent que le clivage de RasGAP n'est pas indispensable pour le développement et l?homéostasie chez la souris. Finalement, nous avons développé une souris transgénique surexprimant le fragment de RasGAP N-terminal dans les cellules ß du pancréas. Les animaux obtenus ne montrent pas de symptômes dans les conditions basales bien qu?ils soient plus résistants au diabète induit expérimentalement. Ces résultats montrent que la surexpression du fragment N-terminal de RasGAP protége efficacement les cellules ß du pancréas de l?apoptose induite par le stress sans pourtant affecter d?autres paramètres physiologiques des Ilot de Langerhans.<br/><br/>Caspases are a series of proteases that are activated during apoptosis. Their activation causes the cleavage of numerous intracellular targets, which leads to cell dismantling and activation of pro-apoptotic pathways. Caspases have been found to be involved in the physiology of numerous cell types including haematopoietic cells, muscle cells, skin cells and neurons. How cells conciliate these two opposite functions? Part of the answer lies in the nature of the substrates they cleave. Some substrates become anti-apoptotic once cleaved by caspases. RasGAP is a caspase substrate that possesses two conserved caspase-cleavage sites. At low caspase activity, RasGAP is first cleaved and the generated N-terminal fragment activates a potent anti-apoptotic signal, mediated by the Ras/PI3K/Akt pathway. At higher caspase activity, the N-terminal fragment is further cleaved thereby losing its anti-apoptotic properties. In the present study we show that the activation of the Ras/PI3K/Akt pathway mediated by RasGAP N-terminal fragment is dependent on RasGAP itself. Moreover, to study the role of RasGAP cleavage in a physiological model, we have developed a knock-in mouse model expressing a RasGAP mutant that is not cleavable by caspases. Preliminary data shows that RasGAP cleavage is not required for normal development and homeostasis in mice. Finally, we have developed a transgenic mouse model overexpressing RasGAP N-terminal fragment in the ß-cell of the pancreas. In basal conditions, these mice show no difference with their wt counterparts. However, they are protected against experimentally induced diabetes. These results indicate that fragment N can protect ? cells from stress-induced apoptosis without affecting other physiological parameters of the Islets.

Use of physiological constraints to identify quantitative design principles for gene expression in yeast adaptation to heat shock

Background: Understanding the relationship between gene expression changes, enzyme activity shifts, and the corresponding physiological adaptive response of organisms to environmental cues is crucial in explaining how cells cope with stress. For example, adaptation of yeast to heat shock involves a characteristic profile of changes to the expression levels of genes coding for enzymes of the glycolytic pathway and some of its branches. The experimental determination of changes in gene expression profiles provides a descriptive picture of the adaptive response to stress. However, it does not explain why a particular profile is selected for any given response. Results: We used mathematical models and analysis of in silico gene expression profiles (GEPs) to understand how changes in gene expression correlate to an efficient response of yeast cells to heat shock. An exhaustive set of GEPs, matched with the corresponding set of enzyme activities, was simulated and analyzed. The effectiveness of each profile in the response to heat shock was evaluated according to relevant physiological and functional criteria. The small subset of GEPs that lead to effective physiological responses after heat shock was identified as the result of the tuning of several evolutionary criteria. The experimentally observed transcriptional changes in response to heat shock belong to this set and can be explained by quantitative design principles at the physiological level that ultimately constrain changes in gene expression. Conclusion: Our theoretical approach suggests a method for understanding the combined effect of changes in the expression of multiple genes on the activity of metabolic pathways, and consequently on the adaptation of cellular metabolism to heat shock. This method identifies quantitative design principles that facilitate understating the response of the cell to stress.

Effect of temperature and moisture on the dormancy and activation pattern of Oxalis latifolia bulbs

Con objeto de buscar información para un mejor control de Oxalis latifolia Kunth, se ha estudiado el efecto que presentan la temperatura y la humedad sobre la activación de sus bulbos, tanto en la forma común como en la forma Cornwall de la misma. Los bulbos de la mala hierba se mantuvieron en un refrigerador a +4°C durante 13, 20, 27, 41, 48 y 55 días. Se sacaron tres grupos de 30 bulbos de cada forma en cada fecha de muestreo y se colocaron a 21°C; 15 de ellos se mantuvieron en condiciones de sequía —no se regaron— y otros 15 en condiciones de humedad —añadiendo el agua necesaria—. Los resultados muestran que la activación ocurre durante un período prolongado de tiempo en ambas formas, tanto en seco como en húmedo. También se observó que los bulbos secos se activaron antes que los húmedos y los de la forma común antes que los Cornwall. Se observaron dos patrones de activación: los bulbos secos de la forma común generalmente presentaron una activación que sigue un patrón logarítmico, mientras que sus bulbos humedecidos mostraron una tendencia linear; los bulbos Cornwall se activaron con una tendencia exponencial en la mayoría de los casos. El tiempo medio requerido para la activación después del almacenamiento en frío fue constante en la forma común, sin embargo la activación de Cornwall fue más rápida cuanto más tiempo permanecieron almacenados en frío.

Earliness per se and its dependence upon temperature in diploid wheat lines differing in the major gene Eps-Am1 alleles

Differences in development among wheat cultivars are not only restricted to photoperiod and vernalization responses. When both requirements are fully satisfied differences may still arise due to earliness per se. It is not clear at present to what extent this trait is ‘ intrinsically ’ expressed (a constitutive trait) independently of the environmental conditions so that it might be selected under any thermal condition or if it may be altered to the extent of showing a crossover interaction with temperature in which the ranking of wheat genotypes may be altered. The present study assessed the influence of temperature on the intrinsic earliness for lines of diploid wheat characterized for their differences in a major gene for intrinsic earliness, but also possibly differing in their genetic background for other factors controlling this polygenic trait. To do so the lines were grown individually in two temperature regimes (16 and 23 xC) under long days having previously been fully vernalized. Multiple comparisons analyses were carried out among lines of the same allelic group for the Eps-Am1 gene. Results indicated that within each group there were lines that did not differ in their earliness per se, others differed but without exhibiting any linertemperature interaction and finally different types of interaction were shown, including cases where the ranking of lines was altered depending on the growing temperature. It is thus possible that the selection of a genotype based on its earliness per se in an environment might not represent the same performance in another location where temperature varied significantly.

Effect of temperature and carpel size during pre-anthesis on potential grain weight in wheat

The effect of environmental conditions immediately before anthesis on potential grain weight was investigated in wheat at the experimental field of the Faculty of Agronomy (University of Buenos Aires, Argentina) during 1995 and 1996. Plants of two cultivars of wheat were grown in two environments (two contrasting sowing dates) to provide different background temperature conditions. In these environments, transparent boxes were installed covering the spikes in order to increase spike temperature for a short period (c. 6 days) immediately before anthesis, i.e. between ear emergence and anthesis. In both environments, transparent boxes increased mean temperatures by at least 3n8 mC. These increases were almost entirely due to the changes in maximum temperatures because minimum temperatures were little affected. Final grain weight was significantly reduced by higher temperature during the ear emergence–anthesis period. It is possible that this reduction could be mediated by the effect of the heat treatment on carpel weight at anthesis because a curvilinear association between final grain weight and carpel weight at anthesis was found. This curvilinear association may also indicate a threshold carpel weight for maximizing grain weight.

Two component systems: physiological effect of a third component

Signal transduction systems mediate the response and adaptation of organisms to environmental changes. In prokaryotes, this signal transduction is often done through Two Component Systems (TCS). These TCS are phosphotransfer protein cascades, and in their prototypical form they are composed by a kinase that senses the environmental signals (SK) and by a response regulator (RR) that regulates the cellular response. This basic motif can be modified by the addition of a third protein that interacts either with the SK or the RR in a way that could change the dynamic response of the TCS module. In this work we aim at understanding the effect of such an additional protein (which we call ‘‘third component’’) on the functional properties of a prototypical TCS. To do so we build mathematical models of TCS with alternative designs for their interaction with that third component. These mathematical models are analyzed in order to identify the differences in dynamic behavior inherent to each design, with respect to functionally relevant properties such as sensitivity to changes in either the parameter values or the molecular concentrations, temporal responsiveness, possibility of multiple steady states, or stochastic fluctuations in the system. The differences are then correlated to the physiological requirements that impinge on the functioning of the TCS. This analysis sheds light on both, the dynamic behavior of synthetically designed TCS, and the conditions under which natural selection might favor each of the designs. We find that a third component that modulates SK activity increases the parameter space where a bistable response of the TCS module to signals is possible, if SK is monofunctional, but decreases it when the SK is bifunctional. The presence of a third component that modulates RR activity decreases the parameter space where a bistable response of the TCS module to signals is possible.

Base temperature and simulation model for nodes appearance in cape gooseberry (Physalis peruviana L.)

Data was analyzed on development of the solanaceen fruit crop Cape gooseberry to evaluate how well a classical thermal time model could describe node appearance in different environments. The data used in the analysis were obtained from experiments conducted in Colombia in open fields and greenhouse condition at two locations with different climate. An empirical, non linear segmented model was used to estimate the base temperature and to parameterize the model for simulation of node appearance vs. time. The base temperature (Tb) used to calculate the thermal time (TT, ºCd) for node appearance was estimated to be 6.29 ºC. The slope of the first linear segment was 0.023 nodes per TT and 0.008 for the second linear segment. The time at which the slope of node apperance changed was 1039.5 ºCd after transplanting, determined from a statistical analysis of model for the first segment. When these coefficients were used to predict node appearance at all locations, the model successfully fit the observed data (RSME=2.1), especially for the first segment where node appearance was more homogeneous than the second segment. More nodes were produced by plants grown under greenhouse conditions and minimum and maximum rates of node appearance rates were also higher.

Equivalent roughness of gravel-bed rivers

The relation between the equivalent roughness and different grain size percentiles of the sediment in gravel-bed rivers was determined under the hypothesis that the vertical distribution of the flow velocity follows a logarithmic law. A set of 954 data points was selected from rivers with gravel size sediment or larger, with a non-sinuous alignment and free of vegetation or obstacles. According to the results, the ks roughness is equivalent to approximately 2.4D90, 2.8D84, and 6.1D50. No correlation was detected between the sediment sorting and the sediment mobility index on one hand, and, on the other, the coefficient of proportionality of each grain size percentile.

Use of the heat dissipation method for sap flow measurement in citrus nursery trees1

Sap flow could be used as physiological parameter to assist irrigation of screen house citrus nursery trees by continuous water consumption estimation. Herein we report a first set of results indicating the potential use of the heat dissipation method for sap flow measurement in containerized citrus nursery trees. 'Valencia' sweet orange [Citrus sinensis (L.) Osbeck] budded on 'Rangpur' lime (Citrus limonia Osbeck) was evaluated for 30 days during summer. Heat dissipation probes and thermocouple sensors were constructed with low-cost and easily available materials in order to improve accessibility of the method. Sap flow showed high correlation to air temperature inside the screen house. However, errors due to natural thermal gradient and plant tissue injuries affected measurement precision. Transpiration estimated by sap flow measurement was four times higher than gravimetric measurement. Improved micro-probes, adequate method calibration, and non-toxic insulating materials should be further investigated.

Excess winter deaths caused by cardiovascular diseases are associated with both mild winter temperature and socio-economic inequalities in the U.S.

Mortality from cardiovascular diseases (CVD) exhibits seasonal variation. For example, 30% more deaths occurred in winter compared to summer in a multicountry study [1]. The effect of cold temperature on several CVD risk factors and on seasonal influenza infection may partially underlie this seasonal variation [2] and [3]. However an unexplained paradox has been observed: seasonality in CVD mortality is larger in temperate mid-latitude countries (e.g. Portugal) than in colder northern countries (e.g. Scandinavian countries) [1]. This paradox has also been previously observed in Europe for overall mortality, and it may relate to uneven proportions between countries of people who are unable to adequately protect themselves against cold due to low socio-economic status (SES), e.g. inadequate clothing, housing insulation and heating systems [4] and [5]. We hypothesized that the seasonal variability in CVD mortality is larger in low socio-economic U.S. states experiencing mild winters compared to high socio-economic states experiencing cold winters.

Gas exchanges in peach palms as a function of the spad chlorophyll meter readings

The close relationship between the chlorophyll-meters readings and the total chlorophyll and nitrogen contents in leaves, has allowed their evaluation both in annual and perennial species. Besides, some physiological events such as the CO2 assimilation have also been estimated by chlorophyll meters. This work was carried out aiming to evaluate the gas exchanges of peach palms as a function of the chlorophyll SPAD-Meter readings. Three year-old peach palms from Yurimaguas, Peru were studied in Ubatuba, SP, Brazil, spaced 2 x 1 m in area under a natural gradient of organic matter which allowed four plots to be considered, according to the peach palms leaves colors, from light yellow to dark green. The SPAD readings and the stomatal frequency of leaflets were evaluated. The photosynthetic photon flux density (PPFD, μmol m-2 s-1), the leaf temperature (Tleaf, ºC), the CO2 assimilation (A, μmol m-2 s-1), the stomatal conductance (g s, mol m-2 s-1), the transpiration (E, mmol m-2 s-1) and the intercellular CO2 concentration (Ci, μmol mol-1) were evaluated with a portable infrared gas analyzer (LCA-4, ADC BioScientific Ltd., Great Amwell, U.K.). A linear increase in the CO2 assimilation as a function of the SPAD readings (y = -0.34 + 0.19x, R² = 0.99), indicates that they can be a rapid and cheap complementary method to evaluate in peach palms some important physiological events, such as CO2 assimilation.

The reproductive cycle of the sea urchin Arbacia lixula in northwest mediterranean: potential influence of temperature and photoperiod

We studied the reproductive cycle of the sea urchin Arbacia lixula in a subtidal population from northeast Spain over four years using a gonadosomatic index (GSI) and gonad histology. Our results show that the GSI of A. lixula follows a seasonal cycle which peaks in May-July and attains its lowest values in October-November every year. The time course of the GSI matched closely the photoperiod cycle. We also found a remarkable inter-annual variability in the maximum value of GSI, which correlated with mean water temperature during the gonad growth period (winter and spring). Gonad histology was also in agreement with a single gametogenic cycle per year in this species. We explored the application of circular statistics to present and analyse gonadal development data, which allowed us to adequately handle the high intra-individual variability detected, with several developmental stages commonly found within the same gonad. The picture that emerged is one of a gametogenic timing driven by photoperiod, while the amount of reproductive output is determined by temperature. This is coherent with the tropical origin of the species and lends support to recent warnings about an increase in the abundance of this species in the Mediterranean as a result of global warming, with associated increased impact potential in sublittoral communities.

Apple biological and physiological disorders in the orchard and in postharvest according to production system

The study aimed to evaluate the incidence of biological and physiological disorders in the field and postharvested apples cvs. Gala, Fuji and Catarina grown in four production systems: conventional, organic transition, integrated and organic. Apples were evaluated for damages related to biological and physiological disorders in the orchard and after harvest. The greatest damages were attributed to pests, especially Anastrepha fraterculus in the organic system and Grapholita molesta in the organic transition. Apples produced in organic orchards had higher damage levels caused by postharvest physiological disorders than those grown in other production systems. For apples becoming from organic orchards most of the damage was due to lenticels breakdown and degeneration ('Gala'), and bitter pit ('Fuji' and 'Catarina'). The incidence of postharvest rot was not influenced by apple production system.