RESUMEN
Cytomegalovirus (CMV) infection is a common, potentially life-threatening complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). We assessed prospectively the safety and efficacy of stem cell-donor- or third-party-donor-derived CMV-specific T cells for the treatment of persistent CMV infections after allo-HSCT in a phase I/IIa trial. Allo-HSCT patients with drug-refractory CMV infection and lacking virus-specific T cells were treated with a single dose of ex vivo major histocompatibility complex-Streptamer-isolated CMV epitope-specific donor T cells. Forty-four allo-HSCT patients receiving a T-cell-replete (D+ repl; n=28) or T-cell-depleted (D+ depl; n=16) graft from a CMV-seropositive donor were screened for CMV-specific T-cell immunity. Eight D+ depl recipients received adoptive T-cell therapy from their stem cell donor. CMV epitope-specific T cells were well supported and became detectable in all treated patients. Complete and partial virological response rates were 62.5% and 25%, respectively. Owing to longsome third-party donor (TPD) identification, only 8 of the 57 CMV patients transplanted from CMV-seronegative donors (D-) received antigen-specific T cells from partially human leukocyte antigen (HLA)-matched TPDs. In all but one, TPD-derived CMV-specific T cells remained undetectable. In summary, adoptive transfer correlated with functional virus-specific T-cell reconstitution in D+ depl patients. Suboptimal HLA match may counteract expansion of TPD-derived virus-specific T cells in D- patients.
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Infecciones por Citomegalovirus/terapia , Citomegalovirus/inmunología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunoterapia Adoptiva/métodos , Linfocitos T/trasplante , Viremia/terapia , Aloinjertos , Antivirales/uso terapéutico , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/etiología , Infecciones por Citomegalovirus/transmisión , Farmacorresistencia Viral , Femenino , Supervivencia de Injerto , Neoplasias Hematológicas/terapia , Histocompatibilidad , Humanos , Huésped Inmunocomprometido , Inmunoterapia Adoptiva/efectos adversos , Depleción Linfocítica , Masculino , Síndromes Mielodisplásicos/terapia , Estudios Prospectivos , Especificidad del Receptor de Antígeno de Linfocitos T , Donantes de Tejidos , Viremia/tratamiento farmacológico , Viremia/etiologíaRESUMEN
Schistosome infections are renowned for their ability to induce regulatory networks such as regulatory T cells (Treg) that control immune responses against homologous and heterologous antigens such as allergies. However, in the case of co-infections with hepatitis C virus (HCV), schistosomes accentuate disease progression and we hypothesized that expanding schistosome-induced Treg populations change their phenotype and could thereby suppress beneficial anti-HCV responses. We therefore analysed effector T cells and n/iTreg subsets applying the markers Granzyme B (GrzB) and Helios in Egyptian cohorts of HCV mono-infected (HCV), schistosome-co-infected (Sm/HCV) and infection-free individuals. Interestingly, viral load and liver transaminases were significantly elevated in Sm/HCV individuals when compared to HCV patients. Moreover, overall Treg frequencies and Helios(pos) Treg were not elevated in Sm/HCV individuals, but frequencies of GrzB(+) Treg were significantly increased. Simultaneously, GrzB(+) CD8(+) T cells were not suppressed in co-infected individuals. This study demonstrates that in Sm/HCV co-infected cohorts, liver disease is aggravated with enhanced virus replication and Treg do not expand but rather change their phenotype with GrzB possibly being a more reliable marker than Helios for iTreg. Therefore, curing concurrent schistosome disease could be an important prerequisite for successful HCV treatment as co-infected individuals respond poorly to interferon therapy.
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Coinfección/inmunología , Hepacivirus/fisiología , Hepatitis C Crónica/inmunología , Schistosoma mansoni/fisiología , Esquistosomiasis mansoni/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Animales , Femenino , Humanos , Interleucina-8/inmunología , Hígado/patología , Hígado/virología , Masculino , Persona de Mediana Edad , Carga ViralRESUMEN
Overall asthmatic symptoms can be controlled with diverse therapeutic agents. However, certain symptomatic individuals remain at risk for serious morbidity and mortality, which prompts the identification of novel therapeutic targets and treatment strategies. Thus, using an adjuvant-free T helper type 2 (Th2) murine model, we have deciphered the role of interleukin (IL)-1 signalling during allergic airway inflammation (AAI). Because functional IL-1ß depends on inflammasome activation we first studied asthmatic manifestations in specific inflammasome-deficient [NACHT, LRR and PYD domains-containing protein 3 (NLRP3(-/-) ) and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC(-/-) )] and IL-1 receptor type 1(-/-) (IL-1R1(-/-) ) mice on the BALB/c background. To verify the onset of disease we assessed cellular infiltration in the bronchial regions, lung pathology, airway hyperresponsiveness and ovalbumin (OVA)-specific immune responses. In the absence of NLRP3 inflammasome-mediated IL-1ß release all symptoms of AAI were reduced, except OVA-specific immunoglobulin levels. To address whether manipulating IL-1 signalling reduced asthmatic development, we administered the IL-1R antagonist anakinra (Kineret®) during critical immunological time-points: sensitization or challenge. Amelioration of asthmatic symptoms was only observed when anakinra was administered during OVA challenge. Our findings indicate that blocking IL-1 signalling could be a potential complementary therapy for allergic airway inflammation.
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Proteínas Portadoras/metabolismo , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Hipersensibilidad Respiratoria/metabolismo , Enfermedad Aguda , Animales , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Adaptadoras de Señalización CARD , Proteínas Portadoras/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Eosinofilia/genética , Eosinofilia/inmunología , Femenino , Células Caliciformes/patología , Hiperplasia , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Ratones , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR , Ovalbúmina/efectos adversos , Neumonía/genética , Neumonía/inmunología , Neumonía/metabolismo , Neumonía/patología , Receptores Tipo I de Interleucina-1/deficiencia , Hipersensibilidad Respiratoria/inducido químicamente , Hipersensibilidad Respiratoria/tratamiento farmacológico , Hipersensibilidad Respiratoria/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismoRESUMEN
T cells have been proven to be therapeutically effective in patients with relapsed leukemias, although target antigens on leukemic cells as well as T-cell receptors (TCRs), potentially recognizing those antigens, are mostly unknown. We have applied an immunopeptidomic approach and isolated human leukocyte antigen (HLA) ligands from primary leukemia cells. We identified a number of ligands derived from different genes that are restrictedly expressed in the hematopoietic system. We exemplarily selected myeloperoxidase (MPO) as a potential target and isolated a high-avidity TCR with specificity for a HLA-B*07:02-(HLA-B7)-restricted epitope of MPO in the single HLA-mismatched setting. T cells transgenic for this TCR demonstrated high peptide and antigen specificity as well as leukemia reactivity in vitro and in vivo. In contrast, no significant on- and off-target toxicity could be observed. In conclusion, we here demonstrate, exemplarily for MPO, that leukemia-derived HLA ligands can be selected for specific effector tool development to redirect T cells to be used for graft manipulation or adoptive T-cell therapies in diverse transplant settings. This approach can be extended to other HLA ligands and HLA molecules in order to provide better treatment options for this life-threatening disease.
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Antígenos HLA/inmunología , Leucemia Mieloide/genética , Leucemia Mieloide/inmunología , Péptidos/inmunología , Peroxidasa/inmunología , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Presentación de Antígeno/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Línea Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Antígenos HLA/metabolismo , Antígeno HLA-B7/inmunología , Antígeno HLA-B7/metabolismo , Xenoinjertos , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Leucemia Mieloide/metabolismo , Leucemia Mieloide/mortalidad , Ligandos , Ratones , Péptidos/metabolismo , Peroxidasa/química , Peroxidasa/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Transducción GenéticaRESUMEN
BACKGROUND: The development of a successful immunotherapy is hampered by an ineffective T-cell repertoire against tumour antigens and the inability of the patient's immune system to overcome tolerance-inducing mechanisms. Here, we test the specific recognition and lytical potential of allo-restricted CD8(+) T cells against Ewing tumour (ET) associated antigens Enhancer of Zeste, Drosophila Homolog 2 (EZH2), and Chondromodulin-I (CHM1) identified through previous microarray analysis. METHODS: Following repetitive CHM1(319) (VIMPCSWWV) and EZH2(666) (YMCSFLFNL) peptide-driven stimulations with HLA-A 0201(+) dendritic cells (DC), allo-restricted HLA-A 0201(-) CD8(+) T cells were stained with HLA-A 0201/peptide multimers, sorted and expanded by limiting dilution. RESULTS: Expanded T cells specifically recognised peptide-pulsed target cells or antigen-transfected cells in the context of HLA-A 0201 and killed HLA-A 0201(+) ET lines expressing the antigen while HLA-A 0201(-) ET lines were not affected. Furthermore, adoptively transferred T cells caused significant ET growth delay in Rag2(-/-)γ(C)(-/-) mice. Within this context, we identified the CHM1(319) peptide as a new candidate target antigen for ET immunotherapy. CONCLUSION: These results clearly identify the ET-derived antigens, EZH2(666) and CHM1(319), as suitable targets for protective allo-restricted human CD8(+) T-cell responses against non-immunogenic ET and may benefit new therapeutic strategies in ET patients treated with allogeneic stem cell transplantation.
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Antígenos de Neoplasias/inmunología , Neoplasias Óseas/patología , Isoantígenos/inmunología , Sarcoma de Ewing/patología , Linfocitos T Citotóxicos/inmunología , Animales , Antígenos de Neoplasias/metabolismo , Neoplasias Óseas/inmunología , Línea Celular Tumoral , Proliferación Celular , Células Cultivadas , Citotoxicidad Inmunológica/inmunología , Proteínas de Unión al ADN/genética , Regulación hacia Abajo , Humanos , Inmunoterapia Adoptiva , Isoantígenos/metabolismo , Células K562 , Ratones , Ratones Noqueados , Sarcoma de Ewing/inmunología , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
The FMS-like tyrosine kinase 3 (FLT3) is highly expressed in acute myeloid leukemia (AML). Internal tandem duplications (ITD) of the juxtamembrane domain lead to the constitutive activation of the FLT3 kinase inducing the activation of multiple genes, which may result in the expression of leukemia-associated antigens (LAAs). We analyzed the regulation of LAA in FLT3-wild-type (WT)- and FLT3-ITD(+) myeloid cells to identify potential targets for antigen-specific immunotherapy for AML patients. Antigens, such as PR-3, RHAMM, Survivin, WT-1 and PRAME, were upregulated by constitutively active FLT3-ITD as well as FLT3-WT activated by FLT3 ligand (FL). Cytotoxic T-cell (CTL) clones against PR-3, RHAMM, Survivin and an AML-directed CTL clone recognized AML cell lines and primary AML blasts expressing FLT3-ITD, as well as FLT3-WT(+) myeloid dendritic cells in the presence of FL. Downregulation of FLT3 led to the abolishment of CTL recognition. Comparing our findings concerning LAA upregulation by the FLT3 kinase with those already made for the Bcr-Abl kinase, we found analogies in the LAA expression pattern. Antigens upregulated by both FLT3 and Bcr-Abl may be promising targets for the development of immunotherapeutical approaches against myeloid leukemia of different origin.
RESUMEN
The non-imprinted in Prader-Willi/Angelman syndrome (NIPA) proteins are highly conserved receptors or transporters. Translocation of NIPA genes were found in patients with Prader-Willi syndrome, and loss-of-function of the NIPA1 gene was identified in hereditary spastic paraplegia. The family of NIPA-like domain containing (NPAL) proteins is closely related to the NIPA proteins, but to date nothing is known about their function. Here, we could demonstrate that both human NPAL3 and mouse NPAL3 are ubiquitously expressed and encode highly conserved proteins. To further elucidate the function of the Npal3 gene, knockout (Npal3(-/-)) mice were generated. Intensive phenotypic analyses revealed that disruption of the Npal3 gene results in a pleiotropic phenotype. The function of the nervous system was impaired in both mutant males and females which could be demonstrated in behavioral tests. In addition, in NPAL3 mutants the number of NK cells was decreased and changes in IgM, IgG(2), and IgA were observed, indicating that the immune system is also affected. Interestingly, increased IgE levels as well as impaired lung functions were observed in mutant males but not in mutant females. It should be noted that the human Npal3 gene is located at 1p36.12-->p35.1, and atopic diseases were previously linked to this genomic region. Thus, the Npal3(-/-) mice could serve as a valuable model system for studying atopic diseases.
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Conducta Animal , Inmunoglobulina E/sangre , Pulmón/fisiología , Proteínas de la Membrana/genética , Secuencia de Aminoácidos , Animales , Proteínas de Transporte de Catión , Membrana Celular/metabolismo , Secuencia Conservada , Evolución Molecular , Femenino , Expresión Génica , Humanos , Inmunoglobulina E/inmunología , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Fenotipo , Filogenia , Alineación de SecuenciaRESUMEN
The German Mouse Clinic (GMC) is a large scale phenotyping center where mouse mutant lines are analyzed in a standardized and comprehensive way. The result is an almost complete picture of the phenotype of a mouse mutant line--a systemic view. At the GMC, expert scientists from various fields of mouse research work in close cooperation with clinicians side by side at one location. The phenotype screens comprise the following areas: allergy, behavior, clinical chemistry, cardiovascular analyses, dysmorphology, bone and cartilage, energy metabolism, eye and vision, host-pathogen interactions, immunology, lung function, molecular phenotyping, neurology, nociception, steroid metabolism, and pathology. The German Mouse Clinic is an open access platform that offers a collaboration-based phenotyping to the scientific community (www.mouseclinic.de). More than 80 mutant lines have been analyzed in a primary screen for 320 parameters, and for 95% of the mutant lines we have found new or additional phenotypes that were not associated with the mouse line before. Our data contributed to the association of mutant mouse lines to the corresponding human disease. In addition, the systemic phenotype analysis accounts for pleiotropic gene functions and refines previous phenotypic characterizations. This is an important basis for the analysis of underlying disease mechanisms. We are currently setting up a platform that will include environmental challenge tests to decipher genome-environmental interactions in the areas nutrition, exercise, air, stress and infection with different standardized experiments. This will help us to identify genetic predispositions as susceptibility factors for environmental influences.
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Investigación Biomédica/métodos , Modelos Animales de Enfermedad , Ratones Mutantes/genética , Fenotipo , Crianza de Animales Domésticos , Animales , Investigación Biomédica/normas , Alemania , Ratones , Ratones Mutantes/crecimiento & desarrollo , Control de CalidadRESUMEN
OBJECTIVE: In a large-scale ENU (N-ethyl-N-nitrosourea) mouse mutagenesis programme, we previously have identified and characterized a novel mutation Ali18 that causes inflammatory arthritis like lesions in peripheral joints. In this study, we analysed the immune system of Ali18 mice to understand mechanisms underlying the spontaneous inflammation. METHODS: Humoral and cellular components of the immune system were phenotyped by ELISA and flow cytometry. The contribution of the immune system for phenotype expression was analysed in disease transfer experiments. The involvement of the adaptive immune system was investigated in Ali18;Rag1 double mutants and the influence of environmental factors was analysed in Ali18 mice reared under germ-free conditions. RESULTS: Bone marrow cells from Ali18 mice were able to transfer the disease phenotype to naïve wild-type recipients suggesting that cellular components of the reconstituted immune system were sufficient to induce arthritis. Ali18 mice revealed abnormal leucocyte populations including lymphocytes and granulocytes, as well as increased plasma IL-5 and IgE levels. Ali18;Rag1 double homozygous mutants, which lack mature lymphocytes, still developed arthritis, suggesting that the phenotype is independent of the adaptive immune system. In addition, the arthritis phenotype appeared to be independent from environmental conditions as demonstrated in mice reared under germ-free conditions. CONCLUSIONS: The Ali18 mutation induces inflammatory arthritis through bone marrow-derived cells. However, non-pro-inflammatory cytokine cascades and mature lymphocyte independent-mechanisms are crucial for initiation and progression of the phenotype. Ali18 mice may thus represent a model to study mechanisms involved in seronegative arthritis induced by cells of the innate immune system.
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Artritis Experimental/inmunología , Artritis Reumatoide/inmunología , Linfocitos B/inmunología , Linfocitos T/inmunología , Animales , Artritis Experimental/genética , Artritis Reumatoide/genética , Linfocitos B/metabolismo , Células de la Médula Ósea/inmunología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Citometría de Flujo , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Probabilidad , Distribución Aleatoria , Valores de Referencia , Sensibilidad y Especificidad , Linfocitos T/metabolismoRESUMEN
This first study of O(2) oxidation (autoxidation) of substituted phenols catalyzed by a dioxygen carrier in supercritical carbon dioxide (scCO(2)) provides additional insights into the established mechanism of reactions that have been much studied in conventional solvents. As has been long believed, the cobalt(II) dioxygen carriers of the class represented by [[N,N'-bis(3,5-di-tert-butylsalicylidene)-1,2-cyclohexanediaminato(2-)]cobalt(II)], Co(salen), show both oxidase and oxygenase activities during oxygenation of substituted phenols in scCO(2). The catalytic autoxidation of 2,6-di-tert-butylphenol (DTBP) and 3,5-di-tert-butylphenol (35-DTBP) in scCO(2) was studied by analysis of products in batch reactions with carefully controlled variables, in the presence of a large excess of O(2), at 207 bar of total pressure and a reaction temperature of 70 degrees C. The oxidation of 35-DTBP yielded only traces of products under the same experimental conditions that converted DTBP totally to a mixture of the oxygenation product 2,6-di-tert-butyl-1,4-benzoquinone (DTBQ) and the related product of radical coupling 3,5,3',5'-tetra-tert-butyl-4,4'-diphenoquinone (TTDBQ). The effects on conversion of DTBP to products and on selectivity between the two products were studied for variations in temperature and the concentrations of catalyst, oxygen, and methylimidazole. Selectivity in favor of the O-transfer product DTBQ over the self-coupling of the phenoxy radical was observed upon changing the oxygen concentration. In contrast, selectivity remained unaffected over a wide range of temperatures and catalyst concentrations. The oxygen dependence of both the conversion and selectivity showed saturation effects identifying the dioxygen complex as the effective oxidant in both the initial radical formation step and the oxygenation of that radical. No direct reaction is observed between the electrophilic phenoxy radical and O(2).
RESUMEN
The crystal structures are reported of trans-dioxocyclam dihydrate, C(10)H(20)N(4)O(2).2H(2)O, a structural isomer of the well known cis-dioxocyclam, and of its novel Ni complex, (1,4,8,11-tetraazacyclotetradecane-2,9-dionato-kappa(4)N)nickel(II) dihydrate, [Ni(C(10)H(18)N(4)O(2))].2H(2)O, the first example of a transition metal complex of this ligand. Both molecules lie on crystallographic centres of inversion. The free ligand has two of its N atoms turned outwards from the ring and hydrogen bonded to water molecules. A major conformational change takes place in the complex in which the ligand binds in a trans tetradentate fashion, as suggested by the electronic spectrum. The nickel(II) ion is low spin, although the electronic spectrum of the complex in water indicates an equilibrium mixture of low-spin and high-spin species. The irreversible electrochemical oxidation of [NiL(1)] (L(1) is deprotonated trans-dioxocyclam, C(10)H(18)N(4)O(2)) in water occurs at a potential of 0.964 V [versus SHE (standard hydrogen electrode)], which is very similar to that for the Ni-cis-dioxocyclam complex.
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We recently constructed IL-15 transgenic (Tg) mice using cDNA encoding a secretable isoform of the IL-15 precursor protein under the control of an MHC class I promoter. The IL-15 Tg mice exhibited resistance against a primary infection with Listeria monocytogenes. The numbers of memory CD8(+) T cells were markedly increased in the IL-15 Tg mice following Listeria infection accompanied by sustained IL-15 production. The increased CD44(+)CD8(+) T cells in the infected IL-15 Tg mice were not specialized to recognize Listeria-specific antigen but produced a large amount of IFN-gamma in response to bystander stimulation exogenous IL-15 in combination with IL-12. Furthermore, Listeria-specific Th1 response by CD4(+) T cells was significantly augmented in the IL-15 Tg mice compared with control mice following Listeria infection. In vivo depletion of the CD8(+) T cells by anti-CD8 monoclonal antibody and adoptive transfer of the T cells from naive IL-15 Tg mice indicated that the CD8(+) T cells functioned not only to eliminate bacteria at the early stage of infection but also to promote Th1 response to L. monocytogenes. Overexpression of IL-15 shed light on a novel role of memory CD8(+) T cells in early protection and promotion of Th1 response against a primary infection with L. monocytogenes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Interleucina-15/fisiología , Listeriosis/inmunología , Traslado Adoptivo , Animales , Antígenos Bacterianos/inmunología , Linfocitos T CD8-positivos/trasplante , Células Cultivadas , Interleucina-12/farmacología , Interleucina-15/biosíntesis , Interleucina-15/genética , Cinética , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/inmunología , Listeriosis/microbiología , Listeriosis/terapia , Ganglios Linfáticos/inmunología , Depleción Linfocítica , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Peritoneo/inmunología , Fenotipo , Tasa de Supervivencia , Subgrupos de Linfocitos T/clasificaciónRESUMEN
A family of Mn3+ and Fe3+ complexes of 4,11-dimethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane (1) and 4,10-dimethyl-1,4,7,10-tetraazabicyclo[5.5.2]tetradecane (2) has been prepared by the chemical oxidation of the divalent manganese and iron analogues. The ligands are ethylene cross-bridged tetraazamacrocycles derived from cylam and cyclen, respectively. The synthesis and characterization of these complexes, including X-ray crystal structure determinations, are described. The structural evidence demonstrates that the tetradentate ligands enforce distorted octahedral geometries on the metal ions, with two cis sites occupied by labile ligands. Magnetic measurements reveal that the complexes are high spin with typical magnetic moments. Cyclic voltammetry shows reversible redox processes for the Fe3+/Fe2+ couples of the iron(III) complexes, while Mn3+/Mn2+ and Mn4+/Mn3+ couples were observed for the complexes with manganese(III). The manganese chemistry of 1 was studied in depth. The dichloro manganese(III) cation of 1 undergoes facile ligand substitution reactions at the labile, monodentate sites, for example substituting azide for chloride ligands. Air oxidation of the dichloro complex of Mn (1)2+ in basic solution does not give the expected mu-oxo dimeric product common to manganese. Instead, an unusual manganese(III)-OH complex has been isolated from this reaction and structurally characterized. A similar reaction under slightly different conditions gives a putative MnIII(OH)2 complex that metathesizes to MnIII(OMe)2 upon recrystallization from methanol.
RESUMEN
NKG2D, a homodimeric lectin-like receptor, is a unique stimulatory molecule that is found on natural killer cells,T cells and activated macrophages. The natural ligands for murine NKG2D are distant major histocompatibility complex homologs, retinoic acid early transcript (Rae1) and H-60 minor histocompatibility antigen. The crystal structure of the extracellular region of murine NKG2D reveals close homology with other C-type lectin receptors such as CD94, Ly49A, rat MBP-A and CD69. However, the precise mode of dimeric assembly varies among these natural killer receptors, as well as their surface topography and electrostatic properties. The NKG2D structure provides the first structural insights into the role and ligand specificity of this stimulatory receptor in the innate and adaptive immune system.
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Cristalografía por Rayos X , Células Asesinas Naturales/inmunología , Receptores Inmunológicos/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Dimerización , Disulfuros/química , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos , Lectinas/química , Lectinas Tipo C , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Subfamilia K de Receptores Similares a Lectina de Células NK , Unión Proteica , Isoformas de Proteínas/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Inmunológicos/inmunología , Receptores de Células Asesinas Naturales , Homología de Secuencia de AminoácidoRESUMEN
H2-M3-restricted presentation of N-formyl methionine (f-Met) peptides to CD8(+) T cells provides a mechanism for selective recognition of bacterial infection. In this report we demonstrate that Listeria monocytogenes infection induces distinct CD8(+) T cell populations specific for each of the known Listeria-derived formyl methionine peptides presented by M3. The sum H2-M3-restricted, Listeria-specific T cell response constitutes a major fraction of the total CD8(+) T cell response to primary infection. H2-M3-restricted T cell populations expand synchronously in vivo and achieve peak frequencies approximately 2 days earlier than MHC class Ia-restricted T cell populations. Although cross-recognition of different f-Met peptides by M3-restricted T cells was previously described, costaining of CD8(+) T cells ex vivo with H2-M3 tetramers complexed with different f-Met peptides shows that the majority of Listeria-specific, M3-restricted CD8(+) T cells are peptide specific. In contrast to the highly predictable size and immunodominance hierarchies of MHC class Ia-restricted T cell responses, the magnitudes of T cell responses specific for H2-M3-restricted peptides are remarkably variable between genetically identical mice. Our findings demonstrate that H2-M3-restricted T cell responses are distinct from classically restricted T cell responses to bacterial infection.
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Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Listeriosis/inmunología , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Animales , Línea Celular , Epítopos de Linfocito T/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Epítopos Inmunodominantes/química , Cinética , Listeria monocytogenes/inmunología , Listeriosis/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , N-Formilmetionina , Fragmentos de Péptidos/química , Pliegue de Proteína , Especificidad de la Especie , Bazo/química , Bazo/citología , Bazo/inmunología , Coloración y Etiquetado , Linfocitos T Citotóxicos/química , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismoRESUMEN
This unit describes methods for infecting mice with L. monocytogenes. Optimal media for growth and methods to maintain bacterial virulence by passage through mice are included. Methods for determining the severity of splenic and hepatic infection are detailed, with strategies for distinguishing innate from specific immune responses following L. monocytogenes infection. This infection induces MHC class I-restricted CD8(+) cytolytic T lymphocytes that clear infection and provide long-term immunity. This unit describes methods that can be used for in vitro expansion of L. monocytogenes-specific T cells.
Asunto(s)
Listeria monocytogenes , Listeriosis , Modelos Animales , Animales , Linfocitos T CD8-positivos/inmunología , Medios de Cultivo , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunidad Celular , Inmunidad Innata , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/inmunología , Listeria monocytogenes/patogenicidad , Listeriosis/inmunología , Listeriosis/microbiología , Listeriosis/patología , Hígado/microbiología , Hígado/patología , Ratones , Especificidad de la Especie , Bazo/microbiología , Bazo/patología , VirulenciaRESUMEN
The novel bicyclic pentadentate ligand 5-methyl-1,5,9,24,25-pentaazapentacyclo[7.7.7.5.5]pentacosane11,13,15,18(25),20,22-hexene (L1) has been synthesized. Because of its cross-bridged topology it exhibits a relatively rigid preorganized conformation especially appropriate to complex formation, as shown by the crystal structure of the monoprotonated ligand salt, HL1ClH2O [orthorhombic, P212121, a = 9.4405(5) A, b = 13.3617(5) A, c = 16.710(1) A]. The complexes of L1 with both iron(II) and manganese(II) have been characterized, including the crystal structures of [FeL1CH3CN][FeCL4] and [MnL1Cl][PF6] [monoclinic, P21/n, a = 10.0460(5) A, b = 19.237(9) A, c = 15.6254(8) A, beta = 95.97(2)degrees and a = 7.745(2) A, b = 22.786(4) A, c = 14.639(4) A, beta = 105.074(10)degrees respectively]. The manganese complex is high spin with mueff = 5.96 and theta = 2.5 +/- 0.8 cm(-1), indicating weak ferromagnetic interactions. The reactions of the complexes with tert-butyl hydroperoxide and hydrogen peroxide have been shown by ESR spectroscopy to produce the tert-butyl peroxyl and hydroperoxyl radicals, as evidenced by their spin adducts with the spin traps N,N-dimethyl-1-pyrroline-N-oxide and N-tert-butyl-phenyl-nitrone.
RESUMEN
Recent studies have demonstrated that viral and bacterial infections can induce dramatic in vivo expansion of Ag-specific T lymphocytes. Although presentation of Ag is critical for activation of naive T cells, it is less clear how dependent subsequent in vivo T cell proliferation and memory generation are upon Ag. We investigated T cell expansion and memory generation in mice infected alternately with strains of Listeria monocytogenes that contained or lacked an immunodominant, MHC class I-restricted T cell epitope. We found substantial differences in the responses of effector and memory T cells to inflammatory stimuli. Although effector T cells undergo in vivo expansion in response to bacterial infection in the absence of Ag, memory T cells show no evidence for such bystander activation. However, Ag-independent expansion of effector T cells does not result in increased memory T cell frequencies, indicating that Ag presentation is critical for effective memory T cell generation. Early reinfection of mice with L. monocytogenes before the maximal primary T cell response induces typical memory expansion, suggesting that the capacity for a memory T cell response exists within the primary effector population. Our findings demonstrate that T cell effector proliferation and memory generation are temporally overlapping processes with differing requirements for Ag.
Asunto(s)
Antígenos Bacterianos/fisiología , Memoria Inmunológica/inmunología , Listeria monocytogenes/inmunología , Activación de Linfocitos/inmunología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/microbiología , Animales , Presentación de Antígeno , Antígenos Bacterianos/genética , Antígenos Bacterianos/metabolismo , División Celular/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Femenino , Inflamación/inmunología , Inflamación/patología , Cinética , Listeria monocytogenes/genética , Listeriosis/inmunología , Listeriosis/patología , Ratones , Ratones Endogámicos BALB C , Subgrupos de Linfocitos T/patologíaRESUMEN
BACKGROUND: Diarrheagenic Escherichia coli may cause serious extraintestinal complications, but there is no specific treatment. METHODS: Patients with diarrhea caused by diarrheagenic E. coli, specifically Shiga toxin-producing E. coli and E. coli-expressing intimin and enterohemorrhagic E. coli-hemolysin were treated by administration of pooled bovine colostrum, rich in antibodies to Shiga toxin and enterohemorrhagic E. coli-hemolysin, in a placebo-controlled, double-blind study. Symptom resolution and fecal excretion of infecting strains were assessed. RESULTS: No side effects were attributable to colostrum. Stool frequencies in the group treated with bovine colostrum were significantly reduced compared with those in the placebo group. No effect of therapy on the carriage of the pathogens or on complications of the infection could be demonstrated. CONCLUSIONS: Bovine colostrum is well tolerated and diminishes frequency of loose stools in children with E. coli-associated diarrhea. A prospective study should be conducted among a larger number of children with Shiga toxin-producing E. coli identified early in illness, to determine the effectiveness of colostrum therapy.
Asunto(s)
Adhesinas Bacterianas , Toxinas Bacterianas/biosíntesis , Proteínas Portadoras , Calostro , Diarrea Infantil/terapia , Enterotoxinas/biosíntesis , Infecciones por Escherichia coli/terapia , Proteínas de Escherichia coli , Proteínas Hemolisinas/biosíntesis , Adolescente , Animales , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Bovinos , Niño , Preescolar , Diarrea Infantil/metabolismo , Diarrea Infantil/microbiología , Método Doble Ciego , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/metabolismo , Femenino , Humanos , Lactante , Masculino , Serotipificación , Toxinas Shiga , VirulenciaRESUMEN
Studies of the murine immune response to infection with the intracellular bacterial pathogen Listeria monocytogenes have provided a wealth of information about innate and acquired immune defenses in the setting of an infectious disease. Our studies have focused on the MHC class I restricted, CD8+ T cell responses of Balb/c mice to L. monocytogenes infection. Four peptides that derive from proteins that L. monocytogenes secretes into the cytosol of infected cells are presented to cytotoxic T lymphocyte (CTL) by the H2-Kd major histocompatibility complex (MHC) class I molecule. We have found that bacterially secreted proteins are rapidly degraded in the host cell cytosol by proteasomes that utilize, at least in part, the N-end rule to determine the rate of degradation. The MHC class I antigen processing pathway is remarkably efficient at generating peptides that bind to MHC class I molecules. The magnitude of in vivo T cell responses, however, is influenced to only a small degree by the amount of antigen or the efficiency of antigen presentation. Measurements of in vivo T cell expansion following L. monocytogenes infection indicate that differences in the sizes of peptide-specific T cell responses are more likely owing to differences in the repertoire of naive T cells than to differences in peptide presentation. This notion is supported by our additional finding that dominant T cell populations express a more diverse T cell receptor (TCR) repertoire than do subdominant T cell populations.