RESUMO
T cells play an important role in the adaptive immune system. After haematopoietic stem cell transplantation (HSCT), T-cell function is impaired. This is reflected by the emergence of opportunistic infections, infections that are often difficult to treat because of the patient's insufficient immune function. T-cell receptor reconstitution was studied using CDR3 spectratyping to analyze the diversity of the T-cell repertoire at 3, 6 and 12 months after myeloablative and reduced intensity conditioning (RIC) HSCT in 23 patients. Immune function in vitro was tested by lymphocyte stimulation at 3, 6 and 12 months after HSCT. Lower diversity in the CDR3 repertoire was demonstrated in CD4+ cells after RIC HSCT at 3 and 6 months and in CD8+ cells at 3 months compared with healthy donors. After myeloablative HSCT, lower diversity was seen at 3, 6 and 12 months in CD4+ cells and at 6 and 12 months in CD8+ cells after HSCT. Acute and chronic graft-versus-host-disease (GVHD) did not affect diversity. Responses to phytohaemagglutinin (PHA), Concanavalin A (Con A) and Staphylococcus aureus protein A were significantly lower compared with healthy donors during the first 6 months after RIC HSCT. After myeloablative HSCT, lymphocyte response to Con A was significantly lower at 3 months compared with healthy donors. Decreased responses to cytomegalovirus and varicella zoster virus antigens were seen in patients suffering from acute GVHD grade II or chronic GVHD. The T-cell repertoire is skewed under the first year after HSCT, and immune reconstitution after HSCT with myeloablative and RIC conditioning seems to be comparable. GVHD, infections and age are more important for immune reconstitution than type of conditioning.
Assuntos
Transplante de Células-Tronco Hematopoéticas , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Condicionamento Pré-Transplante/métodos , Adulto , Antígenos/administração & dosagem , Sequência de Bases , Quimera/genética , Quimera/imunologia , Regiões Determinantes de Complementaridade/genética , DNA/genética , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Mitógenos/farmacologia , Linfócitos T/imunologia , Transplante Homólogo , Resultado do TratamentoRESUMO
The 21Na(p,gamma)22Mg reaction is expected to play an important role in the nucleosynthesis of 22Na in oxygen-neon novae. The decay of 22Na leads to the emission of a characteristic 1.275 MeV gamma-ray line. This report provides the first direct measurement of the rate of this reaction using a radioactive 21Na beam, and discusses its astrophysical implications. The energy of the important state was measured to be E(c.m.)=205.7+/-0.5 keV with a resonance strength omegagamma=1.03+/-0.16(stat)+/-0.14(sys) meV.
RESUMO
Growing experimental evidence supports a broadening role for the caspases; not only do they participate in the process of apoptosis but also in the control of the cell cycle and cellular proliferation. The biological role of the caspases in the process of T-cell activation and proliferation is still not defined. In the present study, we propose a potential role, by demonstrating an association of T-cell receptor-mediated caspase activity with the development of an apoptosis-resistant memory CD45RO+ T-cell population. As previously shown by us, a time-dependent induction of caspase activity, in the absence of apoptosis, can be observed in CD3-stimulated human peripheral blood lymphocytes. We here show that a population of CD45RO+ cells, with activated caspase-3 and with resistance to tributyltin-induced apoptosis, develops after 3 days of stimulation. A concomitant expression of the anti-apoptotic protein Bcl-xL accompanied the caspase activity and the development of the apoptosis-resistant phenotype. Finally, upon co-culturing with dexamethasone (DEX), the CD3-induced caspase-3 activity was blocked. During this condition, the expression of the activation marker HLA-DR as well as the cellular proliferative response was strongly suppressed. The development of memory cells with a CD45RO+ phenotype was also blocked. Our data support the hypothesis that caspase-3 activity, observed in CD3-stimulated cells, may be an important component in the proliferation process and, furthermore, might play a role for the development of memory T cells, and DEX inhibits this process.
Assuntos
Apoptose/imunologia , Regulação da Expressão Gênica/imunologia , Antígenos Comuns de Leucócito/imunologia , Linfócitos T/imunologia , Complexo CD3/imunologia , Caspases/imunologia , Dexametasona/farmacologia , Citometria de Fluxo , Glucocorticoides/farmacologia , Antígenos HLA-DR/farmacologia , Humanos , Memória Imunológica , Ativação Linfocitária/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Compostos de Trialquitina/imunologia , Proteína bcl-XRESUMO
BACKGROUND: Sarcoidosis is a chronic granulomatous lung disease of unknown origin. The accumulation of activated T cells at sites of inflammation represents an early stage in granuloma formation. Since mechanisms governing the normal resolution of inflammatory processes are poorly understood, this study aimed to investigate the apoptotic phenotype of peripheral blood and lung T lymphocytes from patients with sarcoidosis. METHODS: Bronchoalveolar lavage (BAL) was performed in 10 patients with active sarcoidosis and five healthy controls. RESULTS: Virtually no lymphocyte apoptosis, as determined by annexin V or Hoechst staining, was seen in either patients or controls. Sustained caspase-3 activity in non-apoptotic BAL fluid lymphocytes of the patients was detected, however, in agreement with in vitro studies demonstrating caspase activation after T cell receptor (TCR) triggering as a physiological response required for efficient T cell activation. Only 11.0% (range 7.7-17.6) of the BAL lymphocytes from sarcoidosis patients were annexin V positive after exposure to the apoptotic stimulus tributyltin compared with 55.0% (range 42.0-62.0) of BAL lymphocytes from healthy controls (p<0.001). After anti-Fas treatment only 8.5% (range 6-10) of BAL fluid lymphocytes from patients but 45.5% (range 38-62) from healthy controls were apoptotic. CONCLUSION: BAL fluid lymphocytes from patients with sarcoidosis display a non-apoptotic morphology associated with endogenous caspase-3 activity. They seem to be resistant to apoptosis, which might contribute to the accumulation of inflammatory cells in the lungs, persistence of inflammation, and the development and maintenance of granuloma.
Assuntos
Apoptose , Líquido da Lavagem Broncoalveolar/citologia , Sarcoidose Pulmonar/imunologia , Linfócitos T/fisiologia , Adulto , Anexina A5/metabolismo , Lavagem Broncoalveolar , Caspases/metabolismo , Células Cultivadas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfatidilserinas/metabolismo , Sarcoidose Pulmonar/metabolismo , Linfócitos T/enzimologiaRESUMO
Molecular analysis of T-cell receptor (TCR) repertoire, by measuring the CDR3 heterogeneity length of beta-variable regions (spectratyping), is useful for acquiring novel information on the status of immune system in primary immunodeficiency. Here, we evaluate TCR repertoire in a child with trichothiodystrophy (TTD) and combined immunodeficiency (CID). Spectratyping revealed marked alterations of TCR repertoire distribution: 21 and 10 out of 27 TCR Vbeta (TCRBV) families and subfamilies were skewed in CD8+ and CD4+ subsets, respectively. These findings revealed, for the first time in a TTD patient with CID, a marked reduction in the TCR repertoire complexity, which may reflect alterations in the mechanisms regulating the generation and homeostasis of T cells.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Doenças do Cabelo/genética , Doenças do Cabelo/imunologia , Linfopenia/genética , Linfopenia/imunologia , Receptores de Antígenos de Linfócitos T/genética , Transportadores de Cassetes de Ligação de ATP/genética , Estudos de Casos e Controles , Pré-Escolar , Rearranjo Gênico do Linfócito T , Doenças do Cabelo/complicações , Humanos , Linfopenia/complicações , MasculinoRESUMO
Several reports have indicated that cell lineages apart from NK and T cells can also express IFN-gamma. However, the biological relevance of this finding is uncertain. We show in this study that bone marrow-derived macrophages (BMMs) express IFN-gamma at the mRNA and protein level early after infection with Chlamydia pneumoniae. Increased IFN-gamma mRNA accumulation by infected BMMs is early, transient, and requires both bacterial and host protein synthesis. The induction of IFN-gamma mRNA levels is independent of IL-12 and was dramatically enhanced in IL-10(-/-) BMMs. Such IL-10(-/-) BMMs contained less bacteria than the wild-type controls, whereas IFN-gammaR(-/-) BMMs showed increased C. pneumoniae load. Inducible NO synthase (iNOS) also participates in the control of bacterial load, as shown by the enhanced numbers of C. pneumoniae in iNOS(-/-) BMMs. However, the increased accumulation of iNOS mRNA and NO in C. pneumoniae-infected BMMs depended on the presence of IFN-alphabeta, but was independent of IFN-gamma. Interestingly, IFN-alphabeta are also required for increased IFN-gamma mRNA accumulation in C. pneumoniae-infected BMMs. Accordingly, IFN-alphabetaR(-/-) BMMs showed higher levels of C. pneumoniae than wild-type BMMs. Our findings unravel an autocrine/paracrine macrophage activation pathway by showing an IFN-alphabeta-dependent IFN-gamma and iNOS induction in response to infection, which protects macrophages against intracellular bacterial growth.
Assuntos
Células da Medula Óssea/imunologia , Células da Medula Óssea/microbiologia , Chlamydophila pneumoniae/crescimento & desenvolvimento , Chlamydophila pneumoniae/imunologia , Interferon Tipo I/fisiologia , Interferon gama/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Células da Medula Óssea/enzimologia , Células da Medula Óssea/metabolismo , Células Cultivadas , Inibidores do Crescimento/metabolismo , Inibidores do Crescimento/fisiologia , Interferon gama/biossíntese , Interferon gama/genética , Interleucina-10/fisiologia , Interleucina-12/fisiologia , Líquido Intracelular/enzimologia , Líquido Intracelular/imunologia , Líquido Intracelular/microbiologia , Macrófagos/enzimologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
In the present study, we show that the immunotoxicant, tributyltin (TBT), induces a dose-dependent activation of caspases followed by typical apoptotic morphology in resting human peripheral blood lymphocytes. TBT also caused an early loss of mitochondrial membrane potential (Delta(Psi)(m)) and release of cytochrome c, suggesting that apoptosis was triggered by the mitochondrial pathway. When CD4+ T-cells were sorted from peripheral blood and exposed to TBT for 30 min, caspase activation and apoptosis were induced. Interestingly, in the sorted CD8+ T-cell population, caspase activation was not observed until 2 h of TBT exposure, suggesting that these cells were more resistant toward TBT. Moreover, a time-dependent induction of caspase activity was also detected in CD3-stimulated peripheral blood lymphocytes. This caspase activation was not associated with cytochrome c release or loss of mitochondrial Delta(Psi) and did not lead to apoptotic morphology, although it did lead to both PARP and DFF cleavage. We also noticed a concomitant induction of Hsp27, and it awaits to be seen if this chaperone may interfere with the processing of nuclear protein substrates downstream from these primary caspase-3 substrates. Moreover, no increase in caspase activation or induction of apoptosis was observed after TBT treatment in these cells. Instead, the cells were directed toward necrotic deletion. Taken together, these data suggest that TBT-induced deletion of peripheral lymphocytes is likely to be a component in the overall risk for immunotoxic responses in exposed humans.
Assuntos
Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linfócitos/efeitos dos fármacos , Compostos Orgânicos de Estanho/farmacologia , Compostos de Trialquitina/farmacologia , Complexo CD3/sangue , Complexo CD3/imunologia , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Grupo dos Citocromos c/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática , Humanos , Cinética , Linfócitos/sangue , Linfócitos/enzimologia , Potenciais da Membrana , Mitocôndrias/efeitos dos fármacosRESUMO
Our data on peripheral blood T cells from Motor neuron disease (MND) patients indicate major immunological disturbances linked to this disease. Both CD4+ and CD8+ T-cell subsets display an increased fraction of cells showing activation markers compared to controls, indicating an unusually high level of activity in both populations. Likewise, an increased number of T-cell expansions were noted in MND patients compared to controls, most dramatically observed in the CD4+ T-cell subset, where 5/144 T-cell V genes analyzed in eight subjects turned out to be expanded in the peripheral blood. In the CD8+ T-cell subset, four out of eight MND patients had peripheral BV gene expansions, 9/144 V genes analyzed. However, the most interesting result was the observation that in three out eight MND patients, expansions concerning the same BV gene were present in both CD4+ and CD8+ subsets (BV8S1 in two and BV12S1 in one patient). Parallel expansions of BV-gene restricted populations in both CD4+ and CD8+ subsets in the same individual, in an major histocompatibility complex (MHC)-unrestricted manner, are normally limited to situations where superantigens are involved. No known superantigen has to date been described with the capacity to simultaneously stimulate both BV8S1 and BV12S1, suggesting that the postulated 'MND-associated' superantigen is a hitherto undefined molecule.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Ativação Linfocitária/imunologia , Doença dos Neurônios Motores/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Superantígenos/imunologia , Humanos , Leucócitos Mononucleares/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
BACKGROUND: Allergen provocation is a very useful way to study the inflammatory response in asthmatic patients. Although cumulative dose regimens are most often applied, another provocation model with repeated inhalations of low doses of allergens has recently come into use. OBJECTIVE: We were interested to compare these two allergen provocation models. To evaluate the inflammation induced by either model, we examined the mRNA expression of several cytokines that are implicated in the orchestration of the inflammatory response observed in asthma. METHODS: Interleukin (IL)-4, IL-5, IL-13 and interferon (IFN)-gamma mRNA expression was analysed in bronchoalveolar lavage (BAL) cells and peripheral blood (PB) CD4+ and PB CD8+ T cells following any of the two provocation regimens. IL-4 and IFN-gamma mRNA expression was analysed by a competitive reverse transcriptase-polymerase chain reaction (RT-PCR) method, while IL-5 and IL-13 were analysed semiquantitatively, before and after allergen provocation with either model. RESULTS: After low dose provocations none of the cell populations studied showed a clear change in the pattern of IL-4 or IFN-gamma gene expression. In contrast, after cumulative dose provocation there was a clear tendency towards an increased IL-4 mRNA expression in BAL cells, correlating with a significant increase in IL-4 mRNA in PB CD4+ as well as in CD8+ T cells (P = 0.005 and P = 0.04, respectively). Regardless of the allergen provocation method used, in PB IL-4 mRNA was preferentially expressed by CD4+ cells while IFN-gamma was expressed more by CD8+ cells. IL-5 transcripts increased after low dose provocations in PB CD4+ T cells in six of eight patients, while after cumulative dose provocation IL-5 mRNA increased in BAL cells in six out of nine patients but decreased especially in PB CD8+ T cells in six out of eleven patients, suggesting an accumulation of IL-5 expressing cells to the lungs. CONCLUSION: Thus, the cumulative dose provocation regimen can induce a more pronounced Th2-like immune response in asthmatic patients than the low dose provocation model.
Assuntos
Asma/genética , Asma/metabolismo , Testes de Provocação Brônquica , Citocinas/genética , RNA Mensageiro/metabolismo , Adulto , Alérgenos/administração & dosagem , Líquido da Lavagem Broncoalveolar/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Feminino , Expressão Gênica , Humanos , Interferon gama/genética , Interleucina-13/genética , Interleucina-4/genética , Interleucina-5/genética , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Índice de Gravidade de DoençaRESUMO
The objective of this study was to identify disease-associated T cell subsets by characterizing the lung and blood T cell receptor (TCR) repertoires in allergic asthmatics before and after repeated low-dose allergen challenge. Peripheral blood lymphocyte (PBL) and bronchoalveolar lavage (BAL) samples were obtained from eight patients with allergic asthma before and after a period of repeated low-dose allergen inhalations. RT-PCR followed by Southern blot allowed the quantification of relative Vbeta gene segment usage. Thirteen healthy individuals served as controls at PBL level. PBL as well as BAL T cells of asthmatics displayed a higher usage of Vbeta3, Vbeta5.2, and Vbeta6.1-3 and a lower usage of Vbeta16, Vbeta18, and Vbeta19 compared to PBL of healthy controls. Interestingly, TCR Vbeta7 and Vbeta9 usage was significantly higher in BAL than in PBL in asthmatics before as well as after challenge. TCR repertoire alterations after allergen challenge differed between individuals, with relatively mild changes.
Assuntos
Alérgenos , Asma/imunologia , Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Hipersensibilidade Imediata/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Administração por Inalação , Adulto , Alérgenos/administração & dosagem , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , DNA Complementar/genética , Feminino , Humanos , Hipersensibilidade Imediata/patologia , Imunofenotipagem , Pulmão/patologia , Masculino , Pólen/imunologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Immune and inflammatory responses mediated by cytokines are essential in the pathophysiology of asthma. The aim of this study was to analyse the cytokine mRNA profiles in bronchoalveolar lavage (BAL) cells of patients with mild atopic asthma, before and after induction of a subclinical allergic airway inflammation. For this purpose, eight patients with mild atopic asthma received low-dose allergen inhalations equivalent to 10% of a provocational dose causing a 20% fall in forced expiratory flow in 1 sec (PD20) for 7 weekdays. BAL was performed before and after low-dose provocations in patients, and without provocation in five healthy controls. Alveolar macrophages (AM) were enriched by negative selection, using magnetic beads, to enable separate studies of the BAL cells. Using a semiquantitative RT-PCR technique, the mRNA expression of macrophage-derived cytokines interleukin (IL)-1, IL-6, IL-8, IL-10, IL-12, IL-13, interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta was analysed. After low-dose provocations, we observed a significant increase in the expression of IL-13 mRNA (P = 0.01) in BAL cells enriched for AM of the asthmatic patients. The increased IL-13 mRNA positively correlated with the proportion of BAL fluid eosinophils (r = 0.7, P = 0.05). Moreover, a tendency was found towards an increased IL-1 and a reduced IL-6, IL-8, IFN-gamma and TNF-alpha expression by the BAL cells. Comparing asthmatic patients before low-dose provocations and healthy controls, a significantly higher expression of IL-6 (P<0.003), IL-10 (P<0.005) and TGF-beta (P<0.003) and a significantly lower expression of IL-8 (P<0.005) and TNF-alpha (P<0.01) was detected in the patients. In summary, repeated low-dose allergen provocations of asthmatic patients results in a modified BAL cell cytokine mRNA profile with increased production of IL-13, that may be of importance for the development of a Th2-like immune response. A possible source of the increased IL-13 mRNA is AM, which may have a more active function in the allergic inflammation than previously thought.
Assuntos
Asma/metabolismo , Líquido da Lavagem Broncoalveolar/química , Interleucina-13/metabolismo , RNA Mensageiro/metabolismo , Adulto , Testes de Provocação Brônquica , DNA Complementar/metabolismo , Feminino , Volume Expiratório Forçado/fisiologia , Humanos , Masculino , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
By using mice genomically lacking IFN-gammaR, IL-12, perforin, and recombination-activating gene-1 (RAG-1), we analyzed the regulation and importance of IFN-gamma in the control of infection with Chlamydia pneumoniae. IL-12 participates in resistance of mice to C. pneumoniae, probably by regulating the protective levels of IFN-gamma mRNA. In turn, IFN-gamma is necessary for the increased IL-12p40 mRNA accumulation that occurs in lungs during infection with C. pneumoniae, suggesting a positive feedback regulation between these two cytokines. In experiments including RAG-1-/-/IFN-gammaR-/- mice we showed that IFN-gamma produced by innate cells controls the bacterial load and is necessary for the increased accumulation of transcripts for enzymes controlling high output NO release (inducible NO synthase), superoxide production (gp-91 NADPH oxidase), and catalysis of tryptophan (indoleamine 2, 3-dioxygenase (IDO)), mechanisms probably related to bacterial killing. Adaptive immune responses diminish the levels of IFN-gamma and IL-12 mRNA and thereby the levels of inducible NO synthase, IDO, and gp91 NADPH oxidase transcripts. By using RAG-1-/-/perforin-/- mice, we excluded the overt participation of NK cell cytotoxicity in the control of C. pneumoniae. However, NK cells and probably other innate immune cells release IFN-gamma during the bacterial infection.
Assuntos
Infecções por Chlamydia/genética , Infecções por Chlamydia/imunologia , Chlamydophila pneumoniae/imunologia , Interferon gama/biossíntese , Interferon gama/fisiologia , Animais , Infecções por Chlamydia/prevenção & controle , Citotoxicidade Imunológica/genética , Gangliosídeo G(M1)/imunologia , Genes RAG-1/imunologia , Predisposição Genética para Doença , Soros Imunes/administração & dosagem , Imunidade Inata/genética , Imunossupressores/administração & dosagem , Injeções Intravenosas , Interferon gama/genética , Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Interferon/genética , Deleção de Sequência , Receptor de Interferon gamaRESUMO
Using a novel dual-channel FACS methodology, the organotin compound TBT (2 microM) was shown to induce rapid (maximal by 3 min) and sustained elevations in intracellular calcium levels [Ca(2+)](i) in Jurkat T cells. This was preceded by mitochondrial hyperpolarization (maximal at 1 min), with subsequent loss of membrane potential, (Deltapsi(m)) over the next 15 min and was associated with the release of mitochondrial cytochrome c and the activation of type II caspases. The activation of the caspases was blocked by calcium chelation with EGTA and/or BAPTA. Interestingly, changes in Deltapsi(m) caused by TBT were not affected by chelation of intra- and extracellular calcium or by performing the experiments in a Ca(2+)-free medium. TBT also caused rapid elevation of [Ca(2+)](i) in cells lacking glycolytic ATP production. Despite this, the loss of Deltapsi(m) and the activation of type II caspases were delayed (maximal by 2 h) in these cells. Further, there was a failure to activate type II caspases in cells treated with TBT in a Ca(2+)-free medium, despite rapid release of mitochondrial cytochrome c. Consequently, these cells evaded the induction of apoptosis and were diverted to delayed necrotic deletion. Taken together, these data strongly suggest that the rapid rise in [Ca(2+)](i) caused by TBT in Jurkat T cells is not directly coupled to the induction of mitochondrial permeability transition, which rather results from a direct interaction of TBT with mitochondrial component(s) controlling pore transition. However, the rise in [Ca(2+)](i) is a prerequisite for postmitochondrial events involved in caspase activation prior to the induction of apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Canais Iônicos , Células Jurkat/metabolismo , Compostos de Trialquitina/farmacologia , Caspases/metabolismo , Grupo dos Citocromos c/metabolismo , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Citometria de Fluxo , Humanos , Potenciais da Membrana/efeitos dos fármacos , Proteínas de Membrana , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Poro de Transição de Permeabilidade MitocondrialRESUMO
Infection with Chlamydia pneumoniae is a common cause of acute respiratory disease in man and is also associated with atherosclerotic cardiovascular disorder. Herein, we have compared bacterial load and immune parameters of C. pneumoniae-infected mice genomically lacking T cell coreceptors, cytokine receptors, or cytotoxic effector molecules. A protective role for CD8+ cells is shown by the enhanced severity of infection of CD8-/- or TAP-1-/-/beta2-microglobulin -/- mice. CD8+ cells hindered a parasite growth-promoting role of CD4+ T cells, as indicated by the higher sensitivity to early infection of CD8-/- than CD4-/-/CD8-/- mice, which was further confirmed in experiments in which SCID mice were reconstituted with either CD4+ or CD4+ plus CD8+ T cells. Interestingly, CD4+ T cells played a dual role, detrimental early (14 and 24 days) after infection but protective at later time points (60 days after infection). The CD8+ T cell protection was perforin independent. The early deleterious role of CD4+ in the absence of CD8+ T cells was associated with enhanced IL-4 and IL-10 mRNA levels and delayed IFN-gamma mRNA accumulation in lungs. In line with this, IFN-gammaR-/- (but not TNFRp55 -/-) mice showed dramatically increased susceptibility to C. pneumoniae, linked to reduced inducible nitric oxide synthase (iNOS) mRNA accumulation, but not to diminished levels of specific Abs. The increased susceptibility of iNOS-/- mice indicates a protective role for iNOS activity during infection with C. pneumoniae. The higher sensitivity of IFN-gammaR-/- mice to C. pneumoniae compared with that of SCID or recombination-activating gene-1-/- mice suggested a relevant protective role of IFN-gamma-dependent innate mechanisms of protection.
Assuntos
Infecções por Chlamydia/imunologia , Chlamydophila pneumoniae/imunologia , Animais , Antígenos CD/fisiologia , Linfócitos T CD4-Positivos/fisiologia , Linfócitos T CD8-Positivos/fisiologia , Imunidade , Interferon gama/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Óxido Nítrico Sintase/fisiologia , Óxido Nítrico Sintase Tipo II , Receptores de Interferon/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Receptores Tipo I de Fatores de Necrose Tumoral , Receptor de Interferon gamaRESUMO
The idiotypic structures of the myeloma protein might be regarded as tumor-specific antigens. The present study was designed to map T-cell epitopes of the idiotypic myeloma protein to prove the existence of naturally occurring major-histocompatibility-complex-dependent idiotype (peptide)-specific T cells in multiple myeloma. The fine specificity of idiotype-reactive, interferon-gamma-producing blood T cells of a patient with multiple myeloma stage I was characterized by identification of idiotype (heavy and light chains)-derived MHC-restricted T-cell epitopes. T cells specifically reacting with peptides corresponding to each of the 3 complementarity-determining regions (CDRs) of the heavy-chain variable part (V(H)) of the autologous idiotype were found. In contrast, none of the peptides corresponding to the 3 CDRs of the light chain (V(L)) induced a specific T-cell response. The idiotype amino-acid sequence corresponding to the junction of the V(H), diversity (D), and joining (J) gene segments of the VH appeared to be an important target for T cells, since the sequence expressed MHC-class-I- as well as MHC-class-II-restricted epitopes. The study provides further support for the existence of MHC-restricted idiotype-specific T cells, which may target immunogenic CDR peptides in multiple myeloma. Such T cells could be an important part of the specific anti-tumor immune responses induced in idiotype vaccination protocols.
Assuntos
Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/genética , Idiótipos de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Mieloma Múltiplo/imunologia , Linfócitos T/imunologia , Idoso , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Sequência de Bases , Epitopos/genética , Humanos , Região Variável de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Complexo Principal de Histocompatibilidade , Masculino , Dados de Sequência Molecular , Mieloma Múltiplo/genética , Mutação , Alinhamento de SequênciaRESUMO
Earlier, we reported an association between low in vitro and in vivo IL-1 and IL-6 production, decreased IL-1beta and IL-10 mRNA expression and B cell chronic lymphocytic leukemia (B-CLL) disease progression. We have now further investigated cytokine mRNA transcription in B-CLL cells and cytokine serum levels in B-CLL patients. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification of tumor necrosis factor (TNFalpha), IFNgamma, IL-6 and BCGF was equally often seen in non-progressive and progressive patients. However, 4 out of 23 non-progressive cases expressed mRNA for IL-12 while no IL-12 expression was seen in 15 progressive patients. No IL-12 was found in sera or supernatants from in vitro stimulated B-CLL cells, whereas TNFalpha and IL-10 were detected in sera from 51 and 31 of 65 B-CLL patients, respectively. TNFalpha values were significantly high in sera from patients in stages III and IV with disease progression. TNFalpha and IL-10 were also detected in culture supernatants from in vitro stimulated B-CLL cells, whereas IFNgamma was undetectable in these cultures and rarely positive in serum. Although further investigations are required, our data and that from previous reports indicate that B-CLL-derived cytokines are involved in B-CLL disease progression.
Assuntos
Citocinas/biossíntese , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , Ativação Linfocitária , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Citocinas/sangue , Citocinas/genética , DNA de Neoplasias/análise , Progressão da Doença , Feminino , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/imunologia , Ativação Linfocitária/genética , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/análiseRESUMO
Kawasaki disease (KD) is a paediatric multisystem necrotizing vasculitis constituting the most frequent cause of acquired heart disease in childhood. Conflicting data have been reported regarding expanded T-cell populations using particular T-cell receptor (TCR) beta-chain variable (BV) gene segments, suggesting either a superantigen- or a conventional antigen-mediated immune response in this disease. In order to further investigate the role of T lymphocytes, cells were stained with an extensive panel of 21 different TCRBV specific monoclonal antibodies (MoAbs) covering almost 70% of all T-cells. Flow cytometry was employed to analyse the expression of the TCRBV repertoire in the CD4+ and CD8+ subsets separately, and of activation markers, in freshly isolated peripheral blood lymphocytes of 25 Kawasaki disease patients during the acute and convalescent phases of the disease. No abnormal usage of any TCRBV family was found, neither acutely nor during convalescence, compared with a control group of healthy children. However, a significant increase in interleukin-2 receptor (IL-2R)-expressing T lymphocytes restricted to the CD4+ subset was observed in KD patients. Our data confirm a strong immune activation in KD that might be of importance in the pathogenesis of the disease.
Assuntos
Região Variável de Imunoglobulina/imunologia , Síndrome de Linfonodos Mucocutâneos/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Criança , Pré-Escolar , Feminino , Citometria de Fluxo , Genes de Imunoglobulinas , Humanos , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Lactente , Masculino , Receptores de Antígenos de Linfócitos T alfa-beta/genéticaRESUMO
OBJECTIVE: To investigate the T-cell receptor (TCR) repertoire usage in infants born to mothers infected with HIV-1 in order to discern possible perturbations in TCR usage as a consequence of HIV-1 infection. DESIGN: Blood samples from five HIV-1-infected and six non-infected children born to HIV-1-seropositive mothers were collected at two to three timepoints during the first and second year of life and the TCR variable gene usage was determined. METHODS: Triple staining flow cytometry analysis using a panel of monoclonal antibodies (MAb) to TCR V alpha and V beta gene products and antibodies to CD4 and CD8 was performed. RESULTS: Frequent large expansions of CD8+ lymphocyte subpopulations bearing distinct V alpha and V beta gene products was seen in HIV-1-infected children (four out of five) but was rarely detected in uninfected children. CONCLUSION: The study demonstrated the frequent occurrence of persistent and clonal expansions of CD8+ T cells bearing distinct V alpha/V beta gene products in some HIV-1 vertically infected infants similar to those observed during primary infection in adults.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1 , Região Variável de Imunoglobulina/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/transmissão , Humanos , Região Variável de Imunoglobulina/genética , Lactente , Recém-Nascido , Transmissão Vertical de Doenças Infecciosas , Receptores de Antígenos de Linfócitos T/genética , Análise de SequênciaRESUMO
BACKGROUND: The principal symptoms of myasthenia gravis (MG), muscle weakness and fatigue due to impaired neuromuscular transmission, are caused by autoantibodies to the muscle nicotinic acetylcholine receptor (AChR). The mechanisms underlying the autoimmune response, however, appear to be initiated by activation of specific HLA class II-restricted CD4+ T lymphocytes. Thus, central to elucidating the causation of MG is determining how T cells are recruited to contribute to misguided immunological assaults on the major autoantigenic target, AChR. MATERIALS AND METHODS: By combining a polymerase chain reaction (PCR)-based strategy and Southern blot technique, we have analyzed the frequency of expression of 22 individual T cell receptor (TCR) V beta gene subfamilies in CD4+ and CD8+ peripheral blood T cell subsets derived from eight MG patients and seven healthy controls. The quantification of relative usage of individual TCR J beta gene segments was performed by hybridization of PCR-amplified products (specifically V beta 1-C beta) with a complete panel of 32P-5'-end-labeled J beta-specific oligonucleotide probes, followed by scanning analysis of autoradiographs. RESULTS: Comparisons of data obtained from V beta analyses of T cells from MG patients with those from healthy individuals established that MG patients significantly overexpressed V beta 1, V beta 13.2, V beta 17, and V beta 20 gene family members within both CD4+ and CD8+ T cell subpopulations. Moreover, analysis of the relative utilization of individual TCR J beta gene segments in V beta 1+/CD4+ and V beta 1+/CD8+ T lymphocytes revealed distribution patterns in patients indistinguishable from those recorded in the corresponding cell subsets derived from controls. CONCLUSIONS: T lymphocytes from MG patients displayed a biased overexpression of four TCR V beta gene segments: V beta 1, V beta 13.2, V beta 17, and V beta 20. The relative frequencies of association of individual V beta 1 (D beta) J beta combinations revealed that J beta gene usage in the V beta 1-over-represented T cell subsets had normal distribution patterns. It can thus be deduced that J beta gene segment products appear not to have a selective effect on the process leading to overexpression of V beta 1 exons in MG patients. Hence, our observations suggest a possible role for superantigen(s) in the T cell activation in MG patients.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Miastenia Gravis/genética , Receptores de Antígenos de Linfócitos T/genética , Superantígenos/fisiologia , Adulto , Idoso , Southern Blotting , Humanos , Pessoa de Meia-Idade , Miastenia Gravis/sangue , Reação em Cadeia da PolimeraseRESUMO
OBJECTIVE: To investigate T cell receptor (TCR) V alpha/V beta (and in selected cases, J beta) usage in CD4+ and CD8+ peripheral blood lymphocytes of patients with giant cell arteritis (GCA), before and after treatment, as well as to analyze the HLA types of these patients. METHODS: Flow cytometry, with 10 anti-TCR V-specific monoclonal antibodies (MAb), was used. To analyze J beta usage by cell populations expressing certain V beta, we used the polymerase chain reaction (PCR) technique, with V beta- and C beta-specific primers, Southern blotting of PCR products, and subsequent hybridization with radiolabeled J beta-specific probes. HLA typing was performed using the microlymphocytotoxicity technique. RESULTS: Seven of the 9 GCA patients had increased anti-TCR V MAb reactivities (interpreted as T cell expansions), which in many cases, correlated with clinical signs of disease. A strict preference for particular J beta segments was found in 3 of 3 expanded CD4+ T cell populations. CONCLUSION: T lymphocytes expressing specific antigen receptors are implicated in the pathogenesis of GCA.