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Effect of nanoparticles on human cells from healthy individuals and patients with respiratory diseases.Osman, Ilham F. January 2010 (has links)
Ever increasing applications of nanomaterials (materials with one or more dimension
less than 100 nm) has raised awareness of their potential genotoxicity. They have
unique physico¿chemical properties and so could have unpredictable effects. Zinc oxide
(ZnO) and titanium dioxide (TiO2) are widely used in a number of commercial
products. There are published studies indicating that some forms of these compounds
may be photo-clastogenic in mammalian cells. What has not been investigated before is
the effect of nanoparticles from these compounds in human germ cells. Thus the
present study has examined their effects in the presence and absence of UV light in
human sperm and compared responses to those obtained with human lymphocytes using
the Comet assay to measure DNA damage. The effect of nanoparticles (40-70nm range)
was studied in human sperm and lymphocytes in the dark, after pre-irradiation with UV
and simultaneous irradiation with UV. The studies do provide some evidence that there
are photo-genotoxic events in sperm and lymphocytes in the absence of overt toxicity.
The cytotoxic and genotoxic potentials of ZnO and TiO2 as well as their effect on
phosphotyrosine expression, were examined in the human epithelial cervical carcinoma
cells (Hela cells). This was done to try and determine the underlying molecular events
resulting from their exposure to ZnO and TiO2 nanoparticles occurring at the same time
as DNA is damaged. Concentration- and time-dependent cytotoxicity, and an increase
in DNA and cytogenetic damage with increasing nanoparticle concentrations were
reported in this study. Mainly for zinc oxide, genotoxicity was clearly associated with
an increase in tyrosine phosphorylation.
Nanotechnology has raced ahead of nanotoxicology and little is known of the effects of
nanoparticles in human systems, let alone in diseased individuals. Therefore, the effects
of TiO2 nanoparticles in peripheral blood lymphocytes from patients with respiratory
diseases (lung cancer, chronic obstructive pulmonary disease (COPD) and asthma) were
compared with those in healthy individuals using genotoxic endpoints to determine
whether there are any differences in sensitivity to nano-chemical insult between the
patient and control groups. The results have shown concentration dependent genotoxic
effects of TiO2 in both respiratory patient and control groups in the Comet assay and an
increasing pattern of cytogenetic damage measured in the micronucleus assay without
being statistically significant except when compared with the untreated controls of
healthy individuals. Furthermore, modulation of ras p21 expression was investigated.
Regardless of TiO2 treatment, only lung cancer and COPD patients expressed
measurable ras p21 levels that showed modulation as the result of nanoparticle
treatment.
Results have suggested that both ZnO and TiO2 nanoparticles can be genotoxic over a
range of concentrations without either photoa-ctivation or being cytotoxic.
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Mechanisms and consequences of DNA damage, response and apoptosis in spermatozoa.Laubenthal, Julian January 2011 (has links)
DNA damage in spermatozoa is a crucial contributor to spontaneous abortion, severe
genetic disease in the offspring and infertility. The chromatin of spermatozoa is highly
compacted, transcriptionally and translationally silent, hence lacking DNA damage response
(DDR). DDR foci follow within seconds after a DNA double strand break (DSB) and correlate
to an abortive topoisomerase-IIb activity during spermiogenesis.
When comparing the DSB frequencies at the two most fragile genomic loci (fragile sites
FRA3B, FRA16D) in human and murine spermatozoa with lymphocytes, significantly
increased DSB levels were detected in spermatozoa in both species. This corroborates that
spermatozoa are more prone to DSBs than somatic cells. When comparing the DSB
frequencies at FRA3B/FRA16D in spermatozoa of smokers with non-smokers, two-fold
increases were found, probably caused by cigarette smoke components triggering abortive
topoisomerase-II¿ activity. The phosphorylated DDR proteins H2AX and ATM were
identified in human spermatozoa and murine spermatids using multicolour immunostaining
with laser-scanning confocal microscopy (LSCM) and Western blots. Based on significantly
increased DDR foci in spermatozoa of smoking men, but lacking DDR foci in response to in
vitro challenge with H2O2, an abortive topoisomerase-IIb activity is the likely cause of DDR
foci in spermatozoa. As DDR foci are susceptible to cigarette smoke, they can potentially be
used as a novel biomarker. When comparing paternal spermatozoa, and lymphocytes as
well as maternal and cord lymphocytes from 39 families for DSBs (via high-throughput
LSCM pH2AX detection) and DNA fragmentation (Comet assay), significant increases were
found in newborns of mothers exposed to environmental tobacco smoke and smoking
fathers. When challenging lymphocytes and spermatozoa to different genotoxicants,
significantly increased DNA damage in newborns compared to adults was found. This
confirms an exceptional vulnerability in newborns, believed to cause increased susceptibly
to disease in later life, including cancer. / European Union¿s 6th Framework project Newborns and
genotoxic exposure risk (NewGeneris), British Council¿s United Kingdom Indian Education Research Initiative (UKIER)
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Influence of Culture Conditions on Ex Vivo Expansion of T Lymphocytes and Their Function for Therapy: Current Insights and Open QuestionsSudarsanam, Harish, Buhmann, Raymund, Henschler, Reinhard 20 October 2023 (has links)
Ex vivo expansion of T lymphocytes is a central process in the generation of cellular therapies
targeted at tumors and other disease-relevant structures,which currently cannot be reached by
established pharmaceuticals. The influence of culture conditions on T cell functions is, however,
incompletely understood. In clinical applications of ex vivo expanded T cells, so far, a relatively
classical standard cell culture methodology has been established. The expanded cells have
been characterized in both preclinical models and clinical studies mainly using a therapeutic
endpoint, for example antitumor response and cytotoxic function against cellular targets,
whereas the influence of manipulations of T cells ex vivo including transduction and culture
expansion has been studied to a much lesser detail, or in many contexts remains unknown.
This includes the circulation behavior of expanded T cells after intravenous application, their
intracellular metabolism and signal transduction, and their cytoskeletal (re)organization or their
adhesion, migration, and subsequent intra-tissue differentiation. This review aims to provide an
overview of established T cell expansion methodologies and address unanswered questions
relating in vivo interaction of ex vivo expanded T cells for cellular therapy.
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Editorial: Pathogenesis, treatment, and future directions for rare T-cell leukemiasHerling, Marco, Jarjour, Wael, Mishra, Anjali, Brammer, Jonathan E. 15 January 2024 (has links)
Mature T-cell leukemias represent rare, but increasingly recognized diseases of
which, compared to their B-cell counterparts, comparatively little is established on
their pathogenesis, diagnosis, and treatment. These leukemic post-thymic T-cell
neoplasms range from the spectrum of chronic, sometimes debilitating disorders such
as T-large granular lymphocytic leukemia (T-LGLL), and related leukemias such as NKLGLL,
to more aggressive malignancies such as T- prolymphocytic leukemia (T-PLL). In
this series, entitled ‘Pathogenesis, Treatment, and Future Directions for Rare T-cell
Leukemias’ we review the current state of the science of these important T-cell neoplasms
to inform on their treatment, diagnosis, and pathophysiology.
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Chicken infectious anemia virus vaccination induces immune disorders and viral persistency in infectious bursal disease virus-infected young chicksVaziry, Asaad 08 1900 (has links)
La bursite infectieuse aviaire (IBD) est une des causes majeures de pertes économiques pour l’industrie aviaire. La vaccination est le principal outil de contrôle de cette maladie et les oiseaux susceptibles doivent être vaccinés aussitôt que le niveau des anticorps maternels (MA) anti-IBDV est suffisamment bas. L’estimation du moment de vaccination est habituellement déterminée par la formule de Deventer qui utilise le titre initial de MA anti-IBDV et la demi-vie des anticorps pour prédire l’évolution du titre. Dans la présente étude, l’effet du gain de poids sur la vitesse de disparition des MA a été étudié dans le but de l’utiliser pour prédire la détermination du moment de la vaccination. L’analyse des taux d’anticorps neutralisants par ELISA a montré que les poussins avec une forte croissance avaient un taux de disparition plus rapide des MA que ceux à faible croissance. Une formule pour la prédiction du moment de vaccination contre le IBDV, basée sur le gain de poids et le niveau des MA a été développée et vérifiée. La prédiction du moment de vaccination avec cette formule a montré une haute corrélation avec les titres de MA mesurés par ELISA.
Le virus de l’anémie infectieuse aviaire (CIAV) est une cause importante d’immunosuppression chez le poulet augmentant la pathogénicité des infections secondaires et en entraînant une réponse humorale suboptimale et une forte mortalité. D’autre part, l’infections sub-clinique du au CIAV provoque une immunosuppression qui facilite la coinfection par d’autre virus tel que le IBDV. Les effets de la coinfection à J1 avec une souche vaccinale de CIAV CAV-VAC® (Intervet) et à J14 avec une souche faiblement virulente de IBDV isolée au Québec, sur l’état de santé des poussins, sur la persistance virale et sur la réponse immunitaire ont été étudiés autant chez des poussins de 1 jour d’âge exempts d’agents pathogènes specifique (SPF) que ceux provenant d’élevages commerciaux. Les résultats ont montré que l’inoculation de la souche vaccinale du CIAV a entraîné une infection sub-clinique, une persistance virale dans la rate et le thymus, une altération de la thymopoièse et une réponse humorale temporaire chez les poussins SPF. Ces effets ont aussi été mis en évidence chez des poussins d’élevage commerciaux malgré des taux élevés de MA. Lors de l’infection avec la souche de IBDV chez des poussins déjà vaccinés contre le CIAV, la persistance du CIAV dans les organes lymphoïdes a été aggravée par une présence de réponses humorales temporaires contre les deux virus et une altération des populations lymphocytaires dans les organes lymphoïdes. Par contre, la présence des MA contre le CIAV a limité temporairement ces effets. Ces travaux ont mis en évidence des désordres immunitaires cellulaires et humoraux et une persistance virale chez des poussins vaccinés contre le CIAV et co-infectés avec le IBDV. / Infectious bursal disease (IBD) is one of the major causes of economic losses in the chicken industry. Vaccination is the main tool against the disease, and the susceptible birds should be vaccinated as soon as the maternal antibody (MA) becomes low enough to allow the vaccine to break through. Estimation of vaccination time is currently performed by Deventer formula which uses initial anti-IBDV titer and antibody half-life to predict the titer. Considering the increased growth rate of chicken in the last decades and the wide variations of MA, we have examined the effects of chick’s weight gain on MA decline and the use of weight in predicting IBD vaccination time. The virus neutralization test and ELISA results demonstrated that fast-growing birds had a faster rate of antibody decline whereas slow-growing birds demonstrated a slower rate. Based on the effect of weight-gain on maternal antibody decline, a new formula for predicting IBD vaccination time was introduced and tested. The predicted IBD vaccination time made by this weight formula showed higher correlation with the measured ELISA titers in the experiment.
Chicken infectious anemia virus (CIAV) is another cause of immunosuppression in chicken which is characterized by increased pathogenicity of secondary infectious agents, sub-optimal antibody responses and mortality. CIAV subclinical infections can result in immunosuppression and enhancement of pathogenicity of co-infecting agents such as infectious bursal disease virus (IBDV). Effects of pathogenic CIAV and IBDV coinfection on chick’s health and immune responses are investigated in different studies. In this study, newly hatched specific pathogen free (SPF) and commercial chicks were vaccinated with CAV-VAC® (Intervet) vaccine and /or inoculated with a low-virulent Québec isolate of IBDV at 14 days post CIAV vaccination.
Inoculation of the CIAV vaccinal strain at hatch resulted in subclinical infection associated with viral persistency in spleen and thymus, alteration of thymopoiesis and transient humoral response in SPF chicks. Subclinical infection, viral persistency and lack of antibody responses were also shown in CIAV inoculated commercial chicks with high MA. Infection of the low-virulent IBDV in the CIAV vaccinated SPF chicks lead to extended viral persistence of CIAV in lymphoid organs, transient immune responses to both CIAV and IBDV, and alteration of lymphocytes subpopulation in the lymphoid organs. In the coinfected commercial chicks, presence the CIAV in the lymphoid organs was controlled by MA in the first 1-2 weeks after hatch. Thereafter, the immune disorders, viral persistence and lack of humoral responses almost similar to the coinfected SPF chicks were recorded.
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Immunophänotypisierung des entzündlichen Infiltrates der Arthrose assoziierten SynovialitisRistow, Gerhard 07 April 2003 (has links)
Die Entzündungsreaktion der Arthrose wird als eine sekundäre Reaktion auf einen degenerativen Prozeß des Gelenkknorpels angesehen. Die Ursache für die Degeneration kann im Mißverhältnis zwischen Belastbarkeit und Beanspruchung liegen, es können metabolische Störungen (Urämie, Diabetes mellitus) verantwortlich gemacht werden, weswegen von sekundärer Arthrose gesprochen wird. Die Ursache der primären Arthrose bleibt unbekannt. Es kann als bewiesen angesehen werden der Zusammenhang mit Alter und Geschlecht der Patienten, denn Arthrose ist in der Regel eine Erkrankung jenseits des fünfzigsten Lebensjahres und betrifft vornehmlich Frauen. In der vorliegenden Arbeit wurde die Synovialis von 20 Patienten aufgearbeitet und hinsichtlich des enthaltenen entzündlichen Infiltrates untersucht. Unter Anwendung der indirekten Immunperoxidase Technik und der indirekten Immunfluoreszenz Technik wurde die Expression der Antigene CD 20, CD 23, CD 40, CD 27, IgG, IgA, IgM, Kappa, Lambda, CD 3, CD 4, CD 8, Ki M4, CD 68, Ki 67 sowie die Expression der Cytokine IL 2 und IL 10 analysiert. Die Synovialmembran zeigte histologisch eine Verbreiterung der Deckzellschicht, Knorpelfragmente innerhalb der Synovialmembran und ein insgesamt schwach ausgeprägtes entzündliches Infiltrat. In lediglich drei von 20 Fällen fand sich eine stärkere entzündliche Infiltration. Diese entzündlichen Infiltrate wiesen eine perivaskuläre Verteilung auf. Am häufigsten wurden in gefäßnahen Regionen B Lymphozyten identifiziert, Plasmazellen wiesen in der Regel einen deutlich größeren Abstand zum Gefäß auf. Unter den nachgewiesenen Plasmazellen fand sich eine prädominante Expression an IgG bei ausgewogener Anwesenheit sowohl der Kappa- als auch der Lambda- Leichtketten. T Lymphozyten waren ebenfalls zirkulär um die Gefäße anzutreffen und zeigten eine prädominante Interleukin 10 Expression. Lymphozytäre Aggregate, mit follikelähnlicher Struktur ließen sich in lediglich in 4 von 20 Fällen nachweisen. Makrophagen waren sowohl perivaskulär als auch in der Deckzellschicht nachweisbar. Ki M4 positive Retikulumzellen (FDC) waren dagegen nur in einem von 20 Fällen nachweisbar. Alle Zellpopulationen der Membrana synovialis wiesen nur eine schwache Proliferationsaktivität auf. Das Fehlen von dem Keimzentrum des Lymphfollikels vergleichbaren Strukturen, die deutliche Abwesenheit von Ki M4 positiver FDC's sowie die schwache Expression von Ki 67, sprechen trotz Anwesenheit der ebenfalls zur Antigenpräsentation befähigten Makrophagen gegen eine Einwanderung und Maturation nativer B Lymphozyten in die Membrana Synovialis. Wandern dagegen Gedächtniszellen in die Membrana synovialis ein, so ist eine Maturation mit Follikelbildung nicht mehr notwendig. Unter der Mithilfe von T Lymphozyten und Makrophagen können die B Lymphozyten zu Plasmazellen differenzieren. T Lymphozyten zeichnen sich ebenfalls durch eine starke perivaskuläre Verteilung aus. Dabei ist die Expression von IL 10 prädominant, was sich als eine Immunantwort von TH2-Typus interpretieren läßt. Diese ermöglicht eine Differenzierung der B Lymphozyten zu Plasmazellen. Reife B Lymphozyten, die unter dem Einfluß einer TH2 Subpopulation von CD 4 positiven T Lymphozyten ohne Keimzentrum zu Plasmazellen differenzieren, könnten ein Grund dafür sein, daß follikuläre Strukturen fehlen. Vorgereifte B Lymphozyten benötigen auch keine inflammatorisch hochpotenten Zytokine um eine schnelle Reifung und eine Immunantwort zu ermöglichen. Dies könnte ein Grund sein, warum die entzündliche Reaktion bei Arthrose so schwach ausgeprägt ist. / Inflammation in osteoarthritis is a secondary reaction to a degenerating process of the articular cartilage. Cause of Degeneration can be a disproportion of mechanical stress and resistance or metabolic diseases like diabetes mellitus. This kind of osteoarthritis is called "secondary osteoarthritis". Primary osteoarthritis has an unknown cause. Age and sex of the patient are a predictor for osteoarthritis, hense it is a disease of people above the age of 50 and more often it is found in women than in men. This paper investigated the synovial membranes of twenty patients to characterize the inflammatory Infiltrate. It characterized the cell surface antigen CD 20, CD 23, CD 40, CD 27, CD 3, CD 4, CD 8, Ki M4, CD 68, the antibodies IgG, IgA, IgM, Kappa, Lambda, the proliferating antigen Ki 67 and the expression profile of the cytokines IL 2 and IL 10 by using immunohistochemical staining (indirect immunoperoxidase technique and indirect immunofluorescence technique) with monoclonal antibodies. The synovial membrane shows in histology a dissemination of cover cells, fragments of cartilage and a slight expression of inflammatory infiltrate with a perivascular allocation. In only three of twenty cases we detected stronger inflammatory infiltrates. Most of the perivascular cells express CD 20. They are B lymphocytes. Plasma cells have more distance to the blood vessels and showed a predominant expression of IgG. T-lymphocytes were also detected perivascular. The expression of IL 10 was predominant. Lymphocytes aggregates like lymph follicle were detected in four of twenty cases. Macrophages were proved perivascular as well as in the cover cells. Ki M4 positive reticulum cells were found in only one of twenty cases. All kind of cells in the synovial membrane showed a low proliferation activity. The absence of germinal centers or comparable structures, the low expression of Ki M4 and Ki 67 speak against the immigration and maturation of native B lymphocytes in the synovial membrane. Memory B-lymphocytes don't need germinal centers or compatible structures for maturation, they can mature to plasma cells by help of T-lymphocytes, macrophages or other B-lymphocytes. It is more probably that the detected B lymphocytes are memory cells. The perivascular T lymphocytes in combination with the predominant expression of IL 10 may be interpreted as a TH2 immune reaction. This supports the maturation of B-lymphocytes to plasma cells. The maturation of memory B-lymphocytes under influence of TH2 immune reaction can be the reason for the missing of germinal centers or comparable structures. Matured B-lymphocytes don't need high-grade inflammatory cytokines for quick immune response. This is the possible reason for the low-grade inflammatory reaction of osteoarthritis.
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Avaliação da reconstituição imunológica e da resposta anti-citomegalovirus nos receptores de transplante de medula óssea / Anti-cytomegalovirus immunity reconstitution following autologous and allogeneic stem cell and bone marrow transplantation as assessed by CD8+ T cell phenotyping and functioFerrari, Valeria 23 February 2005 (has links)
O citomegalovírus (CMV) é uma séria ameaça aos receptores de transplante de medula óssea. A reativação está associada com uma imunidade mediada por células TCD8+ defeituosa. Nosso objetivo foi correlacionar as diferentes subpopulações de células TCD8+ com a reconstituição imunológica dos pacientes, especificamente a imunidade anti-CMV, analisando as subpopulações de células T infundidas nas diferentes modalidades de transplante de medula óssea. Receptores de transplante alogênico de células tronco mobilizadas para o sangue periférico (n=16) ou coletadas diretamente da medula óssea (n=28) e receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico (n=22) foram avaliados. Verificamos que as transferências de células mobilizadas para o sangue periférico dos doadores, tanto nos transplantes alogênicos como autólogos, são proporcionalmente enriquecidas por subpopulações de células memória efetora e efetora, comparadas às transferências de células procedentes diretamente da medula óssea. Este enriquecimento por subpopulações de células TCD8+ mais diferenciadas foi também correlacionado com maior número de células contendo altos níveis de granzima B, considerado um marcador para linfócitos citotóxicos, sendo também encontrado em maior número nas transferências de células do sangue periférico. Entretanto, no pós-transplante, observou-se que somente os receptores de transplante autólogo de células tronco mobilizadas para o sangue periférico, e não os das outras modalidades de transplante, exibiam números elevados de células T CD8+ de memória-efetora e efetora. Ao mesmo tempo, estes receptores apresentaram menos freqüentemente episódios de reativação pelo CMV, e mais freqüentemente produziram IFN-gama em resposta ao CMV. Portanto, a transferência de células do sangue periférico, desde que em ambiente autólogo, está associada não só com a transferência de células TCD8+ com um fenótipo mais maduro, mas também com uma persistência mais prolongada das mesmas, podendo proporcionar uma resposta imunológica antiviral mais rápida e eficiente, como esperado para as células de memória versus naïve. / Cytomegalovirus (CMV) is a serious threat to the recipients of bone marrow transplantation. Reactivation is associated with defective CD8+ T cell-mediated immunity. We aimed to correlate the different subsets of CD8+ T cells with the patients\' immune reconstitution, specifically anti CMV immunity, by analyzing the CD8+ T cell subsets infused in the different types of bone marrow transplantation. Recipients of allogeneic transplant of peripheral blood stem cells (n=16) or bone marrow (n=28) and recipients of autologous transplant of peripheral blood stem cells (n=22) were evaluated. We show that infusions of stem cells derived from donor\'s peripheral blood, either allogeneic or autologous, are proportionally enriched for the memory-effector and effector phenotypes, compared to the infusions of stem cells of bone marrow origin. This increased number of more differentiated subsets of CD8+ T cells was also correlated with an increased number of cells containing high levels of granzyme B, which is another reliable marker of cytotoxic lymphocyte, and which was also more evident in autologous recipients. However, post-transplant, we observed that only the recipients of autologous peripheral blood cells, and not the recipients of the other transplant modalities, exhibited very high numbers of memory-effector and effector TCD8+ cells. At the same time, they less frequently presented CMV reactivation, and more frequently produced IFN-gama in response to CMV antigens. Thus, transfer of stem cells from peripheral blood, provided in an autologous setting, is associated with transfer and prolonged survival of CD8+ T cells with a more mature phenotype, which may provide a more rapid and efficient anti-viral immune response, as expected for memory versus naïve cells.
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Etude des mécanismes de rupture de tolérance lymphocytaire au cours des déficits immunitaires primitifs de l'adulte avec manifesations auto-immunes / Study of lymphocyte tolerance breakdown in adults primary immunodeficiencies with autoimmunityGuffroy, Aurélien 01 April 2019 (has links)
L’association entre déficits immunitaires primitifs (DIPs) et manifestations auto-immunes peut sembler paradoxale lorsque l’on aborde les DIPs comme des défauts d’immunité opposés à l’autoimmunité vue comme excès d’immunité adaptative à l’encontre du soi. Néanmoins, loin de se résumer à un simple défaut d’une ou plusieurs composantes du système immunitaire qui prédispose aux infections par divers agents pathogènes, les DIPs sont fréquemment associés à une autoimmunité; parfois révélatrice. Ainsi, les données épidémiologiques issues de registres ou de larges séries de patients atteints de DIPs s’accordent sur une prévalence globale de 25 à 30% de complications auto-immunes (au premier rang desquelles figurent les cytopénies auto-immunes). Différentes hypothèses sont avancées pour rendre compte de l’auto-immunité dans les DIPs. On peut citer : 1°) une perturbation profonde de l’homéostasie lymphocytaire, en particulier dans les déficits immunitaires combinés sévères (CID) avec lymphopénies T et B ; 2°) des défauts intrinsèques des lymphocytes B permettant une rupture de tolérance précoce des LB auto réactifs ; 3°) un comportement aberrant des LT (défaut de maturation, excès d’activation) ; 4°) une absence de lymphocytes T ou de B régulateurs ; 5°) une production inappropriée de certaines cytokines proinflammatoires comme dans les interféronopathies. Ces hypothèses concernent surtout les DIPs pédiatriques sévères. Mon travail de thèse explore la rupture de tolérance immunitaire adaptative au cours des DIPs de l’adulte par différentes approches. Nous nous sommes en particulier attachés au plus fréquent, le DICV (Déficit Immunitaire Commun Variable), déficit immunitaire humoral pas toujours bien défini sur le plan génétique et physiopathologique qui constitue un défi thérapeutique lorsqu’il est compliqué d’une auto-immunité nécessitant un traitement immunosuppresseur. / The association between primary immune deficiency (PID) and autoimmunity may seem paradoxical when PID is considered only as an immune response defect against pathogens and autoimmunity only as an excess of immunity. Nevertheless, far from being simple immune defects increasing the risk of infections, DIPs are frequently associated with autoimmunity. Even more, autoimmunes manifestations can sometimes reveal a PID. Thus, epidemiological data from registers or large series of patients with PIDs agree on an overall prevalence of 25 to 30% of autoimmune complications (with auto-immune cytopenias as first causes). Several hypotheses have been proposed with different underlying mechanisms to explain the tolerance breakdown in PIDs. We can cite : 1°) a severe disturbance of lymphocyte homeostasis, for example in severe combined immunodeficiencies ; 2°) an impaired B-cell developpement with earlystage defects of tolerance ; 3°) a dysregulation of T cells (developpement or activation impairments) ; 4°) a dysfunction of T-reg (or B-reg) ; 5°) an excess of production of proinflammatory cytokines. These hypotheses are especially true for early-onset PIDs (in infancy). In this work (PhD), we explore the mechanisms of tolerance breakdown involved in adults PIDs. We use several approaches to describe the pathways leading to autoimmunity, focusing on the most common PID in adult : CVID (common variable immunodeficiency). This syndrome is not well defined on the genetic and physiopathological level. It is still a therapeutic challenge when complicated by autoimmunity (requiring immunosuppressive therapy).
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Chicken infectious anemia virus vaccination induces immune disorders and viral persistency in infectious bursal disease virus-infected young chicksVaziry, Asaad 08 1900 (has links)
La bursite infectieuse aviaire (IBD) est une des causes majeures de pertes économiques pour l’industrie aviaire. La vaccination est le principal outil de contrôle de cette maladie et les oiseaux susceptibles doivent être vaccinés aussitôt que le niveau des anticorps maternels (MA) anti-IBDV est suffisamment bas. L’estimation du moment de vaccination est habituellement déterminée par la formule de Deventer qui utilise le titre initial de MA anti-IBDV et la demi-vie des anticorps pour prédire l’évolution du titre. Dans la présente étude, l’effet du gain de poids sur la vitesse de disparition des MA a été étudié dans le but de l’utiliser pour prédire la détermination du moment de la vaccination. L’analyse des taux d’anticorps neutralisants par ELISA a montré que les poussins avec une forte croissance avaient un taux de disparition plus rapide des MA que ceux à faible croissance. Une formule pour la prédiction du moment de vaccination contre le IBDV, basée sur le gain de poids et le niveau des MA a été développée et vérifiée. La prédiction du moment de vaccination avec cette formule a montré une haute corrélation avec les titres de MA mesurés par ELISA.
Le virus de l’anémie infectieuse aviaire (CIAV) est une cause importante d’immunosuppression chez le poulet augmentant la pathogénicité des infections secondaires et en entraînant une réponse humorale suboptimale et une forte mortalité. D’autre part, l’infections sub-clinique du au CIAV provoque une immunosuppression qui facilite la coinfection par d’autre virus tel que le IBDV. Les effets de la coinfection à J1 avec une souche vaccinale de CIAV CAV-VAC® (Intervet) et à J14 avec une souche faiblement virulente de IBDV isolée au Québec, sur l’état de santé des poussins, sur la persistance virale et sur la réponse immunitaire ont été étudiés autant chez des poussins de 1 jour d’âge exempts d’agents pathogènes specifique (SPF) que ceux provenant d’élevages commerciaux. Les résultats ont montré que l’inoculation de la souche vaccinale du CIAV a entraîné une infection sub-clinique, une persistance virale dans la rate et le thymus, une altération de la thymopoièse et une réponse humorale temporaire chez les poussins SPF. Ces effets ont aussi été mis en évidence chez des poussins d’élevage commerciaux malgré des taux élevés de MA. Lors de l’infection avec la souche de IBDV chez des poussins déjà vaccinés contre le CIAV, la persistance du CIAV dans les organes lymphoïdes a été aggravée par une présence de réponses humorales temporaires contre les deux virus et une altération des populations lymphocytaires dans les organes lymphoïdes. Par contre, la présence des MA contre le CIAV a limité temporairement ces effets. Ces travaux ont mis en évidence des désordres immunitaires cellulaires et humoraux et une persistance virale chez des poussins vaccinés contre le CIAV et co-infectés avec le IBDV. / Infectious bursal disease (IBD) is one of the major causes of economic losses in the chicken industry. Vaccination is the main tool against the disease, and the susceptible birds should be vaccinated as soon as the maternal antibody (MA) becomes low enough to allow the vaccine to break through. Estimation of vaccination time is currently performed by Deventer formula which uses initial anti-IBDV titer and antibody half-life to predict the titer. Considering the increased growth rate of chicken in the last decades and the wide variations of MA, we have examined the effects of chick’s weight gain on MA decline and the use of weight in predicting IBD vaccination time. The virus neutralization test and ELISA results demonstrated that fast-growing birds had a faster rate of antibody decline whereas slow-growing birds demonstrated a slower rate. Based on the effect of weight-gain on maternal antibody decline, a new formula for predicting IBD vaccination time was introduced and tested. The predicted IBD vaccination time made by this weight formula showed higher correlation with the measured ELISA titers in the experiment.
Chicken infectious anemia virus (CIAV) is another cause of immunosuppression in chicken which is characterized by increased pathogenicity of secondary infectious agents, sub-optimal antibody responses and mortality. CIAV subclinical infections can result in immunosuppression and enhancement of pathogenicity of co-infecting agents such as infectious bursal disease virus (IBDV). Effects of pathogenic CIAV and IBDV coinfection on chick’s health and immune responses are investigated in different studies. In this study, newly hatched specific pathogen free (SPF) and commercial chicks were vaccinated with CAV-VAC® (Intervet) vaccine and /or inoculated with a low-virulent Québec isolate of IBDV at 14 days post CIAV vaccination.
Inoculation of the CIAV vaccinal strain at hatch resulted in subclinical infection associated with viral persistency in spleen and thymus, alteration of thymopoiesis and transient humoral response in SPF chicks. Subclinical infection, viral persistency and lack of antibody responses were also shown in CIAV inoculated commercial chicks with high MA. Infection of the low-virulent IBDV in the CIAV vaccinated SPF chicks lead to extended viral persistence of CIAV in lymphoid organs, transient immune responses to both CIAV and IBDV, and alteration of lymphocytes subpopulation in the lymphoid organs. In the coinfected commercial chicks, presence the CIAV in the lymphoid organs was controlled by MA in the first 1-2 weeks after hatch. Thereafter, the immune disorders, viral persistence and lack of humoral responses almost similar to the coinfected SPF chicks were recorded.
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Importance of the HSP90 molecular chaperoning pathway for antibody diversification by determining AID stabilityOrthwein, Alexandre 01 1900 (has links)
La protéine AID (déaminase induite par l’activation) joue un rôle central dans la
réponse immunitaire adaptative. En désaminant des désoxycytidines en désoxyuridines au
niveau des gènes immunoglobulines, elle initie l’hypermutation somatique (SHM), la
conversion génique (iGC) et la commutation isotypique (CSR). Elle est essentielle à une
réponse humorale efficace en contribuant à la maturation de l’affinité des anticorps et au
changement de classe isotypique. Cependant, son activité mutagénique peut être oncogénique et
causer une instabilité génomique propice au développement de cancers et de maladies
autoimmunes. Il est donc critique de réguler AID, en particulier ses niveaux protéiques, pour
générer une réponse immunitaire efficace tout en minimisant les risques de cancer et d’autoimmunité.
Un élément de régulation est le fait qu’AID transite du cytoplasme vers le noyau
mais reste majoritairement cytoplasmique à l’équilibre. AID est par ailleurs plus stable dans le
cytoplasme que dans le noyau, ce qui contribue à réduire sa présence à proximité de l’ADN.
Le but de cette thèse était d’identifier de nouveaux partenaires et déterminants d’AID
régulant sa stabilité et ses fonctions biologiques. Dans un premier temps, nous avons identifié
AID comme une nouvelle protéine cliente d’HSP90. Nous avons montré qu’HSP90 interagit
avec AID dans le cytoplasme, ce qui empêche la poly-ubiquitination d’AID et sa dégradation
par le protéasome. En conséquence, l’inhibition d’HSP90 résulte en une diminution
significative des niveaux endogènes d’AID et corrèle avec une réduction proportionnelle de ses
fonctions biologiques dans la diversification des anticorps mais aussi dans l’introduction de
mutations aberrantes. Dans un second temps, nous avons montré que l’étape initiale dans la
stabilisation d’AID par la voie de chaperonnage d’HSP90 dépend d’HSP40 et d’HSP70. En
particulier, la protéine DnaJa1, qui fait partie de la famille des protéines HSP40s, limite la
stabilisation d’AID dans le cytoplasme. La farnésylation de DnaJa1 est importante pour
l’interaction entre DnaJa1 et AID et moduler les niveaux de DnaJa1 ou son état de farnésylation
impacte à la fois les niveaux endogènes d’AID mais aussi la diversification des anticorps. Les
souris DNAJA1-/- présentent une réponse immunitaire compromise en cas d’immunisation, qui
est dûe à des niveaux réduits d’AID et un défaut de commutation de classe. Dans un troisième
temps, nous avons montré que la protéine AID est intrinsèquement plus instable que sesprotéines paralogues APOBEC. Nous avons identifié l’acide aspartique en seconde position
d’AID ainsi qu’un motif semblable au PEST comme des modulateurs de la stabilité d’AID. La
modification de ces motifs augmente la stabilité d’AID et résulte en une diversification des
anticorps plus efficace.
En conclusion, l’instabilité intrinsèque d’AID est un élément de régulation de la
diversification des anticorps. Cette instabilité est en partie compensée dans le cytoplasme par
l’action protective de la voie de chaperonnage DnaJa1-HSP90. Par ailleurs, l’utilisation
d’inhibiteurs d’HSP90 ou de farnésyltransférases pourrait être un outil intéressant pour la
modulation indirecte des niveaux d’AID et le traitement de lymphomes/leucémies et de
maladies auto-immunes causés par AID. / Activation induced deaminase (AID) plays a central role in adaptive immunity. AID
deaminates deoxycytidine to deoxyuridine in defined regions of the immunoglobulin (Ig) genes
and initiates somatic hypermutation (SHM), gene conversion (iGC) and class switch
recombination (CSR). While being essential for an effective immune response by underpinning
antibody affinity maturation and isotype switching, the mutagenic activity of AID can also be
oncogenic and causes genomic instability leading to the development of cancer, or exacerbate
autoimmune diseases. Therefore, AID regulation, including the control of its protein level, is
central to balancing effective immunity with cancer/autoimmunity. Notably, AID shuttles
between the cytoplasm and the nucleus but is predominantly cytoplasmic at steady-state, with
cytoplasmic AID being much more stable than nuclear AID. These regulatory steps contribute
to limit the exposure of the genome to AID but their mechanisms are unknown.
This thesis aimed at identifying AID partners and intrinsic determinants regulating its
stability and modulating its biological functions. Firstly, we identified AID as a novel HSP90
client protein. We demonstrated that HSP90 interacts with AID in the cytoplasm and prevents
its polyubiquitination and subsequent proteasomal degradation. Consequently, HSP90
inhibition results in a significant reduction of endogenous AID levels and correlates with a
proportional reduction in both AID-mediated antibody diversification and off-target mutations.
Secondly, we showed that the first step in the HSP90 molecular chaperoning pathway and
stabilization is the interaction of AID with the HSP40 and HSP70 system. In fact, a specific
HSP40 protein, DnaJa1, is the limiting step in cytoplasmic AID stabilization. DnaJa1
farnesylation is required for DnaJa1-AID interaction and modulation of DnaJa1 levels or its
farnesylation impacts endogenous AID levels and antibody diversification. In vivo, DnaJa1-
deficient mice display compromized response to immunization, resulting from reduced AID
protein levels and isotype switching. Thirdly, we found that AID is intrinsically less stable
than its APOBEC paralogs. We identified the AID N-terminal aspartic acid residue at position
two and an internal PEST-like motif as destabilizing modulators of AID protein turnover.
Disruption of these motifs increases AID protein stability and antibody diversification.We conclude that AID’s intrinsic instability directly contributes to regulating antibody
diversification. This intrinsic instability is at least partially compensated for in the cytoplasm by
the protective action of the DnaJa1-HSP90 molecular chaperoning pathway. Pharmacologically
targeting AID in an indirect way, by using HSP90 or farnesyltransferase inhibitors, could be
relevant for treating some AID-associated lymphomas/leukemias and/or autoimmune diseases.
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