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1.
Immunity ; 42(5): 929-41, 2015 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-25992863

RESUMEN

It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. We found that self-peptide MHC-specific CD8(+) T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome-encoded SMCY antigen, self-specific T cells exhibited only a 3-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors.


Asunto(s)
Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Supresión Clonal , Animales , Variación Antigénica , Femenino , Citometría de Flujo , Humanos , Masculino , Ratones , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Autotolerancia/inmunología
2.
Immunology ; 152(2): 344-355, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28581024

RESUMEN

Blomia tropicalis is the major asthma allergen in the tropics comparable to Dermatophagoides pteronyssinus. However, little is known about the B. tropicalis epitopes recognized by T cells. Our aim was to identify the T-cell epitopes in the major B. tropicalis allergen, Blo t 5, and investigate the potential of the corresponding peptides to inhibit the allergic inflammatory lung response. C57BL/6 mice were immunized with plasmid DNA encoding Blo t 5 and T-cell epitopes identified using the interferon-γ ELISPOT assay with 15-mer overlapping peptides. C57BL/6 mice were sensitized with bone-marrow-derived dendritic cells (BMDC) pulsed with Blo t 5 allergen followed by intranasal Blo t 5 challenge. Two H-2b restricted epitopes (Bt576-90 and Bt5106-115 ) were recognized by CD4 T cells specific for Blo t 5, but no CD8 epitopes were identified. In mice sensitized with Blo t 5-pulsed BMDC and challenged with intranasal Blo t 5 Bt576-90 and Bt5106-115 , peptide-specific CD4 T cells were found to secrete the T helper type 2 cytokines interleukin-5 and interleukin-13. Intradermal administration of synthetic peptides encoding the identified T-cell epitopes suppressed allergic airway inflammation to further allergen challenges. Hence, we have identified novel CD4 T-cell epitopes specific for Blo t 5 and demonstrated that these peptides could be employed therapeutically to suppress the T-cell response in a murine model of allergic airway inflammation.


Asunto(s)
Alérgenos/inmunología , Antiasmáticos/inmunología , Asma/prevención & control , Linfocitos T CD4-Positivos/inmunología , Epítopos/inmunología , Ácaros/inmunología , Péptidos/inmunología , Neumonía/prevención & control , Vacunas de ADN/inmunología , Alérgenos/administración & dosificación , Alérgenos/genética , Animales , Antiasmáticos/administración & dosificación , Asma/inmunología , Asma/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Ensayo de Immunospot Ligado a Enzimas , Mapeo Epitopo , Inmunización , Inyecciones Intradérmicas , Interferón gamma/inmunología , Interferón gamma/metabolismo , Ensayos de Liberación de Interferón gamma , Activación de Linfocitos , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Péptidos/administración & dosificación , Péptidos/genética , Neumonía/inmunología , Neumonía/metabolismo , Eosinofilia Pulmonar/inmunología , Eosinofilia Pulmonar/metabolismo , Eosinofilia Pulmonar/prevención & control , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética
3.
J Immunol ; 192(2): 641-8, 2014 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-24342804

RESUMEN

Peptide-MHC (pMHC) multimers have become one of the most widely used tools to measure Ag-specific T cell responses in humans. With the aim of understanding the requirements for pMHC-based personalized immunomonitoring, in which individuals expressing subtypes of the commonly studied HLA alleles are encountered, we assessed how the ability to detect Ag-specific T cells for a given peptide is affected by micropolymorphic differences between HLA subtypes. First, analysis of a set of 10 HLA-A*02:01-restricted T cell clones demonstrated that staining with pMHC multimers of seven distinct subtypes of the HLA-A*02 allele group was highly variable and not predicted by sequence homology. Second, to analyze the effect of minor sequence variation in a clinical setting, we screened tumor-infiltrating lymphocytes of an HLA-A*02:06 melanoma patient with either subtype-matched or HLA-A*02:01 multimers loaded with 145 different melanoma-associated Ags. This revealed that of the four HLA-A*02:06-restricted melanoma-associated T cell responses observed in this patient, two responses were underestimated and one was overlooked when using subtype-mismatched pMHC multimer collections. To our knowledge, these data provide the first demonstration of the strong effect of minor sequence variation on pMHC-based personalized immunomonitoring, and they provide tools to prevent this issue for common variants within the HLA-A*02 allele group.


Asunto(s)
Antígenos de Neoplasias/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Complejo Mayor de Histocompatibilidad/inmunología , Péptidos/inmunología , Polimorfismo Genético/genética , Alelos , Secuencia de Aminoácidos , Antígenos de Neoplasias/genética , Células Clonales/inmunología , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Complejo Mayor de Histocompatibilidad/genética , Melanoma/genética , Melanoma/inmunología , Melanoma/metabolismo , Datos de Secuencia Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Péptidos/genética , Péptidos/metabolismo , Polimorfismo Genético/inmunología , Alineación de Secuencia
4.
J Virol ; 88(18): 10613-23, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24990997

RESUMEN

UNLABELLED: Cytotoxic T lymphocytes recognizing conserved peptide epitopes are crucial for protection against influenza A virus (IAV) infection. The CD8 T cell response against the M158-66 (GILGFVFTL) matrix protein epitope is immunodominant when restricted by HLA-A*02, a major histocompatibility complex (MHC) molecule expressed by approximately half of the human population. Here we report that the GILGFVFTL peptide is restricted by multiple HLA-C*08 alleles as well. We observed that M158-66 was able to elicit cytotoxic T lymphocyte (CTL) responses in both HLA-A*02- and HLA-C*08-positive individuals and that GILGFVFTL-specific CTLs in individuals expressing both restriction elements were distinct and not cross-reactive. The crystal structure of GILGFVFTL-HLA-C*08:01 was solved at 1.84 Å, and comparison with the known GILGFVFTL-HLA-A*02:01 structure revealed that the antigen bound both complexes in near-identical conformations, accommodated by binding pockets shaped from shared as well as unique residues. This discovery of degenerate peptide presentation by both HLA-A and HLA-C allelic variants eliciting unique CTL responses to IAV infection contributes fundamental knowledge with important implications for vaccine development strategies. IMPORTANCE: The presentation of influenza A virus peptides to elicit immunity is thought to be narrowly restricted, with a single peptide presented by a specific HLA molecule. In this study, we show that the same influenza A virus peptide can be more broadly presented by both HLA-A and HLA-C molecules. This discovery may help to explain the differences in immunity to influenza A virus between individuals and populations and may also aid in the design of vaccines.


Asunto(s)
Epítopos de Linfocito T/inmunología , Antígenos HLA-A/inmunología , Antígenos HLA-C/inmunología , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Linfocitos T Citotóxicos/inmunología , Proteínas de la Matriz Viral/inmunología , Secuencia de Aminoácidos , Epítopos de Linfocito T/química , Epítopos de Linfocito T/genética , Antígenos HLA-A/química , Antígenos HLA-A/genética , Antígenos HLA-C/química , Antígenos HLA-C/genética , Humanos , Virus de la Influenza A/genética , Gripe Humana/genética , Gripe Humana/virología , Interferón gamma/inmunología , Datos de Secuencia Molecular , Alineación de Secuencia , Linfocitos T Citotóxicos/virología , Proteínas de la Matriz Viral/genética
5.
Cytometry A ; 87(10): 967-75, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26033882

RESUMEN

We designed conditional ligands restricted to HLA-B*08:01, -B*35:01, and -B*44:05 and proved the use of a conditional ligand previously designed for HLA-B*15:02 together with HLA-B*15:01. Furthermore, we compared the detection capabilities of specific HLA-B*15:01-restricted T cells using the HLA-B*15:01 and HLA-B*15:02 major histocompatibility complex (MHC) multimers and found remarkable differences in the staining patterns detected by flow cytometry. These new conditional ligands greatly add to the application of MHC-based technologies in the analyses of T-cell recognition as they represent frequently expressed HLA-B molecules. This expansion of conditional ligands is important to allow T-cell detection over a wide range of HLA restrictions, and provide comprehensive understanding of the T-cell recognition in a given context.


Asunto(s)
Antígeno HLA-B35/inmunología , Antígeno HLA-B8/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Linfocitos T CD8-positivos/inmunología , Humanos , Ligandos , Péptidos/inmunología
6.
J Immunol ; 191(8): 4010-9, 2013 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-24058176

RESUMEN

The identification of virus-specific CD8(+) T cell determinants is a fundamental requirement for our understanding of viral disease pathogenesis. T cell epitope mapping strategies increasingly rely on algorithms that predict the binding of peptides to MHC molecules. There is, however, little information on the reliability of predictive algorithms in the context of human populations, in particular, for those expressing HLA class I molecules for which there are limited experimental data available. In this study, we evaluate the ability of NetMHCpan to predict antiviral CD8(+) T cell epitopes that we identified with a traditional approach in patients of Asian ethnicity infected with Dengue virus, hepatitis B virus, or severe acute respiratory syndrome coronavirus. We experimentally demonstrate that the predictive power of algorithms defining peptide-MHC interaction directly correlates with the amount of training data on which the predictive algorithm has been constructed. These results highlight the limited applicability of the NetMHCpan algorithm for populations expressing HLA molecules for which there are little or no experimental binding data, such as those of Asian ethnicity.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Dengue/inmunología , Hepatitis B/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Síndrome Respiratorio Agudo Grave/inmunología , Algoritmos , Coronavirus/inmunología , Dengue/virología , Virus del Dengue/inmunología , Epítopos de Linfocito T/inmunología , Antígeno HLA-A11/inmunología , Antígeno HLA-A24/inmunología , Hepatitis B/virología , Virus de la Hepatitis B/inmunología , Humanos , Síndrome Respiratorio Agudo Grave/virología , Singapur
7.
Proc Natl Acad Sci U S A ; 109(34): 13739-44, 2012 Aug 21.
Artículo en Inglés | MEDLINE | ID: mdl-22869725

RESUMEN

We used somatic cell nuclear transfer (SCNT) to generate a mouse from the nucleus of an IgG1(+) ovalbumin-specific B cell. The resulting OBI mice show generally normal B-cell development, with elevated percentages of marginal zone B cells and a reduction in B-1 B cells. Whereas OBI RAG1(-/-) mice have exclusively IgG1 anti-ovalbumin in their serum, OBI mice show elevated levels of anti-ovalbumin of nearly all isotypes 3' of the γ1 constant region in the IgH locus, indicating that class switch recombination (CSR) occurs in the absence of immunization with ovalbumin. This CSR is associated with the presence of IgM(+)IgG1(+) double producer B cells that represent <1% of total B cells, accumulate in the peritoneal cavity, and account for near-normal levels of serum IgM and IgG3.


Asunto(s)
Linfocitos B/inmunología , Linfocitos B/metabolismo , Núcleo Celular/metabolismo , Inmunoglobulina G/biosíntesis , Alelos , Animales , Separación Celular , Cruzamientos Genéticos , Femenino , Cambio de Clase de Inmunoglobulina , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Inmunoglobulina M/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Técnicas de Transferencia Nuclear , Recombinación Genética
8.
Infect Immun ; 82(11): 4854-64, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25156726

RESUMEN

CD8(+) T cells play a pathogenic role in the development of murine experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA (PbA) infection in C57BL/6 mice. Only a limited number of CD8(+) epitopes have been described. Here, we report the identification of a new epitope from the bergheilysin protein recognized by PbA-specific CD8(+) T cells. Induction and functionality of these specific CD8(+) T cells were investigated in parallel with previously reported epitopes, using new tools such as tetramers and reporter cell lines that were developed for this study. We demonstrate that CD8(+) T cells of diverse specificities induced during PbA infection share many characteristics. They express cytolytic markers (gamma interferon [IFN-γ], granzyme B) and chemokine receptors (CXCR3, CCR5) and damage the blood-brain barrier in vivo. Our earlier finding that brain microvessels in mice infected with PbA, but not with non-ECM-causing strains, cross-presented a shared epitope was generalizable to these additional epitopes. Suppressing the induction of specific CD8(+) T cells through tolerization with a high-dose peptide injection was unable to confer protection against ECM, suggesting that CD8(+) T cells of other specificities participate in this process. The tools that we developed can be used to further investigate the heterogeneity of CD8(+) T cell responses that are involved in ECM.


Asunto(s)
Linfocitos T CD8-positivos/fisiología , Malaria Cerebral/complicaciones , Especificidad del Receptor de Antígeno de Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Barrera Hematoencefálica , Epítopos , Regulación de la Expresión Génica/inmunología , Malaria Cerebral/inmunología , Malaria Cerebral/parasitología , Ratones , Ratones Endogámicos C57BL , Plasmodium berghei/inmunología
9.
Eur J Immunol ; 43(4): 1109-20, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23280567

RESUMEN

Conditional ligands have enabled the high-throughput production of human leukocyte antigen (HLA) libraries that present defined peptides. Immunomonitoring platforms typically concentrate on restriction elements associated with European ancestry, and such tools are scarce for Asian HLA variants. We report 30 novel irradiation-sensitive ligands, specifically targeting South East Asian populations, which provide 93, 63, and 79% coverage for HLA-A, -B, and -C, respectively. Unique ligands for all 16 HLA types were constructed to provide the desired soluble HLA product in sufficient yield. Peptide exchange was accomplished for all variants as demonstrated by an ELISA-based MHC stability assay. HLA tetramers with redirected specificity could detect antigen-specific CD8(+) T-cell responses against human cytomegalovirus, hepatitis B (HBV), dengue virus (DENV), and Epstein-Barr virus (EBV) infections. The potential of this population-centric HLA library was demonstrated with the characterization of seven novel T-cell epitopes from severe acute respiratory syndrome coronavirus, HBV, and DENV. Posthoc analysis revealed that the majority of responses would be more readily identified by our unbiased discovery approach than through the application of state-of-the-art epitope prediction. This flow cytometry-based technology therefore holds considerable promise for monitoring clinically relevant antigen-specific T-cell responses in populations of distinct ethnicity.


Asunto(s)
Pueblo Asiatico , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA/inmunología , Virosis/inmunología , Secuencia de Aminoácidos , Epítopos de Linfocito T/química , Antígenos HLA/química , Antígenos HLA/genética , Humanos , Ligandos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Multimerización de Proteína , Estabilidad Proteica
10.
Angew Chem Int Ed Engl ; 53(49): 13390-4, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25348595

RESUMEN

Bioorthogonal cleavable linkers are attractive building blocks for compounds that can be manipulated to study biological and cellular processes. Sodium dithionite sensitive azobenzene-containing (Abc) peptides were applied for the temporary stabilization of recombinant MHC complexes, which can then be employed to generate libraries of MHC tetramers after exchange with a novel epitope. This technology represents an important tool for high-throughput studies of disease-specific T cell responses.


Asunto(s)
Compuestos Azo/química , Antígenos HLA-A/química , Péptidos/química , Secuencia de Aminoácidos , Compuestos Azo/inmunología , Ditionita/química , Epítopos/química , Epítopos/inmunología , Antígenos HLA-A/inmunología , Humanos , Ligandos , Modelos Moleculares , Péptidos/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología
11.
J Biol Chem ; 286(32): 28466-75, 2011 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-21680737

RESUMEN

The binding and stabilization capacity of potential T cell epitopes to class I MHC molecules form the basis for their immunogenicity and provide fundamental insight into factors that dictate cellular immune responses. We have developed a versatile high throughput cell-free method to measure MHC stability by capturing a variety of MHC products on the surface of streptavidin-coated particles followed by flow cytometry analysis. Arrays of peptide-MHC combinations, generated by exchanging conditional ligand-loaded MHC, could be probed in a single experiment, thus combining the molecular precision of biochemically purified MHCs with high content multiparametric flow cytometry-based assays. Semiquantitative determination of the peptide affinity for the restriction element could also be accomplished through competition experiments using this bead-based assay. Furthermore, the generated peptide-MHC reagents could directly be applied to antigen-specific CD8(+) T lymphocyte analysis. The combinatorial labeling of beads allowed straightforward identification by their unique fluorescent signatures and provided a convenient means for extended assay multiplexing.


Asunto(s)
Linfocitos T CD8-positivos/química , Epítopos de Linfocito T/química , Citometría de Flujo/métodos , Antígenos de Histocompatibilidad Clase I/química , Biblioteca de Péptidos , Linfocitos T CD8-positivos/citología , Linfocitos T CD8-positivos/inmunología , Línea Celular , Sistema Libre de Células/química , Sistema Libre de Células/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Humanos
12.
J Virol ; 85(20): 10464-71, 2011 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-21813600

RESUMEN

Severe acute respiratory syndrome (SARS) is a highly contagious and life threatening disease, with a fatality rate of almost 10%. The etiologic agent is a novel coronavirus, severe acute respiratory syndrome coronavirus (SARS-CoV), with animal reservoirs found in bats and other wild animals and thus the possibility of reemergence. In this study, we first investigated at 6 years postinfection whether SARS-specific memory T cells persist in SARS-recovered individuals, demonstrating that these subjects still possess polyfunctional SARS-specific memory CD4+ and CD8+ T cells. A dominant memory CD8+ T cell response against SARS-CoV nucleocaspid protein (NP; amino acids 216 to 225) was then defined in SARS-recovered individuals carrying HLA-B*40:01, a HLA-B molecule present in approximately one-quarter of subjects of Asian ethnicities. To reconstitute such a CD8+ T cell response, we isolated the alpha and beta T cell receptors of the HLA-B*40:01-restricted SARS-specific CD8+ T cells. Using T cell receptor gene transfer, we generated SARS-specific redirected T cells from the lymphocytes of normal individuals. These engineered CD8+ T cells displayed avidity and functionality similar to that of natural SARS-specific memory CD8+ T cells. They were able to degranulate and produce gamma interferon, tumor necrosis factor alpha, and macrophage inflammatory proteins 1α and 1ß after antigenic stimulation. Since there is no effective treatment against SARS, these transduced T cells specific for an immunodominant SARS epitope may provide a new avenue for treatment during a SARS outbreak.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Epítopos Inmunodominantes/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Linfocitos T CD4-Positivos/inmunología , Degranulación de la Célula , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Ingeniería Genética , Humanos , Memoria Inmunológica , Interferón gamma/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Factor de Necrosis Tumoral alfa/metabolismo
13.
PLoS Pathog ; 6(3): e1000815, 2010 Mar 19.
Artículo en Inglés | MEDLINE | ID: mdl-20333242

RESUMEN

Production of the pro-inflammatory cytokine IL-12 by innate phagocytes drives the differentiation of IFN-gamma-producing effector T cells during Toxoplasma gondii infection. However, the role of IL-12 in the regulation of memory CD8+ T cell differentiation and function during murine toxoplasmosis is unclear. To track memory CTL development, we identified a novel H-2K(b)-restricted CTL population specific for the Toxoplasma antigen tgd057. Tgd057-specific CTLs were induced by both vaccination and natural peroral infection, and were representative of the polyclonal CTL population. Tgd057-specific primary effector cells required IL-12 for the differentiation of KLRG1+ effector subpopulations and IFN-gamma production in response to restimulation with parasite-infected cells, but not to restimulation with cognate peptide. The effect of IL-12 deficiency during the primary response was profoundly imprinted on memory CTLs, which continued to show defects in cell numbers, KLRG1+ effector memory subpopulation differentiation, and IFN-gamma recall responses. Importantly, isolated CD62L(hi) KLRG1- CD8+ T cells differentiated in the absence of IL-12 were enhanced in their ability to generate IFN-gamma-producing secondary tgd057-specific effector cells. Our data, for the first time, demonstrate the negative impact of IL-12 signaling on the quality of the central memory CTL compartment. Thus, despite the beneficial role of IL-12 in promoting effector differentiation, excessive exposure to IL-12 during CTL priming may limit the development of long-term protective immunity through the decreased fitness of central memory CTL responses.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/parasitología , Memoria Inmunológica/inmunología , Interleucina-12/inmunología , Toxoplasma/inmunología , Toxoplasmosis Animal/inmunología , Animales , Antígenos de Protozoos/inmunología , Diferenciación Celular/inmunología , Epítopos , Inmunofenotipificación , Interleucina-12/genética , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Transducción de Señal/inmunología
14.
Bioorg Med Chem ; 19(11): 3402-9, 2011 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-21561781

RESUMEN

In this paper, we describe the crystal structure of previously reported ring-extended gramicidin S (GS) derivative 2 (GS14K4), containing a d-amino acid residue in one of the ß-strand regions. This structure is in agreement with a previously reported modeling study of the same molecule. The polar side chain of the additional d-amino acid residue is positioned at the same face of the molecule as the hydrophobic side chains, and we believe that because of this compound 2 is considerably less hydrophobic than extended GS derivatives in which the strand regions are exclusively composed of l-amino acids. Using this backbone structure as our benchmark we prepared a small series of ring-extended GS analogues featuring sugar amino acid dipeptide isosteres of varied hydrophobicity at the turn region. We show that via this approach hydrophobicity of extended GS analogues can be tuned without affecting the secondary structure (as observed from NMR and CD spectra). Biological evaluation reveals that hydrophobicity correlates to cell toxicity, but still bacteriolysis is induced with GS analogues that are too hydrophilic to efficiently lyse human red blood cells.


Asunto(s)
Antibacterianos/síntesis química , Gramicidina/análogos & derivados , Antibacterianos/química , Antibacterianos/farmacología , Dicroismo Circular , Cristalografía por Rayos X , Eritrocitos/efectos de los fármacos , Gramicidina/química , Gramicidina/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Pruebas de Sensibilidad Microbiana , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
15.
Proc Natl Acad Sci U S A ; 105(10): 3831-6, 2008 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-18245382

RESUMEN

Class I MHC tetramers allow direct phenotypic identification of CD8(+) T cell populations, but their production remains laborious. A peptide exchange strategy that employs class I MHC products loaded with conditional ligands (caged MHC molecules) provides a fast and straightforward method to obtain diverse arrays of class I MHC tetramers and facilitates CD8(+) T cell epitope discovery. Here, we describe the development of photocleavable analogs of the FAPGNYPAL (SV9) epitope that bind H-2K(b) and H-2D(b) with full retention of their structural and functional integrity. We ranked all possible H-2K(b) octameric and H-2D(b) nonameric epitopes that span the genome of Chlamydia trachomatis and prepared MHC tetramers from approximately 2,000 of the highest scoring peptides by replacement of the SV9 analog with the peptide of choice. The resulting 2,000-member class I MHC tetramer array allowed the discovery of two variants of an epitope derived from polymorphic membrane protein I (PmpI) and an assessment of the kinetics of emergence and the effector function of the corresponding CD8(+) T cells.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Infecciones por Chlamydia/inmunología , Chlamydia trachomatis/inmunología , Epítopos de Linfocito T/inmunología , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/inmunología , Secuencia de Aminoácidos , Animales , Linfocitos T CD8-positivos/efectos de la radiación , Chlamydia trachomatis/efectos de la radiación , Biología Computacional , Cristalografía por Rayos X , Epítopos de Linfocito T/química , Antígenos H-2/química , Antígenos H-2/inmunología , Ligandos , Luz , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/química , Péptidos/inmunología , Estructura Cuaternaria de Proteína , Reproducibilidad de los Resultados , Solubilidad/efectos de la radiación , Estereoisomerismo
16.
Org Biomol Chem ; 8(8): 1881-4, 2010 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-20449493

RESUMEN

Oligoprolines (OPs) are used as rigid backbone scaffolds for the design of oligomeric ligands that target specific G protein-coupled receptors. The OPs were designed to vary in length, the position and number of the ligand-functionalized residues incorporated. For all synthesized compounds a typical PP type II helix was evidenced by circular dichroism indicating that decoration of the helix with large ligands did not affect the helical conformation. Pharmacological evaluation revealed that oligomerization of an agonist with the use of an oligoproline scaffold showed an increase in potency when compared to the monomeric counterparts.


Asunto(s)
Prolina/química , Receptores de HL/agonistas , Receptores de HL/metabolismo , Dicroismo Circular , Humanos , Ligandos , Prolina/síntesis química , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo
17.
J Am Chem Soc ; 131(31): 10800-1, 2009 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-19610623

RESUMEN

The unique reactivity of two sortase enzymes, SrtA(staph) from Staphylococcus aureus and SrtA(strep) from Streptococcus pyogenes, is exploited for site-specific labeling of a single polypeptide with different labels at its N and C termini. SrtA(strep) is used to label the protein's C terminus at an LPXTG site with a fluorescently labeled dialanine nucleophile. Selective N-terminal labeling of proteins containing N-terminal glycine residues is achieved using SrtA(staph) and LPXT derivatives. The generality of N-terminal labeling with SrtA(staph) is demonstrated by near-quantitative labeling of multiple protein substrates with excellent site specificity.


Asunto(s)
Aminoaciltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Cisteína Endopeptidasas/metabolismo , Colorantes Fluorescentes/química , Péptidos/química , Alanina , Secuencia de Aminoácidos , Sitios de Unión , Staphylococcus aureus/enzimología , Streptococcus pyogenes/enzimología , Especificidad por Sustrato
18.
J Virol ; 82(24): 12205-12, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-18922872

RESUMEN

Infection of mice with murine gammaherpesvirus 68 (MHV-68) robustly activates CD8 T cells, but only six class I major histocompatibility complex (MHC)-restricted epitopes have been described to date for the widely used H-2(b) haplotype mice. To explore the specificity and kinetics of the cytotoxic T-lymphocyte response in MHV-68-infected C57BL/6 mice, we screened for H-2K(b)- and H-2D(b)-restricted epitopes using a set of 384 candidate epitopes in an MHC tetramer-based approach and identified 19 new epitopes in 16 different open reading frames. Of the six known H-2K(b)- and H-2D(b)-restricted epitopes, we confirmed a response against three and did not detect CD8 T-cell-specific responses for the remaining three. The peak of the CD8 T-cell response to most peptides occurs between 6 and 10 days postinfection. The respective MHC tetramer-positive CD8 T cells display an activated/effector phenotype (CD62L(lo) and CD44(hi)) and produce gamma interferon upon peptide stimulation ex vivo. MHV-68 infection in vivo elicits a response to multiple viral epitopes, derived from both early and late viral antigens, illustrating a far broader T-cell repertoire and more-rapid activation than those previously recorded.


Asunto(s)
Antígenos/inmunología , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/inmunología , Rhadinovirus/inmunología , Animales , Genoma Viral/inmunología , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Cinética , Ratones , Ratones Endogámicos C57BL , Factores de Tiempo
19.
Nat Chem Biol ; 3(11): 707-8, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17891153

RESUMEN

Genetically encoded reporter constructs that yield fluorescently labeled fusion proteins are a powerful tool for observing cell biological phenomena, but they have limitations. Sortagging (sortase-mediated transpeptidation) is a versatile chemoenzymatic system for site-specific labeling of proteins with small (<2 kDa) probes. Sortagging combines the precision of a genetically encoded tag with the specificity of an enzymatic reaction and the ease and chemical versatility of peptide synthesis. Here we apply this technique to proteins in vitro and on the surface of living cells.


Asunto(s)
Técnicas Biosensibles/métodos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Sondas Moleculares/química , Línea Celular , Humanos , Proteínas de la Membrana/metabolismo , Estructura Molecular
20.
Bioorg Med Chem ; 17(17): 6233-40, 2009 Sep 01.
Artículo en Inglés | MEDLINE | ID: mdl-19679485

RESUMEN

Loloatin C is a cyclic cationic antimicrobial peptide which is active against gram positive as well as certain gram negative bacteria. Unfortunately, it is equally potent against human erythrocytes. To probe the structure-activity relationship of this promising antibiotic peptide, amino acid substitution and/or incorporation of a constraint sugar amino acid dipeptide isoster has been applied. Six new derivatives have been synthesized using SPPS and their solution structure investigated using NMR studies. Finally, the antimicrobial and the hemolytic activities have been determined.


Asunto(s)
Antibacterianos/química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Antibacterianos/síntesis química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/farmacología , Eritrocitos/efectos de los fármacos , Hemólisis , Humanos , Pruebas de Sensibilidad Microbiana , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Relación Estructura-Actividad
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