933 resultados para Lymphocytes T CD4
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The expression of the cell adhesion molecules ICAM-1, ICAM-2, and VCAM-1 and the secretion of the cytokine interleukin 6 have been measured in mouse Sertoli cells cultured in vitro. Cytometric analysis revealed that, in basal conditions, low levels of ICAM-1 and VCAM-1 were present on the surface of the cells, whereas treatment with interleukin 1, tumor necrosis factor alpha, lipopolysaccharide, or interferon gamma induced, with different kinetics, increases in their expression. ICAM-2 was not detectable in basal conditions, nor was it inducible. Electron microscopic analysis and binding experiments using 51Cr-labeled lymphocytes demonstrated that increased expression of ICAM-1 and VCAM-1 on the surface of Sertoli cells, induced by inflammatory mediators, determines an augmented adhesion between the two cell types. The same stimuli, with the exception of interferon gamma, produced a rapid and remarkable increment of interleukin 6 production by Sertoli cells. These results suggest the presence of both direct and paracrine mechanisms of interaction between Sertoli and immune-competent cells, possibly involved in the control of immune reactions in the testis. Such mechanisms are of interest for the understanding of autoimmune pathologies of the testis and, if confirmed in humans, they could be involved in the sexual transmission of human immunodeficiency virus infection.
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Epstein-Barr virus (EBV) is a human DNA tumor virus that efficiently immortalizes human primary B lymphocytes in vitro. Although viral genes that are expressed in latently infected B lymphocytes have been shown to function in cellular growth control, their detailed genetic analysis has been cumbersome for two reasons. The viral genome is too large to permit genetic engineering and human primary B lymphocytes, the only targets for infection by EBV in vitro, are both intractable in culture and recalcitrant to DNA transfection. To overcome these obstacles, we have assembled all the essential genes of EBV on a single recombinant vector molecule in Escherichia coli. We show here that this mini-EBV plasmid can yield immortalized B cells upon transfer of its naked DNA into human primary B lymphocytes. Established cell lines carry recombinant vector DNA and cannot support virus production. Because this DNA can be easily manipulated in E. coli, mutant mini-EBVs as well as foreign genes can now be introduced and studied successfully in recipient B lymphocytes from any human donors. These mini-EBVs therefore are potentially useful for human gene therapy.
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Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.
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The incidence of tuberculosis is increasing on a global scale, in part due to its strong association with human immunodeficiency virus (HIV) infection. Attachment of Mycobacterium tuberculosis to its host cell, the alveolar macrophage (AM), is an important early step in the pathogenesis of infection. Bronchoalveolar lavage of HIV-infected individuals demonstrated the presence of a factor which significantly enhances the attachment of tubercle bacilli to AMs 3-fold relative to a normal control population. This factor is surfactant protein A (SP-A). SP-A levels are increased in the lungs of HIV-infected individuals. SP-A levels and attachment of M. tuberculosis to AMs inversely correlate with peripheral blood CD4 lymphocyte counts. Elevated concentrations of SP-A during the progression of HIV infection may represent an important nonimmune risk factor for acquiring tuberculosis, even before significant depletion of CD4 lymphocytes in the peripheral blood occurs.
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We present data on the decay, after radiotherapy, of naive and memory human T lymphocytes with stable chromosome damage. These data are analyzed in conjunction with existing data on the decay of naive and memory T lymphocytes with unstable chromosome damage and older data on unsorted lymphocytes. The analyses yield in vivo estimates for some life-history parameters of human T lymphocytes. Best estimates of proliferation rates have naive lymphocytes dividing once every 3.5 years and memory lymphocytes dividing once every 22 weeks. It appears that memory lymphocytes can revert to the naive phenotype, but only, on average, after 3.5 years in the memory class. The lymphocytes with stable chromosome damage decay very slowly, yielding surprisingly low estimates of their death rate. The estimated parameters are used in a simple mathematical model of the population dynamics of undamaged naive and memory lymphocytes. We use this model to illustrate that it is possible for the unprimed subset of a constantly stimulated clone to stay small, even when there is a large population of specific primed cells reverting to the unprimed state.
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Several lines of evidence indicate that immunoglobulin-bound prolactin found in human serum is not a conventional complex between an anti-prolactin antibody and prolactin but a different type of association of prolactin with the Fab portion of IgG heavy chains. The complex of prolactin with IgG was purified from serum by anti-human prolactin affinity chromatography and was shown to contain close to 1 mole of N epsilon-(gamma-glutamyl)lysine crosslinks per mole of complex, a characteristic feature in structures crosslinked by transglutaminase. Interestingly, the complex caused a proliferation of cells from a subset of patients with chronic lymphocytic leukemia, while it was inactive in a cell proliferation prolactin bioassay. By contrast, human prolactin stimulated the proliferation of cells in the bioassay but had no effect on the complex-responsive cells from the patients. Competition studies with prolactin and free Fc fragment of IgG demonstrated a necessity for engaging both the prolactin and the immunoglobulin receptors for proliferation. More importantly, competition for the growth response by free prolactin and IgG suggests both possible reasons for the slow growth of this neoplasm as well as avenues for control of the disease.
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The phenotype and antigenic specificity of cells secreting interleukin (IL) 4, IL-6, and interferon gamma was studied in mice during primary and secondary immune responses. T lymphocytes were the major source of interferon gamma, whereas non-B/non-T cells were the dominant source of IL-4 and IL-6 in the spleens of immunized animals. Cytokine-secreting non-B/non-T cells expressed surface receptors for IgE and/or IgG types II/III. Exposing these cells to antigen-specific IgE or IgG in vivo (or in vitro) "armed" them to release IL-4 and IL-6 upon subsequent antigenic challenge. These findings suggest that non-B/non-T cells may represent the "natural immunity" analogue of CD4+ T helper type 2 cells and participate in a positive feedback loop involved in the perpetuation of T helper type 2 cell responses.
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SUMMARY The Porcine Reproductive and Respiratory Syndrome (PRRS) virus is one of the most spread pathogens in swine herds all over the world and responsible for a reproductive and respiratory syndrome that causes severe heath and economical problems. This virus emerged in late 1980s but although about 30 years have passed by, the knowledge about some essential facets related to the features of the virus (pathogenesis, immune response, and epidemiology) seems to be still incomplete. Taking into account that the development of modern vaccines is based on how innate and acquire immunity react, a more and more thorough knowledge on the immune system is needed, in terms of molecular modulation/regulation of the inflammatory and immune response upon PRRSV infection. The present doctoral thesis, which is divided into 3 different studies, is aimed to increase the knowledge about the interaction between the immune system and the PRRS virus upon natural infection. The objective of the first study entitled Coordinated immune response of memory and cytotoxic T cells together with IFN- secreting cells after porcine reproductive and respiratory syndrome virus (PRRSV) natural infection in conventional pigs was to evaluate the activation and modulation of the immune response in pigs naturally infected by PRRSV compared to an uninfected control group. The course of viremia was evaluated by PCR, the antibody titres by ELISA, the number of IFN- secreting cells (IFN- SC) by an ELISPOT assay and the immunophenotyping of some lymphocyte subsets (cytotoxic cells, memory T lymphocytes and cytotoxic T lymphocytes) by flow cytometry. The results showed that the activation of the cell-mediated immune response against PRRSV is delayed upon infection and that however the levels of IFN- SC and lymphocyte subsets subsequently increase over time. Furthermore, it was observed that the course of the different immune cell subsets is time-associated with the levels of PRRSV-specific IFN- SC and this can be interpreted based on the functional role that such lymphocyte subsets could have in the specific production/secretion of the immunostimulatory cytokine IFN-. In addition, these data support the hypothesis that the age of the animals upon the onset of infection or the diverse immunobiological features of the field isolate, as typically hypothesized during PRRSV infection, are critical conditions able to influence the qualitative and quantitative course of the cell-mediated immune response during PRRSV natural infection. The second study entitled Immune response to PCV2 vaccination in PRRSV viremic piglets was aimed to evaluate whether PRRSV could interfere with the activation of the immune response to PCV2 vaccination in pigs. In this trial, 200 pigs were divided into 2 groups: PCV2-vaccinated (at 4 weeks of age) and PCV2-unvaccinated (control group). Some piglets of both groups got infected by PRRSV, as determined by PRRSV viremia detection, so that 4 groups were defined as follows: PCV2 vaccinated - PRRSV viremic PCV2 vaccinated - PRRSV non viremic PCV2 unvaccinated - PRRSV viremic PCV2 unvaccinated - PRRSV non viremic The following parameters were evaluated in the 4 groups: number of PCV2-specific IFN- secreting cells, antibody titres by ELISA and IPMA. Based on the immunological data analysis, it can be deduced that: 1) The low levels of antibodies against PCV2 in the PCV2-vaccinated PRRSV-viremic group at vaccination (4 weeks of age) could be related to a reduced colostrum intake influenced by PRRSV viremia. 2) Independently of the viremia status, serological data of the PCV2-vaccinated group by ELISA and IPMA does not show statistically different differences. Consequently, it can be be stated that, under the conditions of the study, PRRSV does not interfere with the antibody response induced by the PCV2 vaccine. 3) The cell-mediated immune response in terms of number of PCV2-specific IFN- secreting cells in the PCV2-vaccinated PRRSV-viremic group seems to be compromised, as demonstrated by the reduction of the number of IFN- secreting cells after PCV2 vaccination, compared to the PCV2-vaccinated PRRSV-non-viremic group. The data highlight and further support the inhibitory role of PRRSV on the development and activation of the immune response and highlight how a natural infection at early age can negatively influence the immune response to other pathogens/antigens. The third study entitled Phenotypic modulation of porcine CD14+ monocytes, natural killer/natural killer T cells and CD8+ T cell subsets by an antibody-derived killer peptide (KP) was aimed to determine whether and how the killer peptide (KP) could modulate the immune response in terms of activation of specific lymphocyte subsets. This is a preliminary approach also aimed to subsequently evaluate such KP with a potential antivural role or as adjuvant. In this work, pig peripheral blood mononuclear cells (PBMC) were stimulated with three KP concentrations (10, 20 and 40 g/ml) for three time points (24, 48 and 72 hours). TIME POINTS (hours) KP CONCENTRATIONS (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 By using flow cytometry, the qualitative and quantitative modulation of the following immune subsets was evaluated upon KP stimulation: monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and CD4+ and CD8/+ T lymphocyte subsets. Based on the data, it can be deduced that: 1) KP promotes a dose-dependent activation of monocytes, particularly after 24 hours of stimulation, by inducing a monocyte phenotypic and maturation shift mainly involved in sustaining the innate/inflammatory response. 2) KP induces a strong dose-dependent modulation of NK and NKT cells, characterized by an intense increase of the NKT cell fraction compared to NK cells, both subsets involved in the antibody-dependent cell cytotoxicity (ADCC). The increase is observed especially after 24 hours of stimulation. 3) KP promotes a significant activation of the cytotoxic T lymphocyte subset (CTL). 4) KP can modulate both the T helper and T cytotoxic phenotype, by inducing T helper cells to acquire the CD8 thus becoming doube positive cells (CD4+CD8+) and by inducing CTL (CD4-CD8+high) to acquire the double positive phenotype (CD4+CD8+high). Therefore, KP may induce several effects on different immune cell subsets. For this reason, further research is needed aimed at characterizing each effect of KP and thus identifying the best use of the decapeptide for vaccination practice, therapeutic purposes or as vaccine adjuvant. RIASSUNTO Il virus della PRRS (Porcine Reproductive Respiratory Syndrome) uno dei pi diffusi agenti patogeni negli allevamenti suini di tutto il mondo, responsabile di una sindrome riproduttiva e respiratoria causa di gravi danni ad impatto sanitario ed economico. Questo virus emerso attorno alla fine degli anni 80 ma nonostante siano passati circa una trentina di anni, le conoscenze su alcuni punti essenziali che riguardano le caratteristiche del virus (patogenesi, risposta immunitaria, epidemiologia) appaiono ancora spesso incomplete. Considerando che lo sviluppo dei vaccini moderni basato sui principi dellimmunit innata e acquisita essenziale una sempre pi completa conoscenza del sistema immunitario inteso come modulazione/regolazione molecolare della risposta infiammatoria e immunitaria in corso di tale infezione. Questo lavoro di tesi, suddiviso in tre diversi studi, ha lintento di contribuire allaumento delle informazioni riguardo linterazione del sistema immunitario, con il virus della PRRS in condizioni di infezione naturale. Lobbiettivo del primo studio, intitolato Associazione di cellule memoria, cellule citotossiche e cellule secernenti IFN- nella risposta immunitaria in corso di infezione naturale da Virus della Sindrome Riproduttiva e Respiratoria del Suino (PRRSV) stato di valutare lattivazione e la modulazione della risposta immunitaria in suini naturalmente infetti da PRRSV rispetto ad un gruppo controllo non infetto. I parametri valutati sono stati la viremia mediante PCR, il titolo anticorpale mediante ELISA, il numero di cellule secernenti IFN- (IFN- SC) mediante tecnica ELISPOT e la fenotipizzazione di alcune sottopopolazioni linfocitarie (Cellule citotossiche, linfociti T memoria e linfociti T citotossici) mediante citofluorimetria a flusso. Dai risultati ottenuti stato possibile osservare che lattivazione della risposta immunitaria cellulo-mediata verso PRRSV appare ritardata durante linfezione e che landamento, in termini di IFN- SC e dei cambiamenti delle sottopopolazioni linfocitarie, mostra comunque degli incrementi seppur successivi nel tempo. E stato inoltre osservato che gli andamenti delle diverse sottopopolazioni immunitarie cellulari appaiono temporalmente associati ai livelli di IFN- SC PRRSV-specifiche e ci potrebbe essere interpretato sulla base del ruolo funzionale che tali sottopopolazioni linfocitarie potrebbero avere nella produzione/secrezione specifica della citochina immunoattivatrice IFN-. Questi dati inoltre supportano lipotesi che let degli animali alla comparsa dellinfezione o, come tipicamente ipotizzato nellinfezione da PRRSV, le differenti caratteristiche immunobiologiche dellisolato di campo, sia condizioni critiche nell influenzare landamento qualitativo e quantitativo della risposta cellulo-mediata durante linfezione naturale da PRRSV. Il secondo studio, dal titolo Valutazione della risposta immunitaria nei confronti di una vaccinazione contro PCV2 in suini riscontrati PRRSV viremici e non viremici alla vaccinazione ha avuto lo scopo di valutare se il virus della PRRS potesse andare ad interferire sullattivazione della risposta immunitaria indotta da vaccinazione contro PCV2 nel suino. In questo lavoro sono stati arruolati 200 animali divisi in due gruppi, PCV2 Vaccinato (a 4 settimane di et) e PCV2 Non Vaccinato (controllo negativo). Alcuni suinetti di entrambi i gruppi, si sono naturalmente infettati con PRRSV, come determinato con lanalisi della viremia da PRRSV, per cui stato possibile creare quattro sottogruppi, rispettivamente: PCV2 vaccinato - PRRSV viremico PCV2 vaccinato - PRRSV non viremico PCV2 non vaccinato - PRRSV viremico PCV2 non vaccinato - PRRSV non viremico Su questi quattro sottogruppi sono stati valutati i seguenti parametri: numero di cellule secernenti IFN- PCV2 specifiche, ed i titoli anticorpali mediante tecniche ELISA ed IPMA. Dallanalisi dei dati immunologici derivati dalle suddette tecniche stato possibile dedurre che: I bassi valori anticorpali nei confronti di PCV2 del gruppo Vaccinato PCV2-PRRSV viremico gi al periodo della vaccinazione (4 settimane di et) potrebbero essere messi in relazione ad una ridotta assunzione di colostro legata allo stato di viremia da PRRSV Indipendentemente dallo stato viremico, i dati sierologici del gruppo vaccinato PCV2 provenienti sia da ELISA sia da IPMA non mostrano differenze statisticamente significative. Di conseguenza possibile affermare che in questo caso PRRSV non interferisce con la risposta anticorpale promossa dal vaccino PCV2. La risposta immunitaria cellulo-mediata, intesa come numero di cellule secernenti IFN- PCV2 specifiche nel gruppo PCV2 vaccinato PRRS viremico sembra essere compromessa, come viene infatti dimostrato dalla diminuzione del numero di cellule secernenti IFN- dopo la vaccinazione contro PCV2, comparata con il gruppo PCV2 vaccinato- non viremico. I dati evidenziano ed ulteriormente sostengono il ruolo inibitorio del virus della PRRSV sullo sviluppo ed attivazione della risposta immunitaria e come un infezione naturale ad et precoci possa influenzare negativamente la risposta immunitaria ad altri patogeni/antigeni. Il terzo studio, intitolato Modulazione fenotipica di: monociti CD14+, cellule natural killer (NK), T natural killer (NKT) e sottopopolazioni linfocitarie T CD4+ e CD8+ durante stimolazione con killer peptide (KP) nella specie suina ha avuto come scopo quello di stabilire se e come il Peptide Killer (KP) potesse modulare la risposta immunitaria in termini di attivazione di specifiche sottopopolazioni linfocitarie. Si tratta di un approccio preliminare anche ai fini di successivamente valutare tale KP in un potenziale ruolo antivirale o come adiuvante. In questo lavoro, periferal blood mononuclear cells (PBMC) suine sono state stimolate con KP a tre diverse concentrazioni (10, 20 e 40 g/ml) per tre diversi tempi (24, 48 e 72 ore). TEMPI DI STIMOLAZIONE (ore) CONCENTRAZIONE DI KP (g/ml) 24 0-10-20-40 48 0-10-20-40 72 0-10-20-40 Mediante la citometria a flusso stato dunque possibile analizzare il comportamento qualitativo e quantitativo di alcune sottopopolazioni linfocitarie sotto lo stimolo del KP, tra cui: monociti, cellule Natural Killer (NK), cellule T Natural Killer (NKT) e linfociti T CD4 e CD8+. Dai dati ottenuti stato possibile dedurre che: 1) KP promuove unattivazione dei monociti dose-dipendente in particolare dopo 24 ore di stimolazione, inducendo uno shift fenotipico e di maturazione monocitaria maggiormente coinvolto nel sostegno della risposta innata/infiammatoria. 2) KP induce una forte modulazione dose-dipendente di cellule NK e NKT con un forte aumento della frazione delle cellule NKT rispetto alle NK, sottopopolazioni entrambe coinvolte nella citotossicit cellulare mediata da anticorpi (ADCC). Laumento riscontrabile soprattutto dopo 24 ore di stimolazione. 3) KP promuove una significativa attivazione della sottopopolazione del linfociti T citotossici (CTL). 4) Per quanto riguarda la marcatura CD4+/CD8+ stato dimostrato che KP ha la capacit di modulare sia il fenotipo T helper che T citotossico, inducendo le cellule T helper ad acquisire CD8 diventando quindi doppio positive (CD4+CD8+) ed inducendo il fenotipo CTL (CD4-CD8+high) ad acquisire il fenotipo doppio positivo (CD4+CD8+high). Molti dunque potrebbero essere gli effetti che il decapeptide KP potrebbe esercitare sulle diverse sottopopolazioni del sistema immunitario, per questo motivo va evidenziata la necessit di impostare e attuare nuove ricerche che portino alla caratterizzazione di ciascuna abilit di KP e che conducano successivamente alla scoperta del migliore utilizzo che si possa fare del decapeptide sia dal punto di vista vaccinale, terapeutico oppure sotto forma di adiuvante vaccinale.
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O sistema imunolgico materno desempenha um papel importante no estabelecimento da gestao e desenvolvimento de concepto at incio do parto. A hiptese deste projeto que h um recrutamento de clulas Tγδ para o endomtrio ao longo da gestao que expressam citocinas que favorecem o estabelecimento e manuteno da tolerncia materna a antgenos fetais durante a gestao em bovinos. Experimento I Para estudar a dinmica populacional das clulas do sistema imune no sangue perifrico de no lactantes no prenhes (NLNP), vacas lactantes no 1 trimestre e vacas no 3 trimestre da gestao, as PBMCs foram separadas por gradiente de densidade de Ficoll, seguido por protocolo de imunocitoqumica e analisadas em citmetro de fluxo para os anticorpos CD3+, CD4+, CD8+, CD14+, CD25+ e WC1+. As clulas analisadas tanto na regio de linfcitos quanto na regio de moncitos, no apresentaram diferena significativa entre os grupos analisados. Experimento II Para anlise do perfil de expresso gnica das clulas Tγδ, foram coletadas amostras de sangue de vacas no 1 trimestre da gestao, vacas lactantes no prenhes (LNP) e vacas no lactantes no prenhes (NLNP). As clulas mononucleares foram separadas por gradiente de densidade de Ficoll e clulas Tγδ foram analisadas quanto ao perfil de citocinas por qRT-PCR para os genes IFNG; IL10; IL15; IL17; IL18; IL1B; IL4; IL-6; ISG15; PFR; TGFB2 e TNFA. A anlise de expresso gnica mostrou tendncia no aumento na expresso de IL1B, IL6 e TGFB2 em clulas PBMC em vacas NLNP quando comparado com vacas LNP e no 1 trimestre da gestao, enquanto que os outros genes analisados no apresentaram diferena significativa. Experimento III Fragmentos de endomtrio, provenientes de abatedouro, foram coletados de vacas em 1 trimestre (33 a 35 dias de gestao), 2 trimestre (143 a 182 dias de gestao) e 3 trimestre de gestao (228 a 247 dias de gestao). Cortes congelados foram imunolocalizados e quantificados para as clulas do sistema imune CD3+, CD4+, CD8+, CD14+, CD18+, CD25+, CD62L+ e WC1+ por imunofluorescncia. Nossos estudos mostraram aumento das clulas CD25+ e CD62L+ no endomtrio no incio da gestao. No meio da gestao, h um aumento das clulas WC1+ e CD14+. No final da gestao, observamos o aumento de CD14+, CD25+, CD18+ e CD62L+. Em suma, nossos dados sugerem que a modulao do sistema imune materno especfica para cada estgio da gestao, sendo que no incio da gestao h um envolvimento de clulas T ativadas (CD25+) provavelmente para o estabelecimento de uma resposta ativa para tolerncia dos antgenos fetais. J no meio da gestao, h um recrutamento massivo de clulas Tγδ para o endomtrio gravdico provavelmente para manter um microambiente de tolerncia para o desenvolvimento fetal e no final da gestao clulas efetoras como macrfagos so recrutadas para o endomtrio para auxiliar no processo do parto e involuo uterina.
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INTRODUO: Lquen plano (LP) uma doena mucocutnea de natureza inflamatria crnica de etiologia ainda desconhecida. A estimulao da imunidade inata via os receptores Toll-like (TLRs) podem influenciar as clulas dendrticas e direcionar a resposta de clulas T CD4+ e CD8+ efetoras, assim como tambm favorecer o estado inflamatrio do LP. OBJETIVOS: Avaliar o perfil fenotpico de clulas dendrticas mielides (mDCs) e plasmocitides (pDCs) e de linfcitos T CD4+ e CD8+ aps estmulo com agonistas de TLRs no sangue perifrico de pacientes com LP. Alm disto, avaliar a frequncia, perfil de maturao e os subtipos de clulas T CD4+ e TCD8+ reguladores. MTODOS: Foram selecionados 18 pacientes com LP (15 mulheres, 3 homens), com 41,57 4,73 anos de idade e um grupo controle com 22 indivduos sadios (18 mulheres, 4 homens), com 43,92 7,83 anos de idade. As clulas mononucleares (CMNs) de sangue perifrico foram avaliadas por citometria de fluxo quanto : 1) Produo de TNF-? em mDCs e de IFN-? em pDCs em CMNs ativadas por agonistas de TLR 4, 7, 7/8 e 9; 2) Anlise de clulas T CD4+ e CD8+ monofuncionais e polifuncionais aps estmulo com agonistas de TLR 4, 7/8, 9 e enterotoxina B de Staphylococcus aureus (SEB); 3) Avaliao de clulas Th17 e Th22/Tc22 em CMNs aps estmulo com SEB; 4) Frequncia, perfil de maturao e subtipos de clulas T CD4+ e CD8+ reguladoras. RESULTADOS: 1) Nos pacientes com LP foi demonstrado um aumento na frequncia de mDCs TNF-alfa+ aps estmulo com agonistas de TLR4/LPS e TLR7-8/CL097, mas com imiquimode/TLR7 houve diminuio da expresso de CD83. J nas pDCs do grupo LP, o imiquimode foi capaz de diminuir a expresso de CD80 e o CpG/TLR9 diminuiu a expresso de CD83 no LP. 2) As clulas T CD4+ secretoras de IL-10 mostraram aumento da frequncia nos nveis basais, que diminuiu aps estmulo com LPS e SEB. Em contraste, a produo de IFN-y aumentou em resposta ao LPS enquanto diminuiu para CpG. As clulas T CD4+ polifuncionais, secretoras de 5 citocinas simultneas (CD4+IL-17+IL-22+TNF+IL-10+IFN-y+) diminuram no LP aps estmulo com CL097 e CpG. Entretanto, na ausncia de IL-10, houve aumento da frequncia de clulas CD4+IL-17+IL-22+TNF+IFN-y+ em resposta ao LPS. Um aumento na polifuncionalidade foi observado em clulas TCD4+ que expressam CD38, marcador de ativao crnica e na ausncia de IL-10. Similarmente, s TCD4+, uma diminuio de clulas T CD8+ IFN-y+ e TNF+ foram detectadas aps estmulo com CpG. 3) As clulas Th22/Tc22 nos nveis basais e aps estmulo com SEB se mostraram aumentadas. As clulas Th17 no mostraram diferenas entre os grupos. 4) A frequncia das clulas T CD4+ e CD8+ reg totais (CD25+Foxp3+CD127low/-) est elevada no LP. Quanto aos perfis de maturao, h aumento na frequncia de clulas TCD4+ de memria efetora enquanto que para as clulas T CD8+ h predomnio das clulas de memria central. Quanto aos subtipos, h aumento nas clulas T CD4+ regs perifricas (pT reg). CONCLUSES: O estado de ativao das mDCs aps ativao das vias de TLRs 4 e 7/8 pode influenciar na gerao de resposta T efetoras no LP. O perfil de resposta monofuncional e polifuncional aos estmulos TLRs reflete a ativao destas clulas no sangue perifrico. Alm disso, o aumento de Th22/Tc22 e das clulas T regs indicam uma relao entre regulao e clulas efetoras no sangue perifrico evidenciando que existem alteraes extracutneas no LP
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Introduo. Apesar das evidncias dos efeitos imunomodulatrios da morfina, no h na literatura estudos que tenham comparado a interao entre citocinas, imunidade celular (linfcitos T, B e NK) e a administrao prolongada de morfina administrada pelas vias oral ou intratecal em doentes com dor crnica neuroptica no relacionada ao cncer. Foram avaliados de forma transversal e comparativa 50 doentes com diagnstico de dor lombar crnica e com presena de radiculopatia (dor neuroptica) previamente operados para tratar hrnia discal lombar (Sndrome Dolorosa Ps- Laminectomia), sendo 18 doentes tratados prolongadamente com infuso de morfina pela via intratecal com uso de sistema implantvel no compartimento subaracnideo (grupo intratecal); 17 doentes tratados prolongadamente com morfina pela via oral (n=17) e 15 doentes tratados com frmacos mas sem opiides (grupo sem opioide). Foram analisadas as concentrao das citocinas IL-2, IL-4, IL-8, TNFalfa, IFNy, IL-5, GM-CSF, IL-6, IL-10 e IL-1beta no plasma e no lquido cefalorraquidiano; imunofenotipagem de linfcitos T, B e clulas NK e avaliados os ndice de Escalonamento de Opiide (em percentagem de opiide utilizada e em mg), dose cumulativa de morfina (mg), durao do tratamento em meses, dose final de morfina utilizada (em mg), e equivalente de morfina por via oral (em mg). Resultados. No houve diferena estatisticamente significativa entre o nmero de linfcitos T, B e NK nos doentes com morfina administrada pelas vias IT, VO e os no usurios de morfina. Houve correlao positiva entre as concentraes de linfcitos T CD4 e o ndice de Escalonamento de Opiide (em % e mg) nos doentes tratados com morfina por via intratecal. Houve correlao negativa entre as concentraes de clulas NK (CD56+) e o ndice de Escalonamento de Opiide (em % e mg) nos doentes tratados com morfina por via intratecal. Houve correlao positiva entre o nmero de clulas NK (CD56+) e a dose cumulativa de morfina (em mg) administrada pelas vias intratecal e oral. Houve correlao positiva entre as concentraes de linfcitos T CD8 e a durao do tratamento em meses nos doentes tratados com morfina pela via oral. As concentraes de IL-8 e IL-1beta foram maiores no LCR do que no plasma em todos os doentes da amostra analisada. As concentraes de IFNy no LCR foram maiores nos doentes que utilizavam morfina pela via oral e nos no usurios de morfina do que nos que a utilizavam pela via intratecal. As concentraes de plasmticas de IL-5 foram maiores nos doentes utilizavam morfina pela via oral ou intratecal do que nos que no a utilizavam. A concentrao de IL-5 no LCR correlacionou-se negativamente com a magnitude da dor de acordo com a EVA nos doentes tratados com morfina pelas via oral ou intratecal. Nos doentes tratados com morfina pelas via oral ou intratecal, a concentrao de IL-2 no LCR correlacionou-se positivamente com a magnitude da dor de acordo com a EVA e negativamente com o ndice de Escalonamento de Opiide (em % e mg) e a dose cumulativa de morfina (em mg). As concentraes plasmticas de GMCSF foram maiores nos doentes utilizavam morfina pela via oral ou intratecal do que nos no a utilizavam. A concentrao de TNFalfa no LCR nos doentes tratados com morfina pela via intratecal correlacionou-se negativamente com o ndice de Escalonamento de Opiide (em % e mg), a dose cumulativa de morfina (em mg) e dose equivalente por via oral (em mg) de morfina. A concentrao plasmtica das citocinas IL-6 e IL-10 correlacionou-se negativamente com a durao do tratamento (em meses) nos doentes tratados com morfina administrada pela via oral. O ndice de Escalonamento de Opiide (em mg e %) correlacionou-se negativamente com as concentraes no LCR de IL-2 e TNFalfa nos doentes tratados com morfina administrada pela via intratecal. O ndice de Escalonamento de Opiide (em mg e %) correlacionou-se negativamente com as concentraes no LCR de IL-2 e IL-5 nos doentes tratados com morfina administrada pela via oral. Houve correlao negativa entre a intensidade da dor de acordo com a EVA e as concentraes de IL-5 e IL-2 no LCR nos doentes tratados com morfina administrada pelas vias oral e intratecal. Houve correlao negativa entre a intensidade da dor de acordo com a EVA e as concentraes plasmticas de IL-4 nos doentes tratados com morfina administrada pela via intratecal. Houve correlao negativa entre a intensidade da dor de acordo com a EVA e as concentraes plasmticas de IL-1beta nos doentes tratados com morfina administrada pela via intratecal. Concluses: Os resultados sugerem associaes entre citocinas e imunidade celular (clulas T , B e NK) e o tratamento prolongado com morfina administrada pela via oral ou intratecal. Estes resultados podem contribuir para a compreenso da imunomodulao da morfina administrada por diferentes vias em doentes com dor neuroptica crnica no oncolgica . So necessrios mais estudos sobre os efeitos da morfina sobre o sistema imunolgico
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Human adipose mesenchymal stem cells are a heterogeneous population, where cell cultures derived from single cell-expanded clones present varying degrees of differential plasticity. This work focuses on the immunomodulatory/anti-inflammatory properties of these cells. To this end, 5 single cell clones were isolated (generally called 1.X and 3.X) from 2 volunteers. Regarding the expression level of the lineage-characteristic surface antigens, clones 1.10 and 1.22 expressed the lowest amounts, while clones 3.10 and 3.5 expressed more CD105 than the rest and clone 1.7 expressed higher amounts of CD73 and CD44. Regarding cytokine secretion, all clones were capable of spontaneously releasing high levels of IL-6 and low to moderate levels of IL-8. These differences can be explained in part by the distinct methylation profile exhibited by the clones. Furthermore and after lipopolysaccharide stimulation, clone 3.X produced the highest amounts of pro-inflammatory cytokines such as IL-1, while clones 1.10 and 1.22 highly expressed IL-4 and IL-5. In co-culture experiments, clones 1.X are altogether more potent inhibitors than clones 3.X for proliferation of total, CD3+T, CD4+T and CD8+T lymphocytes and NK cells. The results of this work indicates that adipose stem cell population is heterogeneous in cytokine production profile, and that isolation, characterization and selection of the appropriate cell clone is a more exact method for the possible treatment of different patients or pathologies.
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La drgulation du compartiment de cellules B est une consquence importante de linfection par le virus de limmunodficience humaine (VIH-1). On observe notamment une diminution des nombres de lymphocytes B sanguins ainsi quune variation des frquences relatives des diffrentes populations de lymphocytes B chez les individus infects par rapport aux contrles sains. Notre laboratoire a prcdemment dmontr limplication des cellules dendritiques dans la drgulation des lymphocytes B via la roduction excessive de BLyS/BAFF, un stimulateur des cellules B. De plus, lors ltudes menes chez la souris transgnique prsentant une maladie semblable au SIDA, et chez la souris BLyS/BAFF transgnique, linfection au VIH-1 fut associe une expansion de la zone marginale (MZ) de la rate. De faon intressante, nous observons chez les contrleurs lites une diminution de la population B mature de la MZ. Il sagit du seul changement important chez les contrleurs lites et reflte possiblement un recrutement de ces cellules vers la priphrie ainsi quune implication dans des mcanismes de contrle de linfection. Pour tenter dexpliquer et de mieux comprendre ces variations dans les frquences des populations B, nous avons analys les axes chimiotactiques CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 et CCL25-CCR9. Ltude longitudinale de cohortes de patients avec diffrents types de progression clinique ou de contrle de linfection dmontre une modulation des niveaux plasmatiques de la majorit des chimiokines analyses chez les progresseurs rapides et classiques. Au contraire, les contrleurs lites conservent des niveaux normaux de chimiokines, dmontrant leur capacit maintenir lhomostasie. La migration des populations de cellules B semble tre module selon la progression ou le contrle de linfection. Les contrleurs lites prsentent une diminution de la population B mature de la MZ et une augmentation de la frquence dexpression du rcepteur CXCR7 associ la MZ chez la souris, suggrant un rle important des cellules de la MZ dans le contrle de linfection au VIH-1. De faon gnrale, les rsultats dans cette tude viennent enrichir nos connaissances du compartiment de cellules B dans le contexte de linfection au VIH-1 et pourront contribuer laborer des stratgies prventives et thrapeutiques contre ce virus.
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La maladie du greffon contre lhte (GvHD) est un effet secondaire srieux de la transplantation de cellules souches hmatopotiques (HSCT). Cette maladie entraine une haute mortalit et ses symptmes sont dvastateurs. Les traitements actuels de la GvHD comportent plusieurs produits, tels les corticostrodes, mais ces derniers sont immunosuppresseurs et leurs effets secondaires sont aussi trs dommageables pour les patients et leur gurison. Les cellules stromales msenchymateuses (MSC) reprsentent une alternative ou une addition potentielle de traitement pour la GvHD et ces cellules ne semblent pas possder les effets secondaires des traitements classiques. Un nombre important dtudes cliniques faisant lobjet des MSC ont t enregistres. Malgr cet engouement, le mcanisme de leur immunomodulation reste encore lucider. Notre objectif est donc de mieux dfinir ce mcanisme. Nous avons utilis un modle simplifi pour simuler la GvHD in vitro. Ce modle se base sur la stimulation de lymphocytes CD4+ par des cellules dendritiques allogniques. La mesure de la prolifration de ces cellules stimules sert dindicateur de leur ractivit. Selon les rsultats obtenus par la technologie CRISPR de gnie gntique, les MSC exerceraient leur immunosuppression sur les cellules T CD4+ principalement par la scrtion de lenzyme IDO1. Les MSC seraient galement capables dinduire certaines cellules CD4+ en cellules rgulatrices, un processus indpendant de la scrtion dIDO1. Toutefois, ces cellules ne semblent pas correspondre aux cellules Treg conventionnelles.
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4-1BB (CD137) est un membre de la superfamille TNFR qui est impliqu dans la transmission des signaux de survie aux lymphocytes. TRAF1 est une protine adaptatrice qui est recrute par 4-1BB et autres TNFRs et est caractrise par une expression trs restreinte aux lymphocytes, cellules dendritiques et certaines cellules pithliales. TRAF1 est ncessaire pour lexpansion et la survie des cellules T mmoire en prsence d'agonistes anti-4-1BB in vivo. De plus, TRAF1 est requise en aval de 4-1BB pour activer (phosphoryler) la MAP kinase Erk implique dans la rgulation de la molcule pro-apoptotique Bim. Suite lactivation du rcepteur 4-1BB, TRAF1 et ERK sont impliqus dans la phosphorylation de Bim et la modulation de son expression. Lactivation et la rgulation de TRAF1 et Bim ont un rle important dans la survie des cellules T CD8 mmoires. Dans cette tude, nous avons utilis une approche protomique afin de pouvoir identifier de nouveaux partenaires de liaison de TRAF1. Utilisant cette stratgie, nous avons identifi que LSP1 (Leukocyte Specific Protein 1) est recrut dans le complexe de signalisation 4-1BB de manire TRAF1 dpendante. Une caractrisation plus pousse de linteraction entre TRAF1 et LSP1 a montr que LSP1 lie la rgion unique N-terminal de TRAF1 de faon indpendante de la rgion conserve C-terminal. linstar des cellules T dficientes en TRAF1, les cellules T dficientes en LSP1 ne sont pas capables dactiver ERK en aval de 4-1BB et par consquent ne peuvent pas rguler Bim. Ainsi, TRAF1 et LSP1 cooprent en aval de 4-1BB dans le but dactiver ERK et rguler en aval les niveaux de Bim dans les cellules T CD8. Selon la littrature, le rcepteur 4-1BB nest pas exprim la surface des cellules B murines, mais le rcepteur 4-1BB favorise la prolifration et la survie des cellules B humaines. Cependant, il est important d'tudier l'expression du rcepteur 4-1BB dans les cellules B murines afin de disposer d'un modle murin et de prdire la rponse clinique la manipulation de 4-1BB. En utilisant diffrentes stimulations de cellules B murines primaires, nous avons identifi que le rcepteur 4-1BB est exprim la surface des cellules B de souris suite une stimulation avec le LPS (Lipopolysaccharides). Une caractrisation plus pousse a montr que le rcepteur 4-1BB est induit dans les cellules B murines d'une manire dpendante de TLR4 (Toll Like Receptor 4). Collectivement, notre travail a dmontr que la stimulation avec le LPS induit lexpression du rcepteur 4-1BB la surface des cellules B murines, menant ainsi l'induction de TRAF1. De plus, TRAF1 et LSP1 cooprent en aval de 4-1BB pour activer la signalisation de la Map kinase ERK dans les cellules B murines de manire similaire aux cellules T. Les cellules B dficientes en TRAF1 et les cellules B dficientes en LSP1 ne sont pas en mesure d'activer la voie ERK en aval de 4-1BB et montrent un niveau dexpression du rcepteur significativement diminu compar aux cellules B dune souris WT. Ainsi, TRAF1 et LSP1 sont ncessaires pour une expression maximale du rcepteur 4-1BB la surface cellulaire de cellules B murines et cooprent en aval de 4-1BB afin d'activer la cascade ERK dans les cellules B murines.
