RESUMO
Social bacteria use chemical communication to coordinate and synchronize gene expression via the quorum-sensing (QS) regulatory pathway. In Pectobacterium, a causative agent of the blackleg and soft-rot diseases on potato plants and tubers, expression of the virulence factors is collectively controlled by the QS-signals N-acylhomoserine lactones (NAHLs). Several soil bacteria, such as the actinobacterium Rhodococcus erythropolis, are able to degrade NAHLs, hence quench the chemical communication and virulence of Pectobacterium. Here, next-generation sequencing was used to investigate structural and functional genomics of the NAHL-degrading R. erythropolis strain R138. The R. erythropolis R138 genome (6.7 Mbp) contained a single circular chromosome, one linear (250 kbp) and one circular (84 kbp) plasmid. Growth of R. erythropolis and P. atrosepticum was not altered in mixed-cultures as compared with monocultures on potato tuber slices. HiSeq-transcriptomics revealed that no R. erythropolis genes were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the avirulent P. atrosepticum mutant expI, which is defective for QS-signal synthesis. By contrast 50 genes (<1% of the R. erythropolis genome) were differentially expressed when R. erythropolis was cultivated in the presence vs absence of the NAHL-producing virulent P. atrosepticum. Among them, quantitative real-time reverse-transcriptase-PCR confirmed that the expression of some alkyl-sulfatase genes decreased in the presence of a virulent P. atrosepticum, as well as deprivation of organic sulfur such as methionine, which is a key precursor in the synthesis of NAHL by P. atrosepticum.
Assuntos
Genoma Bacteriano , Pectobacterium/patogenicidade , Percepção de Quorum , Rhodococcus/genética , Transcriptoma , Acil-Butirolactonas/metabolismo , Técnicas de Cocultura , DNA Bacteriano/genética , Sequenciamento de Nucleotídeos em Larga Escala , Pectobacterium/metabolismo , Análise de Sequência de DNA , Solanum tuberosum/microbiologiaRESUMO
Background: Amyloidosis is a rare pathology, due to a toxic accumulation of amyloid proteins infiltrating tissues. Published studies have low statistical power. However it seems that ENT localization have favorable prognosis. Management and check up are not well codified. Methods: Bicentric retrospective study conducted between 1987 and 2015, from patient diagnosed with ENT amyloidosis. The study was performed to the database of the pathology department. People concerned, history, symptoms and diagnostic features were analysed. The immunologic and clinical status, locations, extension check, treatment and prognosis have been evaluated. Results: Twenty patients were evaluated, ten men and ten women, average age was 55.5 year of age. Three patients were afflicted with familial amyloidosis. Main localisation was larynx (80%), main type was immunoglobulinic (AL) (80%). Amyloidosis was mostly localised (90%) and primary form (80%). Dysphonia was the most frequently encountered symptom. Most performed examination were local biopsy and creatinine clearance (100%), serum protein electrophoresis (SEP) (89%), myelogram and/or bone marrow aspiration (75%), and trans thoracic echography (TTE) (75%). Surgical removal was performed for 75% of the patients. Global rate of recurrence was 70%, about 4.6 years after diagnosis. In familial forms, overall survival was 66% at ten years. In non-familial forms, overall survival was 100%. Conclusion: ENT amyloidosis are mostly AL, laryngeal, primary and localised. Distant extension check should be managed by internal medicine specialist and associate creatinine clearance, local biopsy, TTE, SEP and myelogram. Head and neck forms treatment is based on surgical removal, familial forms are of poor prognosis.
Assuntos
Amiloidose/diagnóstico , Amiloidose/terapia , Otorrinolaringopatias/diagnóstico , Otorrinolaringopatias/terapia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Estudos RetrospectivosRESUMO
Hematopoietic stem cell transplantation (HSCT) for severe ADs was developed over the past 25years and is now validated by national and international medical societies for severe early systemic sclerosis (SSc) and relapsing-remitting multiple sclerosis (MS) and available as part of routine care in accredited center. HSCT is also recommended, with varying levels of evidence, as an alternative treatment for several ADs, when refractory to conventional therapy, including specific cases of connective tissue diseases or vasculitis, inflammatory neurological diseases, and more rarely severe refractory Crohn's disease. The aim of this document was to provide guidelines for the current indications, procedures and follow-up of HSCT in ADs. Patient safety considerations are central to guidance on patient selection and conditioning, always validated at the national MATHEC multidisciplinary team meeting (MDTM) based on recent (less than 3months) thorough patient evaluation. HSCT procedural aspects and follow-up are then carried out within appropriately experienced and Joint Accreditation Committee of International Society for Cellular Therapy and SFGM-TC accredited centres in close collaboration with the ADs specialist. These French recommendations were performed according to HAS/FAI2R standard operating procedures and coordinated by the Île-de-France MATHEC Reference Centre for Rare Systemic Autoimmune Diseases (CRMR MATHEC) within the Filière FAI2R and in association with the Filière MaRIH. The task force consisted of 3 patients and 64 clinical experts from various specialties and French centres. These data-derived and consensus-derived recommendations will help clinicians to propose HSCT for their severe ADs patients in an evidence-based way. These recommendations also give directions for future clinical research in this area. These recommendations will be updated according to newly emerging data. Of note, other cell therapies that have not yet been approved for clinical practice or are the subject of ongoing clinical research will not be addressed in this document.
Assuntos
Doenças Autoimunes , Transplante de Células-Tronco Hematopoéticas , Escleroderma Sistêmico , Humanos , Condicionamento Pré-Transplante/métodos , Doenças Autoimunes/diagnóstico , Doenças Autoimunes/terapia , Transplante Autólogo , Escleroderma Sistêmico/diagnóstico , Escleroderma Sistêmico/terapiaRESUMO
BACKGROUND: thrombolysis (THL) and primary percutaneous coronary intervention (PCI) are therapeutic options in acute myocardial infarction (MI). These strategies have similar efficiency, particularly in the early phase. However, in these randomized studies, different times to treatment (TT) threshold are recognized as discriminant. OBJECTIVES: to validate, on a one year mortality criteria the best TT threshold in the real life. METHODS: 794 patients, admitted directly in our institution with a Ml< or =12 hours; 299 were treated by THL and 495 by PCI. The primary end-point was 1-year mortality according to TT and strategy of revascularization. Three TT thresholds were tested (120, 150 and 180 min) to define the best strategy of revascularisation. RESULTS: only the 150 min TT threshold showed a significant difference between the two strategies. If TT was less than 150 min, relative risk of 1-year mortality was 1.36 (p=0.62) for patients treated by THL compared to those treated with PCI. By contrast, the relative risk of one year mortality was 1.95 if Tr was greater than 150 min (p=0.02). CONCLUSION: TT is a key factor to define the best strategy of reperfusion. The critical threshold seems to be at 150 min. THL should be considered as a therapeutic choice only if administered within the first 150 min. After this delay, primary PCI should be preferred.
Assuntos
Angioplastia Coronária com Balão , Infarto do Miocárdio/mortalidade , Infarto do Miocárdio/terapia , Terapia Trombolítica , Feminino , França/epidemiologia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de TempoRESUMO
We took advantage of a recently developed system allowing performance of real-time quantitation of polymerase chain reaction to develop a quantitative method of measurement of PML-RARalpha transcripts which are hallmarks of acute promyelocytic leukemia (APL) with t(15;17) translocation. Indeed, although quantitation of minimal residual disease has proved to be useful in predicting clinical outcome in other leukemias such as chronic myeloid leukemia or acute lymphoblastic leukemia, no quantitative data have been provided in the case of APL. We present here a method for quantitation of the most frequent subtypes of t(15;17) transcripts (namely bcr1 and bcr3). One specific forward primer is used for each subtype in order to keep amplicon length under 200 bp. The expression of PML-RARalpha transcripts is normalized using the housekeeping porphobilinogen deaminase (PBGD) gene. This technique allows detection of 10 copies of PML-RARalpha or PBGD plasmids, and quantitation was efficient up to 100 copies. One t(15;17)-positive NB4 cell could be detected among 106 HL60 cells, although quantitation was efficient up to one cell among 105. Repeatability and reproducibility of the method were satisfying as intra- and inter-assay variation coefficients were not higher than 15%. The efficiency of the method was finally tested in patient samples, showing a decrease of the PML-RARalpha copy number during therapy, and an increase at the time of relapse.
Assuntos
Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 17/genética , Leucemia Promielocítica Aguda/diagnóstico , Leucemia Promielocítica Aguda/genética , Receptores do Ácido Retinoico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Translocação Genética , Sondas de DNA , DNA de Neoplasias/análise , DNA de Neoplasias/química , Humanos , Neoplasia Residual/diagnóstico , Neoplasia Residual/genética , RNA Neoplásico/análise , RNA Neoplásico/química , Reprodutibilidade dos Testes , Receptor alfa de Ácido Retinoico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e EspecificidadeRESUMO
UNLABELLED: The IVA/diagonal coronary bifurcation is a high risk area for atheromatous disease. Major technical and strategic risks make the treatment of these lesions a real "challenge" for the interventional cardiologist: angioplasty-stenting and surgery are in direct competition. OBJECTIVES: the aim of this study was to determine the significance of interventional techniques in treating the IVA/diagonal bifurcation, drawing on the experiences of a cardiological haemodynamic laboratory and comparing these results with those obtained with the reference technique, represented by aorto-coronary bypass with the internal mammary artery. METHODS: this was a monocentric retrospective study of a series of 133 patients treated with angioplasty-stenting between January 1997 and December 2002 for a new IVA/Dg bifurcation lesion. Patients admitted for myocardial infarction were excluded. RESULTS: no matter which angioplasty revascularisation technique was used, the angiographic success rate was 95% with 3% occlusions of the diagonal artery. At six months, 72% of patients were asymptomatic, the rate of treated lesion revascularisation (TLR) was 21.9%. At one year 68.8% of patients were asymptomatic, and the TLR was 24.2%. The technique evolved during the six years, especially with the expansion of the radial approach and increasingly frequent use of the "kissing balloon"; at one year the TLR and the rate of major cardiac events was 31% in 1997 and 15% in 2002. CONCLUSIONS: angioplasty-stenting in this at-risk region is thus favourable, and gives results comparable with those of internal mammary/IVA bypass, save on the TLR. However, the development of stents "pre-formed" for the bifurcation and especially "active" endoprostheses could represent a solution to the delicate problem of restenosis.
Assuntos
Angioplastia Coronária com Balão/métodos , Vasos Coronários/cirurgia , Idoso , Arteriosclerose/prevenção & controle , Angiografia Coronária , Feminino , Ventrículos do Coração , Hemodinâmica , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio , Estudos Retrospectivos , Fatores de Risco , Stents , Resultado do TratamentoRESUMO
BACKGROUND: To determine the extent to which major histoincompatibilities are recognized after bone marrow transplantation, we characterized the specificity of the cytotoxic T lymphocytes isolated during graft-versus-host disease. We studied three patients transplanted with marrow from donors who were histoincompatible for different types of HLA antigens. METHODS: Patient 1 was mismatched for one "ABDR-antigen" (HLA-A2 versus A3). Two patients were mismatched for antigens that would usually not be taken into account by standard selection procedures: patient 2 was mismatched for an "HLA-A subtype" (A*0213 versus A*0201), whereas patient 3 was mismatched for HLA-C (HLA-C*0501 versus HLA-C*0701). All three HLA class I mismatches were detected by a pretransplant cytotoxic precursor test. RESULTS: Analysis of the specificity of the cytotoxic T lymphocyte clones isolated after transplantation showed that the incompatibilities detected by the pretransplant cytotoxic precursor assay were the targets recognized during graft-versus-host disease. CONCLUSIONS: Independent of whether the incompatibility consisted of a "full" mismatch, a "subtype" mismatch, or an HLA-C mismatch, all clones recognized the incompatible HLA molecule. In addition, some of these clones had undergone antigen selection and were clearly of higher specificity than the ones established before transplantation, indicating that they had been participating directly in the antihost immune response.
Assuntos
Transplante de Medula Óssea/imunologia , Doença Enxerto-Hospedeiro/imunologia , Antígenos HLA/imunologia , Adolescente , Adulto , Antígeno HLA-A2/análise , Antígeno HLA-A3/análise , Antígenos HLA-B/análise , Antígenos HLA-C/análise , Células-Tronco Hematopoéticas/imunologia , Teste de Histocompatibilidade , Humanos , Masculino , Linfócitos T Citotóxicos/citologiaRESUMO
As compared to bone marrow, transplantation with cord blood (CB) is associated with a lower risk of GVHD. Although some cases of GVHD III have been reported, usually the grade of GVHD remains low, even when donor and recipient are not fully HLA matched. To investigate whether this is due to an intrinsic property of CB T cells, we studied the conditions under which CB CD8+ cells would be able to generate an alloresponse. In addition, we measured the cytokines secreted by the alloreactive cytotoxic T lymphocyte (CTL) clones generated. We show that: (1) the capacity of CB cells to generate alloreactive CTLs in vitro is diminished as compared to adult cells (AB); (2) in the presence of exogenous IL-2, CB cells do generate a normal alloreactive response; (3) although CB-derived CD8+ allospecific clones showed the same T1/T0 cytokine profile as the clones from AB, they secreted a lower amount of IFN-gamma; and (4) in addition, cloned CD4+ CB cells are mainly T2/T0 type, whereas AB CD4+ T cells were mainly of T1/T0 type. These data suggest that the reduced GVHD observed after transplantation with CB might be the result of the naiveness of CB T cells. After allostimulation, CB cytotoxic T cells differ from AB T cells by a higher activation threshold, a lower secretion of IFN-gamma by both CD4+ and CD8+ CB cells and their bias towards a T2 type CD4 response.
Assuntos
Citotoxicidade Imunológica/imunologia , Sangue Fetal/imunologia , Adulto , Citocinas/biossíntese , Citotoxicidade Imunológica/efeitos dos fármacos , Sangue Fetal/efeitos dos fármacos , Doença Enxerto-Hospedeiro/imunologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/imunologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Recém-Nascido , Interleucina-2/farmacologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/imunologiaRESUMO
A 38-year-old man, blood group A+, was allotransplanted for multiple myeloma from his fully matched sister, blood group O+. Anti-A antibodies IgG and IgM titres of the donor were low. Allogeneic peripheral blood stem cells were harvested by leukapheresis after subcutaneous administration of G-CSF. Rapid engraftment occurred since 5.6 x 10(9)/l leukocytes were achieved on day +9 post-transplant. At this time a severe immune haemolytic syndrome occurred and direct antiglobulin test was positive (IgG and C3d). Elution showed an anti-A specificity. Evolution was rapidly unfavourable related to multiorgan failure. The patient died on day +20 post-transplant.
Assuntos
Sistema ABO de Grupos Sanguíneos/imunologia , Incompatibilidade de Grupos Sanguíneos/complicações , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Hemólise , Adulto , Humanos , Masculino , Transplante HomólogoRESUMO
Patients suffering from AIDS develop ocular complications, the most frequent being HIV retinopathy. It is however not clear, if functional visual impairments can be observed as early indicators of ocular complications, before clinical diagnosis of HIV retinopathy is made at fundus examination. To address this issue, we measured colour vision in a group of 49 AIDS subjects with normal clinical fundi using the 'two equation method'. This method, combining red-green Rayleigh and the blue-green Moreland metameric matches, enables more complete and quantitative assessments of colour vision than those based on pigmentary tests. Data were collected on our computer controlled colorimeter and compared to those of normal subjects. While most AIDS subjects without HIV retinopathy demonstrated normal colour vision, a significant portion of them had wider matches than normal subjects (11% for the Rayleigh equation and 16% for the Moreland equation). Furthermore, matching ranges of the Moreland equation were significantly correlated with CD4 lymphocyte counts. Patients with low CD4 values tended to produce larger matching ranges than the patients with high CD4 values. A within subject study on 17 patients confirmed this trend and showed that the patients who increased/decreased their CD4 blood counts generally improved/impaired their colour discrimination in the Moreland match. No such correlation was found between the matching ranges of the Rayleigh equation and the CD4 counts. These results show that colour discrimination is slightly reduced in some AIDS subjects, although there are no detectable ocular complications. They also suggest two different types of colour vision impairments in AIDS patients without retinopathy: one reversible process affecting colour discrimination in the blue-green range; and another irreversible process affecting colour discrimination in the red-green range.
Assuntos
Síndrome da Imunodeficiência Adquirida/fisiopatologia , Percepção de Cores/fisiologia , Síndrome da Imunodeficiência Adquirida/sangue , Adulto , Contagem de Linfócito CD4 , Testes de Percepção de Cores , Defeitos da Visão Cromática/diagnóstico , Feminino , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Rhodococcus equi is the most common infectious cause of mortality in foals between 1 and 6 months of age. Because of an increase in the number of antibiotic-resistant strains, the optimization of a prophylactic strategy is a key factor in the comprehensive management of R. equi pneumonia. The objectives of this study were to assess the safety and immunogenicity of R. equi-secreted proteins (ReSP) co-administered with either the nanoparticular adjuvant Montanide™ IMS 3012 VG, or a new polymeric adjuvant Montanide™ PET GEL A, and to further investigate the most immunogenic proteins for subsequent immunization/challenge experiments in the development of a vaccine against rhodoccocal pneumonia. The approach involved two phases. The first phase aimed to investigate the safety of vaccination in six adult horses. The second phase aimed to determine the safety and immunogenicity of vaccination in twelve 3-week-old foals. We set out to develop a method based on ultrasound measurements for safety assessment in adult horses in order to evaluate any in situ changes at the injection site, in the skin or the underlying muscle, with quantitative and qualitative data revealing that administration of ReSP combined with the Pet Gel A adjuvant led to an increase in local inflammation, associated with 4- to 7-fold higher levels of anti-R. equi IgGa, IgGb and IgGT, compared to administration of ReSP associated with IMS 3012 adjuvant, but without any impact on animal demeanor. Investigations were then performed in foals with serological and clinical follow-up until 6 months of age. Interestingly, we observed in foals a much lower incidence of adverse local tissue reactions at the injection site than in adult horses, with transient and moderate swelling for the group that received ReSP combined with Pet Gel A. Immunized foals with Pet Gel A adjuvant exhibited a similar response in both IgGa and IgGT levels, but a lower response in IgGb levels, compared to adult horses, with a subisotype profile that may however reflect a bias favorable to R. equi resistance. From the crude extract of secreted proteins, dot-blot screening enabled identification of cholesterol oxidase, mycolyl transferase 3, and PSP (probable secreted protein) as the most immunogenic candidates. Taken together, these results are encouraging in developing a vaccine for foals.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Rhodococcus equi/imunologia , Adjuvantes Imunológicos/administração & dosagem , Animais , Vacinas Bacterianas/efeitos adversos , Cavalos , Imunoglobulina G/sangue , Imunoglobulina G/classificação , Nanopartículas/administração & dosagem , Polímeros/administração & dosagemRESUMO
Iatrogenic acute dissection of the ascending aorta following coronary angiography and percutaneous intervention is rare. The options for treatment are dictated by patient stability, nature of dissection of the coronary vessel, ability to restore the coronary circulation and extent of aortic dissection. Usually localized aortic dissections have been managed conservatively or treated by sealing the entry with a coronary stent. Extensive dissections may require a surgical intervention. We report the case of a 52-year-old man with iatrogenic dissection of the right coronary artery ostium and extension of the dissection to the ascending aorta during intraluminal angioplasty of an obstructive lesion in the first portion of the right coronary artery. The patient was managed conservatively without stenting (failure stenting of the right coronary artery) and without surgery. Aortic dissection was monitored by means of transesophageal echocardiography. Serial computed tomography scans demonstrated spontaneous resolution of the dissection. The evolution of the patient was satisfactory. Causes, frequency and treatment procedures of this iatrogeny are discussed.
Assuntos
Angioplastia Coronária com Balão , Aorta/lesões , Vasos Coronários/lesões , Complicações Intraoperatórias , Humanos , Doença Iatrogênica , Complicações Intraoperatórias/terapia , Masculino , Pessoa de Meia-IdadeRESUMO
We have developed an in vitro assay for tetanus toxin (tt) C fragment (C-fr) degradation. Purified endosomes (abbreviated endosomes 1101 or 1104) and lysosomes (abbreviated lysosomes 1101 or 1104) from the DRB1*1101 (Gly 86) and DRB1*1104 (Val 86) B cell lines were used to degrade 125I-labeled C-fr in vitro. Using three distinct methods of analysis, we show that the capacity of endosomes and lysosomes to degrade the tt C-fr or tt synthetic Y-P30 peptide differed. Using sodium dodecylsulfate-polyacrylamide gel electrophoresis, 125I-labeled C-fr degradation patterns observed either with endosomes 1101/1104 or lysosomes 1101/1104 are distinct both in terms of the number of fragments and the kinetics of generation of the fragments. These results were confirmed by high-performance liquid chromatography analysis, where we observed that the elution profiles of the 125I-labeled Y-P30 peptide digested by endosomes 1101/1104 were different compared to those obtained with lysosomes 1101/1104. Furthermore, the kinetics of degradation of 125I-labeled Y-P30 were faster with lysosomes 1104 than with lysosomes 1101. This difference in activity of the 1101 and 1104 organelles was also found in a functional assay where we showed that the activation capacity of the P30 peptide was diminished when digested by lysosome 1104, regardless of the antigen-presenting cell (APC) used, whereas endosomes 1101 or lysosomes 1101 modified P30 peptide in a form that discriminated between presentation by 1101 or 1104 APC. Taken together, these results suggest that the differential processing and presentation displayed by the DRB1*1101 and DRB1*1104 APC is due partly to a different enzymatic content and partly to the dimorphism at position DR beta 86.
Assuntos
Células Apresentadoras de Antígenos/metabolismo , Antígenos/metabolismo , Antígenos HLA-DR/genética , Organelas/imunologia , Sequência de Aminoácidos , Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/ultraestrutura , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Endossomos/imunologia , Cadeias HLA-DRB1 , Humanos , Lisossomos/imunologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Toxina Tetânica/metabolismoRESUMO
Phospholipase A(2) (PLA(2)) is one of the major honey bee venom allergens for humans. To assess the long-term prevention of allergic reactions by DNA vaccination, a PLA(2)-CBA/J mouse model was employed using empty or PLA(2) sequence-carrying DNA plasmids. Early skin application of either DNA construct before (prophylactic approach) or after (therapeutic approach) sensitization with PLA(2)/alum led to reduced PLA(2)-specific IgE and IgG1 titers at 7 mo, with concomitant rise in IgG2a and IgG3. Splenocytes recovered at 5-6 mo after the last DNA administration exhibited a sustained IFN-gamma and IL-10 secretion and reduced IL-4 production. Recall challenge with PLA(2) boosted IFN-gamma and IL-10 secretion, suggesting the reactivation of quiescent memory Th1 lymphocytes. Mice from the prophylactic groups were fully protected against anaphylaxis, whereas 65% of the animals recovered in the therapeutic groups. Th1-polarized immune responses were also active in mice vaccinated with an empty plasmid 32 wk before sensitization with another Ag (OVA). This is the first demonstration that the Ag-coding sequence in DNA vaccine is not necessary to promote immune modulation in naive and sensitized animals for a prolonged period, and has relevance for the understanding of the innate and induced mechanisms underlying gene immunotherapy in long-term treatment of allergy.
Assuntos
Antígenos/fisiologia , Venenos de Abelha/imunologia , Dessensibilização Imunológica/métodos , Imunossupressores/administração & dosagem , Fosfolipases A/imunologia , Vacinas de DNA/imunologia , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/genética , Adjuvantes Imunológicos/uso terapêutico , Anafilaxia/imunologia , Anafilaxia/prevenção & controle , Animais , Especificidade de Anticorpos/genética , Antígenos/administração & dosagem , Venenos de Abelha/administração & dosagem , Células CHO , Células Cultivadas , Cricetinae , Citocinas/biossíntese , Citocinas/metabolismo , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/imunologia , Vetores Genéticos/uso terapêutico , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos CBA , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Fosfolipases A/administração & dosagem , Fosfolipases A/genética , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Células Th1/imunologia , Células Th1/metabolismo , Transfecção , Regulação para Cima/genética , Regulação para Cima/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/uso terapêuticoRESUMO
The response to tetanus toxoid (TT) was studied in immune donors that carry two alleles of DR5 that differ only at DR beta residue 86: DRB1*1101 (G86, abbreviated 1101) and DRB1*1104 (V86, abbreviated 1104). A large number of TT-specific T cell clones was isolated and the epitopes recognized in association with 1101 and 1104 were mapped. We found that two epitopes (p2 and p32) can be recognized in association with both 1101 and 1104 while three epitopes (p23, p27 and p30) are recognized in association with 1101, but never in association with 1104. The sets of naturally processed self peptides displayed by 1101 and 1104 were characterized using alloreactive T cell clones. We found that all 1104 alloreactive clones recognize both 1104 and 1101, while approximately 30% of the alloreactive 1101 clones fail to recognize 1104. Thus it is apparent that both naturally processed TT and self peptides displayed on 1104 molecules represent a fraction of those displayed on 1101 molecules. The mechanism responsible for this differential presentation was investigated by comparing the capacity of 1101 and 1104 antigen-presenting cells to present TT or synthetic peptides to specific T cells and by measuring the binding of these peptides to DR molecules. Three sets of results suggest that V86 acts as a constraint to the binding of naturally processed peptides: (i) all 1104-restricted or alloreactive T cell clones recognize TT- or allo-epitopes presented by 1101 as well, thus ruling out a major effect of V86 as a residue determining T cell restriction specificity; (ii) presentation of naturally processed peptides correlates in general with the capacity of long synthetic peptides to bind to 1101 or 1104 and (iii) while the naturally processed p30 epitope discriminates between 1101 and 1104, a short synthetic peptide binds equally well to and is comparably recognized in association with both 1101 and 1104. Taken together these results suggest that the natural polymorphism at residue 86 might be a molecular switch that finely tunes the complexity of the peptide collection presented on DR molecules.
Assuntos
Epitopos/imunologia , Antígeno HLA-DR1/imunologia , Peptídeos/imunologia , Polimorfismo Genético , Alelos , Sequência de Aminoácidos , Células Apresentadoras de Antígenos/imunologia , Sequência de Bases , Linhagem Celular , Células Cultivadas , Humanos , Ativação Linfocitária/imunologia , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Ligação Proteica , Linfócitos T/imunologia , Toxoide Tetânico/imunologiaRESUMO
BACKGROUND: Nasal administration of major peptide T cell epitopes gives contradictory data on the induction of peripheral tolerance. OBJECTIVE: To compare the prophylactic effect of intranasal treatment (INT) on the development of an allergic response, using either ovalbumin (OVA) or its major T cell epitope OVA 323-339 (OVAp). METHODS: BALB/c mice were treated intranasally with OVA or OVAp and subsequently immunized s.c. with OVA. Anti-OVA-specific antibody, T cell proliferation and cytokine responses were analysed. In an adoptive transfer model using OVAp specific TCR transgenic (Tg) T cells from D011.10 mice, in vivo tracking and characterization of transferred T cells in the cervical, inguinal and bronchial lymph nodes (BLN) and in the spleen were determined by FACS analysis. RESULTS: Prophylactic INT with OVA induced T cell tolerance towards subsequent OVA s.c. immunizations, inhibiting OVA specific T cell proliferation, IgE and IgG1 production, in contrast to INT with OVAp, which was unable to induce tolerance. In vivo analysis of transferred OVA-specific TCR Tg T cells showed that INT with OVA induced a preferential activation of T cells in BLN, as opposed to a broad, systemic activation with OVAp. In vivo, OVAp INT led to faster and more sustained cell division cycles than OVA INT. Ex vivo, tolerance to OVA was associated with the generation of IL-10 secreting CD4(+) T cells in BLN of OVA-treated mice only. CONCLUSION: INT with OVA but not with OVAp led to regional (as opposed to systemic) T cell activation and the induction of IL-10 secreting CD4(+) T cells in BLN, potentially critical steps in the induction of T cell-specific tolerance via the nasal route.
Assuntos
Hipersensibilidade/prevenção & controle , Tolerância Imunológica/imunologia , Interleucina-10/biossíntese , Ovalbumina/uso terapêutico , Subpopulações de Linfócitos T/imunologia , Administração Intranasal , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/imunologia , Divisão Celular/imunologia , Epitopos de Linfócito T/uso terapêutico , Feminino , Hipersensibilidade/imunologia , Imunoglobulina E/biossíntese , Imunoglobulina G/biossíntese , Ativação Linfocitária/imunologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/imunologia , Fragmentos de Peptídeos/uso terapêuticoRESUMO
The capacity of peripheral blood lymphocytes (PBL) or Epstein-Barr virus (EBV)-transformed B cell lines from CGD patients to process and present tetanus toxin (tt)-specific epitopes was assessed using various tt preparations and human tt-specific T cell clones. PBL from all of the donors were able to process and present either native tt and/or denatured tt to human T cell clones specific for various tt epitopes. Furthermore, no difference was found in the antigen requirement when normal or CGD EBV-B cell lines were used as antigen-presenting cells (APC). These results suggest that the deficiency in oxygen metabolism in CGD cells does not affect tt processing and presentation.
Assuntos
Apresentação de Antígeno , Células Apresentadoras de Antígenos/fisiologia , Doença Granulomatosa Crônica/imunologia , Linfócitos T/imunologia , Toxina Tetânica/imunologia , Sequência de Aminoácidos , Linfócitos B/fisiologia , Linhagem Celular Transformada , Células Clonais , Herpesvirus Humano 4 , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Dados de Sequência MolecularRESUMO
MHC class II molecules present to CD4+ T cells protein fragments which mostly derive from the extracellular and from the endosomal compartments. Determinants of cytosolic proteins are, however, also displayed by MHC class II molecules following pathways which are still not yet fully characterized. Here we describe the isolation of DRB1*1103-restricted T cell clones specific for the measles virus (MV) nucleoprotein peptide 185-199 (N185). Experiments were then conducted to delineate how this determinant is assembled with DR molecules. In vitro binding analyses indicated that complexes between the N185 peptide and DRB1*1103 protein are optimally constituted at pH 4-4.5. In cellular experiments it was observed that chloroquine, leupeptin and emetine, which are classical inhibitors of presentation of MHC class II-restricted antigens, when added during infection of B cells with MV, prevent presentation of the N185 determinant. In addition, it was found that the N185 determinant is efficiently presented when the nucleoprotein is exogenously provided to B cells, either by blocking MV fusion with the peptide FFG or by the use of purified nucleoprotein. In contrast, it was observed that nucleoprotein recombinant vaccinia virus (vv-N)-infected B cells weakly stimulated N185-specific T cells, indicating that the restricted localization of the nucleoprotein in the cytosol resulted in a poor presentation of the N185 determinant. Taken together, these findings suggest that it is prior to delivery of the nucleoprotein into the cytosol that the N185 determinant is efficiently assembled with newly synthesized DR molecules in the acidic environment of the endosomal compartment.
Assuntos
Apresentação de Antígeno/imunologia , Endossomos/metabolismo , Epitopos de Linfócito T/imunologia , Antígenos HLA-DR/imunologia , Vírus do Sarampo/imunologia , Nucleoproteínas/imunologia , Proteínas Virais/imunologia , Sequência de Aminoácidos , Cadeias HLA-DRB1 , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Proteínas do Nucleocapsídeo , Peptídeos/imunologia , Linfócitos T/imunologiaRESUMO
We have determined the mechanism of neutralization of influenza virus infectivity by three antihemagglutinin monoclonal antibodies, the structures of which we have analyzed before as complexes with hemagglutinin. The antibodies differ in their sites of interaction with hemagglutinin and in their abilities to interfere in vitro with its two functions of receptor binding and membrane fusion. We demonstrate that despite these differences all three antibodies neutralize infectivity by preventing virus from binding to cells. Neutralization occurs at an average of one antibody bound per four hemagglutinins, a ratio sufficient to prevent the simultaneous receptor binding of hemagglutinins that is necessary to attach virus to cells.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza A/patogenicidade , Animais , Sítios de Ligação de Anticorpos , Linhagem Celular , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Testes de NeutralizaçãoRESUMO
We have studied the alterations in CD45R phenotypes of CD4(+)CD45RA(-)RO(+) T cells in recipients of T cell-depleted bone marrow grafts. These patients are convenient models because early after transplantation, their T cell compartment is repopulated through expansion of mature T cells and contains only cells with a memory phenotype. In addition, re-expression of CD45RA by former CD4(+)CD45RA(-) T cells can be accurately monitored in the pool of recipient T cells that, in the absence of recipient stem cells, can not be replenished with CD45RA(+) T cells through the thymic pathway. We found that CD4(+)CD45RA(-)RO(+) recipient T cells could re-express CD45RA but never reverted to a genuine CD4(+)CD45RA(+)RO(-) naive phenotype. Even 5 years after transplantation, they still co-expressed CD45RO. In addition, the level of CD45RA and CD45RC expression was lower ( approximately 35 %) than that of naive cells. In contrast, the level of CD45RB expression was comparable to that of naive cells. We conclude that CD4(+)CD45RA(-)RO(+) T cells may re-express CD45(high) isoforms but remain distinguishable from naive cells by their lower expression of CD45RA / RC and co-expression of CD45RO. Therefore, it is likely that the long-lived memory T cell will be found in the population expressing both low and high molecular CD45 isoforms.