Immunotherapeutic targeting of LIGHT/LTβR/HVEM pathway fully recapitulates the reduced cytotoxic phenotype of LIGHT-deficient T cells.

Tumor necrosis factor (TNF)/TNF receptor (TNFR) superfamily members play essential roles in the development of the different phases of the immune response. Mouse LIGHT (TNFSF14) is a type II transmembrane protein with a C-terminus extracellular TNF homology domain (THD) that assembles in homotrimers and regulates the course of the immune responses by signaling through 2 receptors, the herpes virus entry mediator (HVEM, TNFSFR14) and the lymphotoxin β receptor (LTβR, TNFSFR3). LIGHT is a membrane-bound protein transiently expressed on activated T cells, natural killer (NK) cells and immature dendritic cells that can be proteolytically cleaved by a metalloprotease and released to the extracellular milieu. The immunotherapeutic potential of LIGHT blockade was evaluated in vivo. Administration of an antagonist of LIGHT interaction with its receptors attenuated the course of graft-versus-host reaction and recapitulated the reduced cytotoxic activity of LIGHT-deficient T cells adoptively transferred into non-irradiated semiallogeneic recipients. The lack of LIGHT expression on donor T cells or blockade of LIGHT interaction with its receptors slowed down the rate of T cell proliferation and decreased the frequency of precursor alloreactive T cells, retarding T cell differentiation toward effector T cells. The blockade of LIGHT/LTβR/HVEM pathway was associated with delayed downregulation of interleukin-7Rα and delayed upregulation of inducible costimulatory molecule expression on donor alloreactive CD8 T cells that are typical features of impaired T cell differentiation. These results expose the relevance of LIGHT/LTβR/HVEM interaction for the potential therapeutic control of the allogeneic immune responses mediated by alloreactive CD8 T cells that can contribute to prolong allograft survival.

Interaction between the mineral content and the occurrence number of aquatic fungi in leaves submerged in a stream in the Atlantic rainforest, São Paulo, Brazil

Leaves of Ficus microcarpa L. f., Quercus robur L., and Alchornea triplinervia (Spreng.) Muell. Arg., submerged in a stream of the Atlantic rainforest in the "Reserva Biológica do Alto da Serra de Paranapiacaba", State of São Paulo, Brazil, were collected monthly, from April to November 1990, in order to determine the number of fungal occurrences (zoosporic fungi and aquatic Hyphomycetes), and the content of total N (%), total P (%), K+ (%), Ca+2 (%), Mg+2 (%), S+3 (%), Fe+3 (ppm), Cu+3 (ppm), Mn+2 (ppm), Zn+2 (ppm), Bo (ppm), Na+2 (ppm) and Al+3 (ppm). According to the tests of Kruskal-Wallis, Mann-Whitney and Wilcoxon, the means of the mineral content of the three types of leaves were significantly different, except for Mg+2 (%), Mn+2 (ppm), Zn+2 (ppm) and Na+2 (ppm). On comparing the mineral content with the number of fungal occurrence, an independence test showed a positive correlation between the presence of zoosporic fungi on the leaves of A. triplinervia and the total nitrogen, phosphorus and S+3 content, whereas the aquatic Hyphomycetes depended on the amount of Ca+2 available. Regarding leaves of F. microcarpa, the occurrence of zoosporic fungi was linked to the S+3 level, and the presence of aquatic Hyphomycetes, to the content of K+, Ca+2, S+3 and Bo. On Q. robur leaves, zoosporic fungi showed a positive correlation with the Ca+2 content, but a negative one with Fe+3 and Al+3 levels, while the occurrence of aquatic Hyphomycetes was influenced by the content of Ca+2, Mg+2, Fe+3, Al+3, Mn+2, Zn+2 and Na+2. The correlation between the occurrence number of aquatic Hyphomycetes and a high mineral content indicates that their nutritional requirements may be more complex than those of zoosporic fungi. Further studies are still required to understand the implications of this tendency on the diversity of aquatic native mycota.

Effects of using different host plants on the detected biodiversity of arbuscular mycorrhizal fungi from an agroecosystem

The influence of peanut (Arachis hypogaea L.), sorghum (Sorghum bicolor (L.) Moench) and maize (Zea mays L.) on the development and diversity of arbuscular mycorrhizal fungi (AMF) from an agrosystem was investigated. Soil collected from an agricultural field where maize had been grown was inserted into sowing holes, under the seeds of peanut, sorghum and maize those were subsequently grown in sterilised quartz sand separately in plastic boxes for five months. After this period, collections of roots and rhizospheric soil were made to evaluate the percentages of root colonization (RC), number of spores (NS) and species of AMF. Peanut showed the highest average values for RC and NS: 24.5% and 547.8/100 g of soil, respectively. Maize had an average RC of 19.7% and 415.2 spores/100g soil. Sorghum showed the lowest values: 15.9% for average RC and 349.8 spores/100 g soil. A total of fourteen species of AMF were identified. Seven species were identified from peanut rhizospheres, Entrophospora colombiana being the most abundant and frequent. In sorghum rhizospheres, twelve species were found, Glomus geosporum was the dominant taxon in terms of number of spores and frequency. Ten species were detected in maize with Acaulospora longula being the most abundant and the most frequent. It was observed that peanut was the best plant for promoting the sporulation of AMF, while sorghum favoured the establishment of most AMF species, followed by maize.

Development of new heparin-like compounds and other antithrombotic drugs and their interaction with vascular endothelial cells

The anticlotting and antithrombotic activities of heparin, heparan sulfate, low molecular weight heparins, heparin and heparin-like compounds from various sources used in clinical practice or under development are briefly reviewed. Heparin isolated from shrimp mimics the pharmacological activities of low molecular weight heparins. A heparan sulfate from Artemia franciscana and a dermatan sulfate from tuna fish show a potent heparin cofactor II activity. A heparan sulfate derived from bovine pancreas has a potent antithrombotic activity in an arterial and venous thrombosis model with a negligible activity upon the serine proteases of the coagulation cascade. It is suggested that the antithrombotic activity of heparin and other antithrombotic agents is due at least in part to their action on endothelial cells stimulating the synthesis of an antithrombotic heparan sulfate.

Stromal interaction molecule 1 and its role in the regulation, proliferation and protein expression in follicular ML-1 thyroid cancer cells

Calcium (Ca2+) is involved in the regulation of variety of cellular functions including hallmarks of cancer development such as cellular migration and cellular proliferation. Store-operated calcium entry (SOCE) is a central mechanism in cellular calcium signaling and in maintaining the cellular calcium balance. Stromal interaction molecule 1(STIM1) has been identified as an important constituent of SOCE. In this thesis , the STIM1 proteins are studied for their importance in cellular processes and their effects on the expression of S1P1, S1P2, S1P3, VEGFR-2, and TRPC-1 in follicular ML-1 thyroid cancer cells. The results show the importance of STIM1 proteins in SOCE in these cells. The SOCE is significantly reduced in the STIM1 knockdown cells. The results also show the importance of STIM1 proteins in the expression of S1P2 and VEGFR-2 in these cells, as knockdown of STIM1 was shown to upregulate the expression of S1P2 and VEGFR-2. The migration and proliferation is also considerably reduced in the cells in which STIM1 has been knocked down showing the significance of STIM1 in the migration and proliferation in these cells.

Immuno-gluorescence study of five zygomycetous fungi with two chitin-binding probes

A polyclonal antiserum was prepared against a purified microsomal chitinase isolated from the fungus Choanephora cucurbitarum. Indirect immunofluorescence was used to localize chitinase at various developmental stages of five zygomycetous fungi and during abiotrophic mycoparasite interaction with a susceptible and resistant host. This was compared to localization of oligomers of N-acetylglucosamine with the lectin wheat germ agglutinin (WGA). Dotimmunoblot and Western blot techniques revealed that the anti-serum reacted strongly with the antigen from which it was derived. Cross reactivity of the antiserum was found with WGA and another chitin binding lectin, Phyto/acca americana agglutinin (PAA). Immuno-fluorescence results showed the direct involvement of chitinase in spore swelling, germination, sporangium development and response during mechanical injury. There appeared to be no involvement of chitinase during apical hyphal growth or new branch initiation in any of the fungi tested despite mild proteolysis and permeabilization of the cell surface prior to labelling. Binding with WGA revealed similar patterns of fluorescence to that of chitinase localization but differed by showing fluorescence and therefore chitin localization at the apex and new branch initiation when tested at different developmental stages. There was no difference between chitinase localization and binding with WGA in a susceptible host and resistant host challenged with the mycoparasite, Piptocephalis virginiana. Differences in binding ability of antichitinase and lectin WGA suggests that the latter is not a suitable indicator for indirect localization of the lytic enzyme, chitinase.

Probing cell surfaces of host and nonhost species of a mycoparasite, using FITC-labeled lectins

Cell surfaces of susceptible host species (Mortierella pusllla and Cboanepilora cucurbitarum ), resistant host (Pilascolomyces articulosus ), nonhost (Mortierella candelabrum ) and the mycoparasite (Piptocepilalis virginiana) were examined for sugar distribution patterns using light and fluorescent microscopy techniques. The susceptible host, resistant host and the mycoparasite species exhibited a similar sugar distribution profile; they all showed N-acetyl glucosamine and D-glucose on their cell wall surfaces. The nonhost cell wall surface showed a positive binding reaction to FITClectins specific for N-acetyl glucosamine and also for OI.-fucose, N-acetyl galactosamine and galactose. Treatment of these fungi with mild concentrations of proteinases (both commercial as well as the mycoparasiteproteinase) resulted in the revelation of additional sugars on the fungal cell walls. The susceptible host treated with proteinase expressed higher levels of N-acetyl glucosamine and D-glucose. The susceptible host also showed the presence of OI.-fucose, N-acetyl galactosamine and galactose. The proteinasetreated susceptible host cell walls also showed an increase in the levels of attachment with the mycoparasite. Treatment of the resistant host with proteinases revealed OI.-fucose in addition to N-acetyl glucosamine and D-glucose. Treatment of the nonhost cell wall with proteinase resulted in the exposure of low levels of D-glucose, in addition to sugars found on the untreated nonhost cell wall surface. The mycoparasite treated with proteinase revealed OI.-fucose, N-acetyl galactosamine and galactose on its cell surface in addition to the sugars N-acetyl glucosamine and D-glucose. Protoplasts were isolated from hosts and nonhost fungi and their surfaces were examined for sugar distribution patterns. The susceptible host and nonhost protoplast membranes showed all the sugars (N-acetyl glucosamine, D-glucose, (It.-fucose, N-acetyl galactosamine and galactose) tested for. The resistant host protoplast membrane however, had only N-acetyl glucosamine and D-glucose exposed. This sugar distribution profile resembles that exhibited by the untreated resistant host cell wall, as well as that shown by the untreated mycoparasite cell surface. Only susceptible host protoplasts were successful in attaching to the mycoparasite surface. Resistant host protoplasts did not show any interaction with the i mycoparasite cell surface. Both susceptible as well as resistant host protoplasts were incapable of attaching to agarose beads surface-coated with specific carbohydrates. The mycoparasite however, did attach to agarose beads surface-coated with either N-acetyl glucosamine, D-glucose/Dmannose or o:,- methyl-D-mannose. The relevance of the cell wall and the protoplast membrane in the light of the present results, in reacting appropriately to bring about either a susceptible, a resistant or a nonhost response has been discussed.

Interaction entre le virus SRRP et Actinobacillus pleuropneumoniae dans un modèle d'infection en culture cellulaire

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est un pathogène d’importance dans l’industrie porcine et est responsable d’importantes pertes économiques. Il n’existe pas d’antiviral efficace contre celui-ci. Il a récemment été mis en évidence que le surnageant de culture d’Actinobacillus pleuropneumoniae, l’agent étiologique de la pleuropneumonie porcine, possédait une activité antivirale in vitro contre le VSRRP dans la lignée cellulaire SJPL. Les objectifs de mon projet sont (i) d’étudier les mécanismes cellulaires menant à l’activité antivirale causée par le surnageant de culture d’A. pleuropneumoniae, et (ii) de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae. Dans un premier temps, des analyses de protéome ont été effectuées et ont permis d’observer que le surnageant de culture modulait la régulation du cycle cellulaire. Dans le but d’analyser le cycle cellulaire des cellules SJPL, la cytométrie en flux a été utilisée et a permis de démontrer que le surnageant de culture induisait un arrêt du cycle cellulaire en phase G2/M. Deux inhibiteurs de la phase G2/M ont alors été utilisé. Il s'est avéré que ces inhibiteurs avaient la capacité d’inhiber le VSRRP dans les cellules SJPL. Enfin, la spectrométrie de masse a été utilisée dans le but de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae et d’identifier deux molécules. Ce projet a permis de démontrer pour la première fois qu’A. pleuropneumoniae est capable de perturber le cycle cellulaire et que ce dernier était un élément important dans l’effet antiviral contre le VSRRP.

Interaction entre ventilation mécanique et traumatisme à la moelle épinière : réponses inflammatoires pulmonaires et médullaires

Le traumatisme de la moelle épinière est à l’origine d’une inflammation locale importante caractérisée par l’augmentation massive des cellules inflammatoires et la présence de réactions oxydatives. Cette inflammation locale peut déclencher une réponse inflammatoire systémique par voie hématogène. Au niveau cervical, les lésions médullaires peuvent entraîner des faiblesses ou la paralysie des muscles respiratoires. Le patient, qui ne peut plus respirer de façon autonome, doit avoir recours à un support respiratoire. Bien que la ventilation mécanique soit la thérapie traditionnellement appliquée aux blessés médullaires souffrant d’insuffisance respiratoire, les études ont démontré qu’elle pouvait contribuer à promouvoir une réponse inflammatoire ainsi que des dommages pulmonaires. L’interaction entre le traumatisme médullaire et la ventilation mécanique, indispensable au maintien de l’équilibre des échanges respiratoires, est inconnue à ce jour. En voulant protéger les tissus, cellules et organes, l’organisme met en œuvre toute une panoplie de réponses inflammatoires à différents endroits. Nous pensons que ces réponses peuvent être altérées via l’interaction entre ce traumatisme et cette ventilation mécanique, sous l’influence de la principale source cellulaire de cytokines pour la défense de l’hôte, le macrophage, récemment classé en deux phénotypes principaux: 1) l’activation classique de type M1 et 2) l’activation alternative de type M2. Le phénotype M1 est conduit par le facteur GM-CSF et induit par l’interféron IFN-ɣ ainsi que le lipopolysaccharide. Le phénotype M2 quant à lui, est conduit par le facteur M-CSF et induit par les interleukines IL-4, IL-13 ou IL-21. M1 relâche principalement IL-1β, IL-6, TNF-α et MIP-1α tandis que M2 principalement IL-10 et MCP-1. Toutefois, nous ignorons actuellement par quel type d’activation se manifestera cette réponse immunitaire et si l’application de support respiratoire pourrait entraîner un risque inflammatoire additionnel au site du traumatisme. Nous ignorons également si la ventilation mécanique affecterait, à distance, les tissus de la moelle épinière via une inflammation systémique et amplifierait alors le dommage initial. Il n’existe pas à ce jour, de thérapie qui ait montré d’effet bénéfique réel envers une récupération fonctionnelle des patients blessés médullaires. Il paraît donc essentiel de déterminer si la ventilation mécanique peut moduler l’inflammation post-traumatique à la fois au niveau pulmonaire et au site de la lésion. Ce travail visait à caractériser les liens entre l’inflammation issue du traumatisme médullaire et celle issue de la ventilation, dans le but de fournir une meilleure compréhension des mécanismes inflammatoires activés dans ce contexte. L’étude a été menée sur un modèle animal. Elle consistait à évaluer : 1) si le traumatisme médullaire influençait les réponses inflammatoires pulmonaires induites par la ventilation mécanique, y compris le phénotype des macrophages alvéolaires et 2) si la ventilation pouvait altérer à distance, les tissus de la moelle épinière. L’impact de la blessure médullaire sur l’inflammation pulmonaire et locale, induite par la ventilation fut interprété grâce à l’analyse des cellules inflammatoires dans les lavages broncho-alvéolaires et dans les tissus prélevés à l’endroit de la blessure après 24 heures. Ces analyses ont démontré un profil spécifique des cytokines pulmonaires et médullaires. Elles ont révélé que la ventilation mécanique a engendré un environnement pro-inflammatoire en faveur d’un phénotype M1 chez les animaux ayant bénéficié de la thérapie respiratoire. Inversement, l’atteinte thoracique chez les animaux sans ventilation, a montré qu’une réponse immunitaire avait été activée en faveur d’un environnement anti-inflammatoire de phénotype M2. La lésion cervicale quant à elle a induit un profil de cytokines différent et les réponses au stress oxydatif dans le poumon induites par la ventilation ont été réduites significativement. De plus, une lésion médullaire a augmenté l’expression d’IL-6 et la ventilation a diminué l’IL-1β et augmenté le TNF-α dans les tissus de la moelle. Finalement, ces données ont fourni les premières évidences que la ventilation a induit d’avantage à un phénotype pulmonaire M1 et que le traumatisme médullaire a impacté spécifiquement les réponses inflammatoires et oxydatives dans le poumon. La ventilation a contribué non seulement à distance à une inflammation des tissus médullaires lésés mais aussi des tissus sains.

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growthmedium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml-1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGFII had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells

The role of fungi and bacteria on the organic matter decomposition process in streams: interaction and relevance in biofilms

L'objectiu d'aquest estudi és el d'investigar sobre l'ús de matèria orgànica per part dels fongs i bacteris que colonitzen diferents substrats bentònics en rius Mediterranis i analitzar l'efecte dels factors ambientals i antròpics sobre l'estabilitat estructural i funcional de les comunitats del biofilm. La metodologia emprada en aquest estudi consisteix en: i) anàlisi de la biomassa bacteriana i fúngica, ii) anàlisi de la composició de les comunitats bentòniques (identificació d'hifomicets aquàtics i anàlisi del 16S rDNA bacterià), i iii) anàlisi de l'activitat enzimàtica extracel·lular relacionada amb el reciclatge de matèria orgànica en rius.

Host-symbiont interactions of the primary endosymbiont of human head and body lice

The first mycetome was discovered more than 340 yr ago in the human louse. Despite the remarkable biology and medical and social importance of human lice, its primary endosymbiont has eluded identification and characterization. Here, we report the host-symbiont interaction of the mycetomic bacterium of the head louse Pediculus humanus capitis and the body louse P. h. humanus. The endosymbiont represents a new bacterial lineage in the -Proteobacteria. Its closest sequenced relative is Arsenophonus nasoniae, from which it differs by more than 10%. A. nasoniae is a male-killing endosymbiont of jewel wasps. Using microdissection and multiphoton confocal microscopy, we show the remarkable interaction of this bacterium with its host. This endosymbiont is unique because it occupies sequentially four different mycetomes during the development of its host, undergoes three cycles of proliferation, changes in length from 2–4 µm to more than 100 µm, and has two extracellular migrations, during one of which the endosymbionts have to outrun its host’s immune cells. The host and its symbiont have evolved one of the most complex interactions: two provisional or transitory mycetomes, a main mycetome and a paired filial mycetome. Despite the close relatedness of body and head lice, differences are present in the mycetomic provisioning and the immunological response.—Perotti, M. A., Allen, J. M., Reed, D. L., Braig, H. R. Host-symbiont interactions of the primary endosymbiont of human head and body lice.

A qualitative host-pathogen interaction in the Theobroma cacao-Moniliophthora perniciosa pathosystem

The aim of this study was to test whether resistance of clones of Theobroma cacao ( cocoa) varied between isolates of Moniliophthora (formerly Crinipellis) perniciosa, the cause of witches' broom disease. Developing buds of vegetatively propagated T. cacao grown in greenhouses in the UK were inoculated with 16 000 spores of M. perniciosa per meristem in water, under conditions where water condensed on the inoculated shoot for at least 12 h after inoculation. The proportion of successful inoculations varied between clones and was inversely correlated with time to symptom production or broom formation. A specific interaction was demonstrated among three single-spore isolates of M. perniciosa and the clone Scavina 6 (SCA 6) and a variety of susceptible clones. Isolates Castenhal-I and APC3 were equally likely to infect SCA 6 and the other clones, but isolate Gran Couva A9 never infected SCA 6, although it was as virulent on the other clones. The interaction was maintained when the wetness period was extended to 70 h. Offspring of SCA 6 x Amelonado matings were all susceptible to both Castenhal-I and GC-A5, with no evidence of greater variability in susceptibility to GC-A5 than Castanhal-I. This suggests recessive inheritance of a single homozygous factor conferring resistance to GC-A5, from SCA 6. The progenies were slightly more susceptible to Castanhal-I than GC-A5. The implications for managing the disease are discussed.