RESUMO
HLA class II α and ß chains form receptors for antigen presentation to CD4(+) T cells. Numerous pairings of class II α and ß subunits from the wide range of haplotypes and isotypes may form, but most of these combinations, in particular those produced by isotype mixing, yielded mismatched dimers. It is unclear how selection of functional receptors is achieved. At the atomic level, it is not known which interactions of class II residues regulate selection of matched αß heterodimers and the evolutionary origin of matched isotype mixed dimer formation. In this study we investigated assembly of isotype-mixed HLA class II α and ß heterodimers. Assembly and carbohydrate maturation of various HLA-class II isotype-mixed α and ß subunits was dependent on the groove binding section of the invariant chain (Ii). By mutation of polymorphic DPß sequences, we identified two motifs, Lys-69 and GGPM-(84-87), that are engaged in Ii-dependent assembly of DPß with DRα. We identified five members of a family of DPß chains containing Lys-69 and GGPM 84-87, which assemble with DRα. The Lys/GGPM motif is present in the DPß sequence of the Neanderthal genome, and this ancient sequence is related to the human allele DPB1*0401. By site-directed mutagenesis, we inspected Neanderthal amino acid residues that differ from the DPB1*0401 allele and aimed to determine whether matched heterodimers are formed by assembly of DPß mutants with DRα. Because the *0401 allele is rare in the sub-Saharan population but frequent in the European population, it may have arisen in modern humans by admixture with Neanderthals in Europe.
Assuntos
Evolução Molecular , Antígenos HLA-DP/genética , Antígenos HLA-DR/genética , Immunoblotting , Mutação , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Sítios de Ligação/genética , Células COS , Linhagem Celular , Chlorocebus aethiops , Antígenos HLA-DP/química , Antígenos HLA-DP/metabolismo , Cadeias alfa de HLA-DP/química , Cadeias alfa de HLA-DP/genética , Cadeias alfa de HLA-DP/metabolismo , Cadeias beta de HLA-DP/química , Cadeias beta de HLA-DP/genética , Cadeias beta de HLA-DP/metabolismo , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Cadeias alfa de HLA-DR/química , Cadeias alfa de HLA-DR/genética , Cadeias alfa de HLA-DR/metabolismo , Cadeias beta de HLA-DR/química , Cadeias beta de HLA-DR/genética , Cadeias beta de HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Modelos Moleculares , Homem de Neandertal , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de ProteínaRESUMO
The histone variant H2A.Z has been implicated in many biological processes, such as gene regulation and genome stability. Here, we present the identification of H2A.Z.2.2 (Z.2.2), a novel alternatively spliced variant of histone H2A.Z and provide a comprehensive characterization of its expression and chromatin incorporation properties. Z.2.2 mRNA is found in all human cell lines and tissues with highest levels in brain. We show the proper splicing and in vivo existence of this variant protein in humans. Furthermore, we demonstrate the binding of Z.2.2 to H2A.Z-specific TIP60 and SRCAP chaperone complexes and its active replication-independent deposition into chromatin. Strikingly, various independent in vivo and in vitro analyses, such as biochemical fractionation, comparative FRAP studies of GFP-tagged H2A variants, size exclusion chromatography and single molecule FRET, in combination with in silico molecular dynamics simulations, consistently demonstrate that Z.2.2 causes major structural changes and significantly destabilizes nucleosomes. Analyses of deletion mutants and chimeric proteins pinpoint this property to its unique C-terminus. Our findings enrich the list of known human variants by an unusual protein belonging to the H2A.Z family that leads to the least stable nucleosome known to date.
Assuntos
Processamento Alternativo , Histonas/genética , Histonas/fisiologia , Nucleossomos/metabolismo , Adenosina Trifosfatases/metabolismo , Linhagem Celular , Cromatina/metabolismo , DNA/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Lisina Acetiltransferase 5 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Individual doses of dual-dose vaccine-regimens are sequentially administered into the deltoid muscle, but little attention has so far been paid to the immunological effects of choosing the ipsilateral or the contralateral side for the second dose. METHODS: In an observational study, 303 previously naive individuals were recruited, who received the second dose of the COVID-19 vaccine BNT162b2 on either the ipsilateral (n = 147) or the contralateral side (n = 156). Spike-specific IgG, IgG-avidity, and neutralizing antibodies were quantified using ELISA and a surrogate assay 2 weeks after dose 2. A subgroup of 143 individuals (64 ipsilateral, 79 contralateral) was analysed for spike-specific CD4 and CD8 T-cells using flow-cytometry. FINDINGS: Median spike-specific IgG-levels did not differ after ipsilateral (4590 (IQR 3438) BAU/ml) or contralateral vaccination (4002 (IQR 3524) BAU/ml, p = 0.106). IgG-avidity was also similar (p = 0.056). However, neutralizing activity was significantly lower after contralateral vaccination (p = 0.024). Likewise, median spike-specific CD8 T-cell levels were significantly lower (p = 0.004). Consequently, the percentage of individuals with detectable CD8 T-cells was significantly lower after contralateral than after ipsilateral vaccination (43.0% versus 67.2%, p = 0.004). Spike specific CD4 T-cell levels were similar in both groups, but showed significantly higher CTLA-4 expression after contralateral vaccination (p = 0.011). These effects were vaccine-specific, as polyclonally stimulated T-cell levels did not differ. INTERPRETATION: Both ipsilateral and contralateral vaccination induce a strong immune response, but secondary boosting is more pronounced when choosing vaccine administration-routes that allows for drainage by the same lymph nodes used for priming. Higher neutralizing antibody activity and higher levels of spike-specific CD8 T-cells may have implications for protection from infection and severe disease and support general preference for ipsilateral vaccination. FUNDING: Financial support was provided in part by the State chancellery of the Saarland to M.S.
Assuntos
Vacinas contra COVID-19 , COVID-19 , Humanos , SARS-CoV-2 , Vacina BNT162 , COVID-19/prevenção & controle , Vacinação , Anticorpos Neutralizantes , Imunidade Celular , Imunoglobulina G , Anticorpos AntiviraisRESUMO
The enormous number of allelic MHC class II glycoproteins provides the immune system with a large set of heterodimeric receptors for the binding of pathogen-derived peptides. How do inherited allo- or isotypic subunits of MHC class II combine to produce such a variety of functional peptide receptors? We propose a new mechanism in which pairing of matched MHC class II alpha- and beta-subunits is coordinated by the invariant chain chaperone. The assembly is proposed to occur in a sequential fashion, with a matched beta-chain being selected by the alpha-chain-invariant chain 'scaffold' complex that is formed first. This sequential assembly is a prerequisite for subsequent intracellular transport of the alpha-chain-invariant chain-beta-oligomer and its maturation into a functional peptide receptor.
Assuntos
Antígenos de Histocompatibilidade Classe II/química , Modelos Moleculares , Receptores de Peptídeos/química , Sítios de Ligação , Haplótipos , Antígenos de Histocompatibilidade Classe II/metabolismo , Ligação Proteica , Receptores de Peptídeos/metabolismoRESUMO
Spatially addressable arrays of molecules embedded in or anchored to supported lipid bilayers are important for on-chip screening and binding assays; however, methods to sort or accumulate components in a fluid membrane on demand are still limited. Here we apply in-plane surface acoustic shear waves (SAWs) to laterally accumulate double-stranded DNA segments electrostatically bound to a cationic supported lipid bilayer. The fluorescently labeled DNA segments are found to segregate into stripe patterns with a spatial frequency corresponding to the periodicity of the standing SAW wave (~10 µm). The DNA molecules are accumulated 10-fold in the regions of SAW antinodes. The superposition of two orthogonal sets of SAW sources creates checkerboard like arrays of DNA demonstrating the potential to generate arrayed fields dynamically. The pattern relaxation time of 0.58 s, which is independent of the segment length, indicates a sorting and relaxation mechanism dominated by lipid diffusion rather than DNA self-diffusion.
Assuntos
DNA/metabolismo , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos/métodos , DNA/química , Difusão , Fluorescência , Bicamadas Lipídicas/química , Análise de Sequência com Séries de Oligonucleotídeos/instrumentação , Som , Eletricidade Estática , Xantenos/análiseRESUMO
Tryptophan catabolism through IDO activity can cause nonresponsiveness and tolerance acting on T cells. Given the crucial importance of dendritic cells (DCs) in the initiation of a T cell response, surprisingly little is known about the impact of IDO activity and tryptophan deprivation on DCs themselves. In the present study, we show that human DCs differentiated under low-tryptophan conditions acquire strong tolerogenic capacity. This effect is associated with a markedly decreased Ag uptake as well as the down-regulation of costimulatory molecules (CD40, CD80). In contrast, the inhibitory receptors ILT3 and ILT4 are significantly increased. Functionally, tryptophan-deprived DCs show a reduced capacity to stimulate T cells, which can be restored by blockade of ILT3. Moreover, ILT3(high)ILT4(high) DCs lead to the induction of CD4(+)CD25(+) Foxp3(+) T regulatory cells with suppressive activity from CD4(+)CD25(-) T cells. The generation of ILT3(high)ILT4(high) DCs with tolerogenic properties by tryptophan deprivation is linked to a stress response pathway mediated by the GCN2 kinase. These results demonstrate that tryptophan degradation establishes a regulatory microenvironment for DCs, enabling these cells to induce T regulatory cells. The impact of IDO thus extends beyond local immune suppression to a systemic control of the immune response.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Glicoproteínas de Membrana/biossíntese , Receptores de Superfície Celular/biossíntese , Receptores Imunológicos/biossíntese , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Triptofano/metabolismo , Antígenos CD4/biossíntese , Células Cultivadas , Técnicas de Cocultura , Células Dendríticas/enzimologia , Fatores de Transcrição Forkhead/biossíntese , Humanos , Tolerância Imunológica , Imunofenotipagem , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Subunidade alfa de Receptor de Interleucina-2/biossíntese , Monócitos/enzimologia , Monócitos/imunologia , Monócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Superfície Celular/fisiologia , Linfócitos T Reguladores/citologia , Triptofano/deficiência , Regulação para Cima/imunologiaRESUMO
In the past decades supported lipid bilayers (SLBs) have been an important tool in order to study the physical properties of biological membranes and cells. So far, controlled manipulation of SLBs is very limited. Here we present a new technology to create lateral patterns in lipid membranes controllable in both space and time. Surface acoustic waves (SAWs) are used to generate lateral standing waves on a piezoelectric substrate which create local "traps" in the lipid bilayer and lead to a lateral modulation in lipid concentration. We demonstrate that pattern formation is reversible and does not affect the integrity of the lipid bilayer as shown by extracting the diffusion constant of fluid membranes. The described method could possibly be used to design switchable interfaces for the lateral transport and organization of membrane bound macromolecules to create dynamic bioarrays and control biofilm formation.
Assuntos
Acústica , Dispositivos Lab-On-A-Chip , Bicamadas Lipídicas/química , Técnicas Analíticas Microfluídicas/instrumentação , Desenho de Equipamento , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência , Modelos TeóricosRESUMO
The interaction of targeted drug carriers with epithelial and endothelial barriers in vivo is largely determined by the dynamics of the body fluids. To simulate these conditions in binding assays, a fully biocompatible in vitro model was developed which can accurately mimic a wide range of physiological flow conditions on a thumbnail-format cell-chip. This acoustically-driven microfluidic system was used to study the interaction characteristics of protein-coated particles with cells. Poly(D,L-lactide-co-glycolide) (PLGA) microparticles (2.9 +/- 1 microm) were conjugated with wheat germ agglutinin (WGA-MP, cytoadhesive protein) or bovine serum albumin (BSA-MP, non-specific protein) and their binding to epithelial cell monolayers was investigated under stationary and flow conditions. While mean numbers of 1500 +/- 307 mm(-2) WGA-MP and 94 +/- 64 mm(-2) BSA-MP respectively were detected to be cell-bound in the stationary setup, incubation at increasing flow velocities increasingly antagonized the attachment of both types of surface-modified particles. However, while binding of BSA-MP was totally inhibited by flow, grafting with WGA resulted in a pronounced anchoring effect. This was indicated by a mean number of 747 +/- 241 mm(-2) and 104 +/- 44 mm(-2) attached particles at shear rates of 0.2 s(-1) and 1 s(-1) respectively. Due to the compactness of the fluidic chip which favours parallelization, this setup represents a highly promising approach towards a screening platform for the performance of drug delivery vehicles under physiological flow conditions. In this regard, the flow-chip is expected to provide substantial information for the successful design and development of targeted micro- and nanoparticulate drug carrier systems.
Assuntos
Portadores de Fármacos/metabolismo , Análise Serial de Tecidos/métodos , Acústica , Adesão Celular , Portadores de Fármacos/química , Humanos , Tamanho da Partícula , Análise Serial de Tecidos/instrumentação , Células Tumorais CultivadasRESUMO
The highly polymorphic major histocompatibility complex class II (MHCII) polypeptides assemble in the ER with the assistance of invariant chain (Ii) chaperone. Ii binds to the peptide-binding pocket of MHCII heterodimers. We explored the mechanism how MHCII subunits attach to Ii. Expression with single alpha or beta subunits from three human HLA and two mouse H2 class II isotypes revealed that Ii co-isolates predominantly with the alpha polypeptide. Co-isolation with alpha chain requires the groove binding Ii-segment and depends on M91 of Ii. Immunoprecipitation of Ii from pulse chase labeled cells showed sequential assembly of alpha and beta chains.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Chaperonas Moleculares/metabolismo , Animais , Antígenos HLA-DP/metabolismo , Antígenos HLA-DR/metabolismo , Cadeias HLA-DRB1 , Humanos , Imunoprecipitação , Camundongos , Peptídeos/metabolismoRESUMO
Previously, two mouse monoclonal antibodies (12B8 and 6D4) were raised against the alpha- and beta-subunits of the rat non-classical MHC class-II heterodimer RT1.DM. Here, I describe the epitope mapping of mAb 12B8 to amino acids alpha3-14 (EASPQAWWDESQ) and mAb 6D4 to amino acids beta35-44 (WDPEEGQIVP). Epitope mapping was conducted by preparing fusion proteins between the alpha and beta chain of RT1.DM for Western detection with mAb 12B8 and 6D4. By mutating non-conserved amino acids of the human orthologue of RT1.DM, the rat epitopes were introduced, thereby making the alpha and beta polypeptides sensitive for mAb 12B8 and 6D4 detection. The epitopes, designated as 12B8 and 6D4, were tested for protein tagging. They were appended to the N- or C-terminus of four human proteins, the tumour suppressor protein VHL (von Hippel-Lindau), SUMO4, MHC class-II DQbeta and -DPbeta for expression in mammalian cells. Western detection, immunoprecipitation and localisation of the tagged proteins were successfully demonstrated. Thus, the 12B8 and 6D4 epitope tag can be universally used for the immunodetection of recombinant proteins and to study protein-protein interactions.
Assuntos
Anticorpos/imunologia , Western Blotting/métodos , Antígenos de Histocompatibilidade/análise , Antígenos de Histocompatibilidade/imunologia , Lisossomos/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/imunologia , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Mapeamento de Epitopos , Epitopos/química , Epitopos/imunologia , Escherichia coli/genética , Antígenos de Histocompatibilidade/química , Antígenos de Histocompatibilidade/genética , Dados de Sequência Molecular , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
The term correlative microscopy denotes the sequential visualization of one and the same cell using various microscopic techniques. Correlative microscopy provides a unique platform to combine the particular strength of each microscopic approach and compensate for its specific limitations. As an example, we report results of a correlative microscopic study exploring features of the nuclear landscape in HeLa cells. We present a detailed protocol to first investigate distinct structural features of a living cell in space and time (4D) using spinning disk laser scanning microscopy (SDLSM). Then, after fixation and staining of selected structures (e.g., by means of immunodetection), details of these structures are explored at increasingly higher resolution using three-dimensional (3D) confocal laser scanning microscopy (CLSM); super-resolution fluorescence microscopy, such as three-dimensional structured illumination microscopy (3D-SIM); and transmission electron microscopy (TEM). We discuss problems involved in the comparison of images of a given cell nucleus recorded with different microscopic approaches, which requires not only a compensation for different resolutions but also for various distortions.
Assuntos
Núcleo Celular/metabolismo , Microscopia Confocal/métodos , Microscopia Eletrônica de Transmissão/métodos , Microscopia de Fluorescência/métodos , Análise de Célula Única/métodos , Linhagem Celular Tumoral , Células HeLa , Humanos , Imageamento Tridimensional/métodos , Proteínas Luminescentes/química , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: The HLA gene complex encodes three class II isotypes, DR, DQ, and DP. HLA class II molecules are peptide receptors that present antigens for recognition by T lymphocytes. In antigen presenting cells, the assembly of matched α and ß subunits to heterodimers is chaperoned by invariant chain (Ii). Ii forms a homotrimer with three binding sites for class II heterodimers. The current model of class II and Ii structure states that three αß heterodimers bind to an Ii trimer. METHODOLOGY/PRINCIPAL FINDINGS: [corrected] We have now analyzed the composition and size of the complexes of class II and Ii using epitope tagged class II subunits and density gradient experiments. We show here that class II-Ii oligomers consist of one class II heterodimer associated with one Ii trimer, such that the DR, DQ and DP isotypes are contained within separate complexes with Ii. CONCLUSION/SIGNIFICANCE: We propose a structural model of the class II-Ii oligomer and speculate that the pentameric class II-Ii complex is bent towards the cell membrane, inhibiting the binding of additional class II heterodimers to Ii.
Assuntos
Antígenos de Diferenciação de Linfócitos B/análise , Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos HLA-D/análise , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/metabolismo , Multimerização Proteica/fisiologia , Sequência de Aminoácidos , Animais , Células Apresentadoras de Antígenos/química , Células Apresentadoras de Antígenos/imunologia , Antígenos de Diferenciação de Linfócitos B/química , Células COS , Fracionamento Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Antígenos HLA-D/química , Antígenos HLA-DP/análise , Antígenos HLA-DP/química , Antígenos HLA-DP/metabolismo , Antígenos HLA-DQ/análise , Antígenos HLA-DQ/química , Antígenos HLA-DQ/metabolismo , Antígenos HLA-DR/análise , Antígenos HLA-DR/química , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Concentração Osmolar , Estrutura Quaternária de Proteína , UltracentrifugaçãoRESUMO
Human HLA-DM or mouse H2-DM plays a vital role for presentation of antigenic sequences by MHC class II peptide receptors. These non-classical MHC class II molecules catalyze the release of the invariant chain (Ii) fragment CLIP from the class II cleft and facilitate acquisition of antigenic peptides by MHC class II peptide receptors. H2-DM- or Ii-deficient mice display an impaired ability of their antigen-presenting cell to present peptides to CD4+ T cells and a molecular link between the immunodeficiencies of these mouse strains may exist. We show that in transfected cells the presence of HLA-DM molecules in endocytic vesicles was strongly reduced when HLA-DM was accompanied by HLA-DR. Exclusion of HLA-DM from endocytic vesicles is explained by mixed association of HLA-DM with HLA-DR subunits and retention of the aggregates in the endoplasmic reticulum. Expression of Ii, however, impairs formation of mixed HLA-DR and HLA-DM complexes and directs matched pairing of HLA-DR and HLA-DM heterodimers. In Ii gene-deficient mice, aberrant association of H2A with H2-DM polypeptides was detected. Low expression of Ii in transgenic mice inhibits interaction of H2A with H2-DM subunits and facilitates formation of H2-DM alphabeta heterodimers. A role of Ii for assembly of H2-DM heterodimers partially explains the immunodeficient phenotype of Ii-/- mice.
Assuntos
Antígenos de Diferenciação de Linfócitos B/genética , Antígenos HLA-D/metabolismo , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Animais , Células COS , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Dimerização , Antígenos HLA-D/genética , Antígenos HLA-DR/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Transfecção , Vesículas Transportadoras/metabolismoRESUMO
The human lymphocyte antigen (HLA) class II region encodes highly polymorphic peptide receptors, which associate in the ER to the chaperone invariant chain (Ii). Ii facilitates assembly of class II subunits to functional peptide receptors. We searched for a conserved structure on HLA-DR polypeptides that mediates contact to a previously identified proline-rich class-II-binding sequence of Ii. Major histocompatibility complex (MHC) class II beta chain sequences exhibit two conserved tryptophan residues separated by 22 amino acids. Inspection of this motif in the X-ray structure of DR3 showed TrpTyr residues in the vicinity of the Ii-derived fragment CLIP. Five DRbeta mutants were produced. Mutation at Tyr123, Trp153 and Asp152 residues abolished interaction to the proline-rich sequence of Ii. All mutants formed heterodimers with DRalpha, were capable of binding an antigenic sequence and were expressed on the cell surface of transfected cells. In the presence of endogenous DRbeta chain however, the TyrAspTrp mutant was not cell-surface exposed and did not co-isolate with Ii or DRalpha. The competition of the mutant with the endogenous DRbeta for binding to DRalpha indicates that a structure on DRbeta chain regulates assembly of DR subunits. Hence, the chaperone function of Ii is mediated through a conserved region on the beta2 domain of class II.
Assuntos
Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos HLA-DR/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Chaperonas Moleculares/fisiologia , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/metabolismo , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Cristalografia por Raios X , Dimerização , Antígenos HLA-DR/química , Antígenos HLA-DR/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mutagênese/genética , Mutação/genética , Reação em Cadeia da Polimerase/métodos , Prolina/química , Prolina/genética , Prolina/metabolismo , Ligação Proteica , Homologia de Sequência de Aminoácidos , Triptofano/química , Triptofano/genética , Triptofano/metabolismoRESUMO
Dendritic cells (DC) change their phenotype and functional properties during maturation. CD83 cell surface expression is induced on mature DC (mDC). In this study, we investigated intracellular CD83 localization and transport in human monocyte-derived DC. The enhanced level of CD83 cell surface expression in mDC resulted predominantly from increased protein synthesis, and in addition from regulated intracellular transport of CD83 protein. An internal pool of CD83 protein is present in immature DC (iDC). Although CD83 protein in iDC and in mDC was localized in the Golgi compartment and in recycling endosomes, only in mature cells did CD83 co-localize with MHC class II molecules in endocytic vesicles. CD83 cell surface expression on iDC was induced by inhibition of endocytosis. This result could be explained by CD83 cycling between endosomes and the cell surface in iDC. The mDC also rapidly internalized membrane-bound CD83 protein. Furthermore, a thiol protease inhibitor and specific cathepsin inhibitors impaired CD83 up-regulation in DC, indicating a role of endosomal proteases in the maturation-induced exposure of CD83 on the plasma membrane.
Assuntos
Células Dendríticas/imunologia , Imunoglobulinas/imunologia , Glicoproteínas de Membrana/imunologia , Antígenos CD , Endossomos/imunologia , Complexo de Golgi/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Fatores de Tempo , Antígeno CD83RESUMO
HSV type 1 (HSV-1) has evolved numerous strategies for modifying immune responses that protect against infection. Important targets of HSV-1 infection are the MHC-encoded peptide receptors. Previous studies have shown that a helper T cell response and Ab production play important roles in controlling HSV-1 infection. The reduced capacity of infected B cells to stimulate CD4(+) T cells is beneficial for HSV-1 to evade immune defenses. We investigated the impact of HSV-1 infection on the MHCII processing pathway, which is critical to generate CD4(+) T cell help. HSV-1 infection targets the molecular coplayers of MHC class II processing, HLA-DR (DR), HLA-DM (DM), and invariant chain (Ii). HSV-1 infection strongly reduces expression of Ii, which impairs formation of SDS-resistant DR-peptide complexes. Residual activity of the MHC class II processing pathway is diminished by viral envelope glycoprotein B (gB). Binding of gB to DR competes with binding to Ii. In addition, we found gB associated with DM molecules. Both, gB-associated DR and DM heterodimers are exported from the endoplasmic reticulum, as indicated by carbohydrate maturation. Evaluation of DR, DM, and gB subcellular localization revealed abundant changes in intracellular distribution. DR-gB complexes are localized in subcellular vesicles and restrained from cell surface expression.
Assuntos
Apresentação de Antígeno , Herpesvirus Humano 1/imunologia , Herpesvirus Humano 1/patogenicidade , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Transdução de Sinais/imunologia , Animais , Antígenos de Diferenciação de Linfócitos B/biossíntese , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linfócitos B/virologia , Ligação Competitiva/imunologia , Células COS , Linhagem Celular Transformada , Linhagem Celular Tumoral , Chlorocebus aethiops , Dimerização , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Complexo de Golgi/imunologia , Complexo de Golgi/metabolismo , Complexo de Golgi/virologia , Antígenos HLA-D/metabolismo , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidade Classe II/biossíntese , Humanos , Peptídeos/metabolismo , Ligação Proteica/imunologia , Transporte Proteico/imunologia , Proteínas do Envelope Viral/metabolismo , Virulência/imunologiaAssuntos
Dor Abdominal/etiologia , Tecido Adiposo/irrigação sanguínea , Apendicite/diagnóstico , Apêndice/irrigação sanguínea , Anormalidade Torcional/diagnóstico , Tiflite/diagnóstico , Doença Aguda , Adulto , Apendicite/cirurgia , Apêndice/patologia , Diagnóstico Diferencial , Feminino , Humanos , Laparoscopia , Necrose , Sensibilidade e Especificidade , Anormalidade Torcional/cirurgia , Ultrassonografia , Ultrassonografia Doppler em CoresRESUMO
The stage of a colorectal carcinoma represents the most important prognostic factor regarding the probability of survival. The primary objective of this study was to document the management of patients with colorectal carcinoma after onset of symptoms. Factors influencing the delay in definitive therapy should thus be determined. Anthropometric, social, and operative data were obtained by standardized questionnaires from 40 patients with colonic cancer and 30 patients with rectal cancer. The influence of delayed treatment on outcome was analyzed. A significant correlation was found for the time between onset of first symptoms and definitive surgical therapy with tumor stage (colon cancer: r = 0.52, p < 0.05; colorectal cancer: r = 0.62, p < 0.05). The time delay in rectal carcinoma patients averaged 224 days and in patients with colonic carcinoma 149 days. Social influences such as profession, type of education, marital status, and quality of health insurance had a significant influence on treatment delay, as did the clinical experience of the physician first contacted. The leading symptom in patients with rectal cancer was peranal hemorrhage, and in patients with colonic cancer it was abdominal pain. The main causes of iatrogenic delay were insufficient clinical investigation and a lack of awareness when typical first symptoms were present. Delayed treatment of colorectal cancer seems to be a multifactorial problem. Causes for such delay are found not only in the patients and their social environments but also in the type and quality of their medical care systems. Intensified education and earlier prevention are the major aims for patients and their physicians.
Assuntos
Neoplasias do Colo/diagnóstico , Neoplasias Retais/diagnóstico , Idoso , Neoplasias do Colo/cirurgia , Feminino , Humanos , Cobertura do Seguro , Modelos Lineares , Masculino , Neoplasias Retais/cirurgia , Fatores SocioeconômicosRESUMO
Major histocompatibility complex class I (MHCI) molecules are major targets of virus evasion strategies because they introduce antigens from the biosynthesis pathway into the antigen-processing and presentation pathways for immune recognition by CD8+ T cells. Little is known about viral strategies that interfere with the MHC class II (MHCII) antigen presentation pathway. We identified a six amino acid sequence from type I herpes simplex virus (HSV-1) glycoprotein B (gB) that is identical to a sequence of human leucocyte antigen D (HLA-D) -associated invariant chain (Ii). In addition, this gB sequence is adjacent to a highly conserved HLA-DR1 binding motif. Both viral sequences together resemble the class II binding site of human Ii, consisting of a MHCII groove binding segment and a promiscuous binding site. We cloned gB from HSV-1 strain 17 and demonstrate association of the virus envelope protein to three HLA-DR allotypes. With chimeric Ii/gB fusion proteins we identified gB sequences that mediate promiscuous or allotype-specific binding to the HLA-DR peptide-binding domain. Mutation of two Lys residues in the viral segment of Ii/gB abolished promiscuous binding to HLA-DR heterodimers. The result indicates promiscuous binding of the virus sequence to HLA-DR molecules and suggests a potential for HSV-1 to manipulate antigen processing and presentation.