RESUMO
PURPOSE: Growing evidence suggests different systemic exposure of anti-vascular endothelial growth factor (anti-VEGF) agents with repeated intravitreal application. Since the penetration of anti-VEGF agents through vascular barrier was reported, the interaction of anti-VEGF with nonresident platelets has become a topic of interest. The purpose of this study was to evaluate, with the help of visualization techniques, whether platelets take up the anti-VEGF agents ranibizumab, aflibercept, and bevacizumab. METHODS: The uptake of anti-VEGF agents with or without VEGF treatment was investigated using immunofluorescence and immunogold staining in human platelets. The role of actin filaments and clathrin-coated vesicles in the transport of ranibizumab, aflibercept, and bevacizumab was evaluated by two pharmacologic inhibitors: staurosporine (protein kinase C inhibitor) and cytochalasin D. RESULTS: All three anti-VEGF agents were taken up by platelets and colocalized with VEGF. Ranibizumab and aflibercept were mainly detected in alpha-granules; however, bevacizumab was equally localized in alpha-granules and in platelet vesicles. Both staurosporine and cytochalasin D completely inhibited the uptake of aflibercept into platelets. Both pharmacological inhibitors also decreased the transport of ranibizumab and bevacizumab into platelets. Bevacizumab was significantly more frequently colocalized within clathrin-coated vesicles than ranibizumab and aflibercept. CONCLUSION: All three anti-VEGF agents are taken up by platelets and internalized in alpha-granules, which may result in a higher local exposure of anti-VEGF after the activation of platelets, potentially contributing to arterial thromboembolic events. Clathrin-coated vesicles seem to be more prominent in the transport of bevacizumab than ranibizumab and aflibercept. Nevertheless, whether the different localization and transport of bevacizumab are truly related to specific differences of receptor-mediated endocytosis has to be revealed by further research.
RESUMO
Peptide fragments of foreign and self-proteins are of great immunologic importance as their binding to major histocompatibility complex (MHC) class I or II molecules makes an interaction with a corresponding T cell receptor possible. Recently, allele-specific peptide sequence motifs proved to be responsible for MHC binding, no matter whether self- or non-self-antigens were involved. Up to now, all investigated human class II-associated peptides were derived from foreign antigenic proteins. Therefore, we undertook sequence and binding analyses with a 16-mer self-peptide (SP3) that has been eluted from HLA-DR1. Here we demonstrate, by synthetic polyalanine-based 13-mer analogues of SP3, that two bulky hydrophobic anchor residues with relative spacing i, i + 8 are sufficient for high affinity binding. This is consistent with the hydrophobic i, i + 8 binding pattern recently found for DR-restricted T cell epitopes. Nevertheless, highly helical alanine-based design peptides with anchor spacing i, i + 9 exhibit maximal affinity, whereas replacement of alanine by helix destabilizing proline abrogates binding. Thus, a two-residue contact motif is the common minimal requirement of self- and foreign peptides for high affinity anchoring to HLA-DR1. In contrast to class I, the anchor spacing of DR1-associated peptides seems to bear some variability due to conformational diversity.
Assuntos
Antígeno HLA-DR1/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Sequência de Aminoácidos , Linhagem Celular Transformada , Epitopos/análise , Antígeno HLA-DR1/química , Herpesvirus Humano 4/genética , Humanos , Cinética , Complexo Principal de Histocompatibilidade , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Homologia de Sequência do Ácido NucleicoRESUMO
Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Defensinas/farmacologia , Lisina/metabolismo , Neutrófilos/imunologia , Fosfatidilgliceróis/metabolismo , Staphylococcus aureus/metabolismo , Sequência de Aminoácidos , Aminoaciltransferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Sequência de Bases , Membrana Celular/metabolismo , DNA Bacteriano , Resistência Microbiana a Medicamentos , Esterificação , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Peptídeos/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Suínos , Virulência , alfa-Defensinas/farmacologiaRESUMO
Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373 glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20% of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake.
Assuntos
Cátions/metabolismo , Compostos Heterocíclicos/metabolismo , Compostos Organometálicos/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Transporte Biológico/fisiologia , Cátions/química , Linhagem Celular Tumoral , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Compostos Heterocíclicos/química , Humanos , Dados de Sequência Molecular , Ácido Mirístico/metabolismo , Compostos Organometálicos/química , Peptídeos/química , Peptídeos/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Cytotoxic T lymphocytes (CTLs) recognize foreign peptides bound to major histocompatibility complex (MHC) class I molecules. MHC molecules can also bind endogenous self peptides, to which T cells are tolerant. Normal mice contained CTLs specific for self peptides that were from proteins of ubiquitous or tissue-restricted expression. In vivo, these endogenous self peptides are not naturally presented in sufficient density by somatic cells expressing MHC class I molecules. They can, however, be presented if added exogenously. Thus, our data imply that CTLs are only tolerant of those endogenous self peptide sequences that are presented by MHC class I-positive cells in a physiological manner.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Complexo Principal de Histocompatibilidade , Fragmentos de Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Autoimunidade , Linhagem Celular , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Peptide conjugates derived from the SV 40 T antigen nuclear localisation sequence (NLS) have been successfully used to translocate both fluorescein isothiocyanate (FITC) and Gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) into the cytoplasm and nucleus of glioma cells. However, uptake occurred only in up to 35% of cells. To improve cellular uptake, we designed three novel FITC-labelled Gd-DOTA conjugates. In the first conjugate, the commonly used Gd-DOTA-complex was coupled to the nuclear localization sequence (NLS) of the Simian Virus (SV) 40 T antigen alone as a control. In the second conjugate, the Gd-DOTA-coupled SV 40 T antigen NLS was elongated by the HIV-1 tat peptide (HIV-NLS). A third conjugate, in which the Gd-DOTA-complex was coupled to the SV 40 T antigen NLS elongated by a peptide containing seven arginines and six aminohexanoic acids (Ahx6R7) was also synthesized (AHX-NLS). By means of confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests we were able to demonstrate that the first conjugate containing only the NLS of the SV 40 T antigen stained the nuclei of no more than 10-12% of U373 and LN18 glioma cells, resulting in low signal intensity in MRI. The stained cells remained viable. After incubation with conjugates HIV-NLS and AHX-NLS the nuclei of up to 73% of U373 and LN18 glioma cells were stained. This was associated with high signal intensity in MRI and cell death. As previously shown, the gadolinium ion reduces cellular uptake of DOTA conjugates. To confirm this, the conjugates were produced with or without gadolinium. The gadolinium-free DOTA conjugates showed a higher cellular uptake rate and an increased cytotoxic potential.
Assuntos
Núcleo Celular/metabolismo , Corantes Fluorescentes/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Transporte Ativo do Núcleo Celular , Antígenos Transformantes de Poliomavirus/química , Sobrevivência Celular/efeitos dos fármacos , Citoplasma/metabolismo , Citometria de Fluxo , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes/química , Glioma/patologia , Compostos Heterocíclicos/metabolismo , Compostos Heterocíclicos/farmacologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Humanos , Imageamento por Ressonância Magnética , Microscopia Confocal , Sinais de Localização Nuclear/química , Sinais de Localização Nuclear/metabolismo , Compostos Organometálicos/metabolismo , Compostos Organometálicos/farmacologia , Peptídeos/síntese química , Espectrometria de Massas por Ionização por Electrospray , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The molecular basis of high versus low frequency hearing loss and the differences in the sensitivity of outer hair cells depending on their cochlear localization are currently not understood. Here we demonstrate the existence of two different outer hair cell phenotypes along the cochlear axis. Outer hair cells in low frequency regions exhibit early sensitivity for loss of Ca(v)1.3 (alpha1 subunit 1.3 forming the class D L-type voltage-gated Ca(2+) channel), while high frequency regions display a progressive susceptibility for loss of the Ca(2+)-activated large conductance K(+) (BK) channel. Despite deafness, young Ca(v)1.3-deficient mice displayed distortion-product otoacoustic emissions (DPOAEs), indicating functional outer hair cells in the higher frequency range of the cochlea. Considering that DPOAEs are also found in the human deafness syndrome DFNB9 caused by mutations in the synaptic vesicle protein otoferlin, we tested the expression of otoferlin in outer hair cells. Surprisingly, otoferlin showed a distinct tonotopic expression pattern at both the mRNA and protein level. Otoferlin-expressing, Ca(v)1.3 deletion-sensitive outer hair cells in the low frequency range could be clearly separated from otoferlin-negative, BK deletion-sensitive outer hair cells in the high frequency range. In addition, BK deletion led to a higher noise vulnerability in low frequency regions, which are normally unaffected by the BK deletion alone, suggesting that BK currents are involved in survival mechanisms of outer hair cells under noise conditions. Our findings propose new mechanisms and candidate genes for explaining high and low frequency hearing loss.
Assuntos
Cóclea/citologia , Células Ciliadas Auditivas Externas/citologia , Células Ciliadas Auditivas Externas/fisiologia , Estimulação Acústica/métodos , Oxirredutases do Álcool , Animais , Animais Recém-Nascidos , Limiar Auditivo/fisiologia , Canais de Cálcio Tipo L/deficiência , Proteínas Correpressoras , Cóclea/crescimento & desenvolvimento , Proteínas de Ligação a DNA/metabolismo , Potenciais Evocados Auditivos do Tronco Encefálico/genética , Regulação da Expressão Gênica/genética , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Perda Auditiva Neurossensorial/fisiopatologia , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/deficiência , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/fisiologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Emissões Otoacústicas Espontâneas/genética , Fosfoproteínas/metabolismo , RNA Mensageiro , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de TempoRESUMO
Endogenous peptides bound to the constitutively expressed MHC class II molecules HLA-DR and HLA-DQ of the melanoma cell line FM3 were examined. By a combination of analytical methods (narrow bore and capillary reversed-phase high-performance liquid chromatography with subsequent spotting on polyvinylidene difluoride membranes, matrix-assisted laser desorption ionization mass spectrometry, and Edman microsequencing), we were able to isolate and identify a panel of HLA-DR4/2 (HLA-DRB1*0401/0201/DRB5*0101)-associated self-peptides from the melanoma cell line FM3. Among ubiquitously HLA-DR-associated peptides such as peptides from the class II-associated invariant chain peptide region of the invariant chain, HLA-class I, the transferrin receptor, and the IFN-gamma receptor, we identified several potential tumor-associated antigens stemming from the MHC class I-restricted tumor antigen gp100, the Ca2(+)-binding protein annexin II, and proteins from the hsp70 family. Chinese hamster ovary cells cotransfected with HLA-DRA, DRB1*0401, and CD80 genes were shown specifically to prime T lymphocytes from HLA-DRB1*0401 donors to the annexin II and gp100 peptides. These results demonstrate that MHC class II molecules expressed by melanoma cells potentially present a variety of novel antigens to the immune system, some of which could be exploited for immunotherapy.
Assuntos
Antígenos de Neoplasias/isolamento & purificação , Antígenos HLA-DR/isolamento & purificação , Melanoma/imunologia , Animais , Anexina A2/imunologia , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Antígenos HLA-DQ/imunologia , Humanos , Concentração de Íons de Hidrogênio , Glicoproteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Fragmentos de Peptídeos/metabolismo , Células Tumorais Cultivadas , Antígeno gp100 de MelanomaRESUMO
The function of the steel factor receptor, p145c-kit, in patient-derived acute myeloblastic leukemia (AML) cells was investigated. Steel factor stimulation of AML cells coexpressing p145c-kit and the progenitor cell antigen CD34 resulted in complete receptor down-regulation, a marked decrease of CD34 antigen expression, and the induction of the granulocyte lineage antigen CD15. These changes in surface marker expression paralleled morphological differentiation to granulated blasts and promyelocytes. Interestingly, the same phenotype was achieved by IL-3 stimulation of AML cells. p145c-kit extracellular domain-specific antibodies had either blocking or enhancing effects on ligand binding, receptor phosphorylation and down-regulation, and induction of cell proliferation. Correlations of these phenomena with distinct effects of antibody stimulation on cell substrate phosphorylation provide clues for the dissection of the p145c-kit signal and the analysis of its relevance for AML.
Assuntos
Leucemia Mieloide Aguda/patologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Diferenciação Celular , Regulação para Baixo , Fatores de Crescimento de Células Hematopoéticas/fisiologia , Humanos , Interleucina-3/farmacologia , Leucemia Mieloide Aguda/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-kit , Fator de Células-TroncoRESUMO
Melanoma and renal cell carcinoma (RCC) are thought to be the most immunogenic human tumors. Presently a series of tumor-specific peptides of melanoma is being tested in clinical trials with different immunotherapy protocols. In contrast, only one decameric peptide (SPSSNRIRNT) derived from one (ORF2) of three possible open reading frames (ORFs) of a gene named RAGE (Renal tumor AntiGEn) was shown to be the target for tumor-specific CTLs on renal carcinoma cells. One reason for the lack of identification of tumor antigens on RCC compared with melanoma may be the difficulty in generating tumor-specific CTLs as screening instruments. Therefore, our approach was directly to isolate and identify peptides bound to HLA class I molecules of the HLA-A2 and -B8 homozygous RCC line A-498. High performance liquid chromatography-fractionated peptides eluted with acid from immunoaffinity-purified HLA class I-peptide complexes were sequenced and identified for the first time by the novel and highly sensitive mass spectrometric method matrix-assisted laser desorption ionization-post source decay (MALDI-PSD) from minute amounts of 100 fmol to 1.5 pmol of the fractionated peptide samples. Fourteen peptide sequences first deduced from interpretations of the mass spectra were also shown to fulfill other reliability criteria such as matching the mass spectra of the respective synthetic peptides. Some peptides were identified to be derived from genes preferentially activated in malignant tissues or resulted from a possibly mutated gene. The most promising candidate for a CTL epitope is a decameric peptide (PASKKTDPQK) derived from another possible ORF (ORF5) of the RAGE gene and probably presented in association with HLA-B8. This peptide was synthesized and used for the in vitro induction of CTLs that lysed the A-498 cells and another HLA-B8-positive RCC line significantly more strongly than either other RAGE-positive but HLA-B8-negative RCC lines or K562 cells. Sensitive sequencing by MALDI-PSD thus may provide a powerful method of identifying potentially tumor-specific and HLA-restricted antigens, even on native malignant cells and tissues.
Assuntos
Carcinoma/imunologia , Antígenos de Histocompatibilidade Classe I/análise , Neoplasias Renais/imunologia , Espectrometria de Massas/métodos , Peptídeos/análise , Antígenos de Neoplasias/química , Cromatografia Líquida de Alta Pressão , Humanos , Peptídeos/síntese química , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The requirements for insulin presentation and recognition by A alpha b A beta b- and A alpha b A beta k-restricted mouse T cells were studied using a variety of derivatives of the insulin A chain. It was found that A chain peptides with irreversibly blocked Cys residues are non-stimulatory for the T cells. This suggests that at least one of the Cys residues is essential for recognition. On the other hand, all A chain peptides containing Cys residues modified in a way reversible by reaction with thiols are stimulatory yet differ in antigenic potency. All these A chain derivatives including a 14 amino acid fragment require uptake by antigen presenting cells (APC) for efficient presentation. Differences in stimulatory potency between the A chain peptides derived from the same insulin appear to be mainly due to the efficiency of uptake and/or processing by APC. Based on these findings we propose that processing in the case of insulin and its A chain derivatives involves the reductive deblocking of Cys residues or the rearrangement of disulfide bonds apart from a possible proteolytic cleavage. The same may apply to other proteins if Cys residues in the presented peptides are important for the interaction with Ia or the T cell receptor.
Assuntos
Insulina/imunologia , Peptídeos/imunologia , Linfócitos T/imunologia , Animais , Células Apresentadoras de Antígenos/imunologia , Bovinos , Linhagem Celular , Cloroquina/farmacologia , Cisteína/química , Epitopos , Antígenos de Histocompatibilidade Classe II/imunologia , Técnicas In Vitro , Insulina/química , Interleucina-3/biossíntese , Camundongos , Camundongos Endogâmicos , Receptores de Antígenos de Linfócitos T/imunologia , SuínosRESUMO
Endothelial cells of the blood-brain barrier form complex tight junctions, which are more frequently associated with the protoplasmic (P-face) than with the exocytoplasmic (E-face) membrane leaflet. The association of tight junctional particles with either membrane leaflet is a result of the expression of various claudins, which are transmembrane constituents of tight junction strands. Mammalian brain endothelial tight junctions exhibit an almost balanced distribution of particles and lose this morphology and barrier function in vitro. Since it was shown that the brain endothelial tight junctions of submammalian species form P-face-associated tight junctions of the epithelial type, the question of which molecular composition underlies the morphological differences and how do these brain endothelial cells behave in vitro arose. Therefore, rat and chicken brain endothelial cells were investigated for the expression of junctional proteins in vivo and in vitro and for the morphology of the tight junctions. In order to visualize morphological differences, the complexity and the P-face association of tight junctions were quantified. Rat and chicken brain endothelial cells form tight junctions which are positive for claudin-1, claudin-5, occludin and ZO-1. In agreement with the higher P-face association of tight junctions in vivo, chicken brain endothelia exhibited a slightly stronger labeling for claudin-1 at membrane contacts. Brain endothelial cells of both species showed a significant alteration of tight junctions in vitro, indicating a loss of barrier function. Rat endothelial cells showed a characteristic switch of tight junction particles from the P-face to the E-face, accompanied by the loss of claudin-1 in immunofluorescence labeling. In contrast, chicken brain endothelial cells did not show such a switch of particles, although they also lost claudin-1 in culture. These results demonstrate that the maintenance of rat and chicken endothelial barrier function depends on the brain microenvironment. Interestingly, the alteration of tight junctions is different in rat and chicken. This implies that the rat and chicken brain endothelial tight junctions are regulated differently.
Assuntos
Encéfalo/metabolismo , Endotélio Vascular/metabolismo , Junções Íntimas/fisiologia , Animais , Anticorpos/metabolismo , Encéfalo/irrigação sanguínea , Galinhas , Claudina-1 , Técnica de Fratura por Congelamento , Imuno-Histoquímica , Proteínas de Membrana/metabolismo , Microscopia Eletrônica , Microscopia de Fluorescência , Ratos , Ratos Wistar , Fatores de TempoRESUMO
From a high-affinity Ins-P4 (inositol 1,3,4,5-P4) receptor purified from pig cerebellum, digested with the protease Lys C peptide sequences were obtained. Synthetic peptide-3 (19 amino acid residues) was used to generate an antiserum. Reaction of the affinity-purified antibodies with the purified pig receptor protein in ELISA or Western blot was completely inhibited by peptide-3. In cerebellar membranes, the antibodies clearly recognized the 42 kDa Ins-P4 receptor protein and two additional proteins (25 kDa, 37 kDa) which still have to be identified. The anti-peptide antibodies could selectively immunoprecipitate the Ins-P4 receptor protein. The antiserum was used (i) to demonstrate that in brain from different species (human, pig, beef, rat, mouse and sheep) a similar 42 kDa Ins-P4 receptor protein is contained, and (ii) to obtain indications for the existence of a related soluble form of the 42 kDa Ins-P4 receptor besides the membrane-associated receptor.
Assuntos
Química Encefálica , Soros Imunes/farmacologia , Fosfatos de Inositol/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/imunologia , Sequência de Aminoácidos , Animais , Anticorpos/isolamento & purificação , Afinidade de Anticorpos , Especificidade de Anticorpos , Western Blotting , Bovinos , Cerebelo/química , Fracionamento Químico , Cromatografia/métodos , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida/métodos , Ensaio de Imunoadsorção Enzimática , Feminino , Imunoglobulinas/sangue , Imunoglobulinas/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia , Testes de Precipitina , Coelhos , Receptores Citoplasmáticos e Nucleares/metabolismo , SuínosRESUMO
In order to obtain specific antibodies against thymosin beta 9 showing minimal cross-reactivity with the highly homologous peptide thymosin beta 4, the N-terminal fragment 1-14 of thymosin beta 9 was used for immunization. These antibodies have been tested in a competitive ELISA and show less than 1% cross-reactivity with thymosin beta 4. On the other hand, antibodies raised against the native thymosin beta 9 (1-14) cross-react 35% with thymosin beta 4. Specific antibodies against thymosin beta 9 are important for studying the concentration and localization of thymosin beta 9 in thymus and other bovine tissues because thymosin beta 9 is always accompanied by thymosin beta 4. Using N-terminal fragments of thymosin beta 4-like peptides may be a general approach for obtaining specific antibodies since this part of sequence is less conserved in thymosin beta 4-like peptides.
Assuntos
Timosina/análise , Sequência de Aminoácidos , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Dados de Sequência Molecular , Fragmentos de Peptídeos/imunologia , Timosina/análogos & derivados , Timosina/sangue , Timosina/classificação , Timosina/imunologia , Timo/análiseRESUMO
Here we describe a fast and efficient subcellular fractionation procedure that permits lysosomes to be separated from endosomes. Differential centrifugation is used to isolate a subcellular fraction containing both endosomes and lysosomes. Because lysosomes are sensitive to osmotic stress, hypotonic conditions destroy them, whereas endosomes, which are osmotically insensitive, stay intact. We demonstrate that hypotonic lysis of an endosome-lysosome-pool releases 85% of the lysosomes into the supernatant as measured by the activity of the lysosomal marker enzyme N-acetyl-beta-D-glucosaminidase (beta-AGA). The endosomal fraction is thoroughly characterised using a variety of subcellular markers. After pulsing cells with fluorescein isothiocyanate labelled transferrin (FITC-Tf), only about 12% of the marker is released under hypotonic conditions. A typical fractionation procedure takes about 1-2 h from initial cell homogenisation. The fractionation gives a pure lysosomal fraction (fraction L) containing high activities of lysosomal enzymes and an endosomal fraction (fraction E) reflecting different stages of endosomes.
Assuntos
Fracionamento Celular/métodos , Endossomos/química , Lisossomos/química , Antígenos de Diferenciação de Linfócitos B/análise , Linhagem Celular , Centrifugação , Endopeptidases/análise , Antígenos de Histocompatibilidade Classe II/análise , HumanosRESUMO
We report for the first time the immunoadjuvant effects of lipoconjugation of peptide antigens in an in vitro system by using CD4+ T-cells. The lipopeptides obtained by conjugating a palmitoyl moiety at the N(alpha)-terminal of Gln(74) or at the epsilon-NH(2) of Lys(75) of GpMBP(74-85) induced increased T-cell responsiveness compared to the native nonlipidated peptide. On the other hand, lipoderivatives of GpMBP(82-98) did not increase the T-cell response, demonstrating that the superagonist inducing effect of lipoconjugation is epitope-specific. Digestion of the two native peptides with cathepsin D and L, both implicated in antigen processing, and with a complete lysosomal fraction of a EBV-transformed B cell line shows that GpMBP(74-85) is resistant to cellular proteases, while GpMBP(82-98) is easily digested by these enzymes. These results suggest that the first prerequisite for increasing the T-cell response by lipoconjugation is the stability of the native peptide to peptidases, providing an important insight into the understanding of the immunoadjuvant effect of lipoderivative antigens.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Lipoproteínas/síntese química , Proteína Básica da Mielina/imunologia , Ácido Palmítico/química , Fragmentos de Peptídeos/imunologia , Peptídeo Hidrolases/química , Animais , Linfócitos T CD4-Positivos/imunologia , Catepsina D/química , Catepsina L , Catepsinas/química , Divisão Celular , Cisteína Endopeptidases , Epitopos , Feminino , Técnicas In Vitro , Lipoproteínas/química , Lipoproteínas/farmacologia , Lisossomos/enzimologia , Ratos , Ratos Endogâmicos Lew , Relação Estrutura-AtividadeRESUMO
In this study, we evaluated the role of the two functional HLA-DR heterodimers, DR2a (DR alpha paired with the beta chain encoded by DRB5*0101) and DR2b (DR alpha paired with the beta chain encoded by DRB1*1501), that are coexpressed in the multiple sclerosis (MS)-associated haplotype HLA-DR15 Dw2, in presenting myelin basic protein (MBP) peptides to MBP-specific T cell lines (TCL). Our results show that both HLA-DR molecules serve as restriction elements for HLA-DR15-restricted TCL. Slightly higher numbers of TCL use DR2a as restriction element, and the epitopes contained in the immunodominant C-terminal region (131-159) are uniquely restricted by DR2a. The immunodominant middle epitope (81-99) is recognized in the context of both DR2a and DR2b, but this specificity strongly dominates the DR2b-restricted T cell response. Overall, immunodominance in the MBP-specific T cell response correlated well with peptide binding to DR2a or DR2b, demonstrating that the affinity of MHC-peptide interactions is important for shaping the T cell response to this autoantigen. Furthermore, we show that binding of the middle MBP peptide to HLA-DR15 molecules prevents cleavage by cathepsin D, a protease abundantly found in endosomal processing compartments, and thus contributes to its immunodominance. Surprisingly, the restriction element employed by MBP-specific T cell clones influenced the effector function (i.e., cytotoxic activity) of T cells irrespective of their peptide fine specificity.
Assuntos
Citotoxicidade Imunológica , Antígenos HLA-DR/genética , Haplótipos/imunologia , Epitopos Imunodominantes/imunologia , Esclerose Múltipla/imunologia , Proteína Básica da Mielina/imunologia , Peptídeos/imunologia , Subpopulações de Linfócitos T/imunologia , Alelos , Sequência de Aminoácidos , Catepsina D/metabolismo , Linhagem Celular , Células Clonais , Antígenos HLA-DR/metabolismo , Subtipos Sorológicos de HLA-DR , Antígeno HLA-DR2/genética , Antígeno HLA-DR2/metabolismo , Humanos , Hidrólise , Epitopos Imunodominantes/genética , Dados de Sequência Molecular , Esclerose Múltipla/genética , Proteína Básica da Mielina/metabolismo , Peptídeos/metabolismo , Ligação Proteica/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
Exogenous antigens are internalized by antigen-processing cells and processed within vesicular compartments to produce antigenic peptides that bind to newly synthesized MHC II molecules. These MHC class II peptide complexes are displayed at the plasma membrane and stimulate specific CD4+ T cells. In the present study, we established a method to isolate intracellular MHC molecules in a preparative scale (2-3 mg HLA-DR1) from endosomal compartments by Percoll density-gradient centrifugation. Peptides associated with HLA-DR1 in these intracellular fractions were released, purified by microbore HPLC, characterized by sequencing, and compared with the amino acid composition of peptides derived from MHC class II molecules obtained by solubilization of the plasma membrane. The binding affinity of these MHC fractions was analyzed by our highly sensitive binding assay using different DR1-restricted IM and Ii peptides. The results indicate that (a) intracellular MHC molecules show higher peptide-binding capacity, (b) peptides that are about 18-25 amino acids long need only a core region of 11 amino acids for binding, (c) specific positions of the peptides are important for DR1 binding, (d) most of the naturally processed peptides show a proline at position 2 or 3 that may represent a stop signal for trimming, and (e) Ii peptides are very abundant in DR1 peptide pools derived from intracellular compartments.
Assuntos
Antígeno HLA-DR1/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Linhagem Celular Transformada , Centrifugação com Gradiente de Concentração , Humanos , Dados de Sequência Molecular , Peptídeos/imunologia , Frações Subcelulares/imunologiaRESUMO
The human Ca(2+)-binding (storage) protein calreticulin, located in the lumen of the endoplasmic reticulum, is proposed to play a role as autoantigen: anticalreticulin autoantibodies occur in the sera of patients with SLE and patients with onchocerciasis (calreticulin shows a high sequence homology to the Onchocerca volvulus antigen RAL-1). Here we present sequencing data of a HLA-DR4Dw4-associated calreticulin peptide fragment, Cal(295-310), purified from a DR4Dw4 self-peptide pool. Cal(295-310) proved to be one of three commonest self-peptides associated with DR4Dw4 molecules that were isolated from the EBV-transformed B-cell line BSM (DR4Dw4, DRw53). We tested the binding of Cal(295-309) and the analogous RAL-1 peptide to HLA-DR molecules: Cal(295-309) exhibited specific binding characteristics for DR4Dw4. Binding assays using self-peptide analogues with replaced amino acids led us to a DR4Dw4-binding motif with anchor residues at relative positions 1 and 6. The sequencing data suggest that calreticulin is a frequently processed intracellular protein. The abundance of calreticulin makes the presentation of different calreticulin peptides associated with HLA-D molecules likely to occur, supporting the immunologic relevance of this molecule.
Assuntos
Autoantígenos/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/imunologia , Antígeno HLA-DR4/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/imunologia , Sequência de Aminoácidos , Linfócitos B , Sítios de Ligação , Calreticulina , Linhagem Celular Transformada , Cromatografia Líquida de Alta Pressão , Humanos , Dados de Sequência MolecularRESUMO
Alloreactivity remains an important barrier to organ transplantation and is caused by T cell recognition of foreign histocompatibility antigens (HAg) in two ways: (1) indirect recognition, in which processed HAg peptides are presented by self MHC like any other foreign antigen, and (2) direct recognition, where the foreign MHC itself is recognized in contravention of the T cell recognition rule of self restriction. Whereas the role of endogenous peptides in direct MHC class I specific recognition is now established, their role in class II specific direct alloreactivity remains controversial, since no defined endogenous peptide has been shown to be required for alloreactivity. That mutations resulting in defective antigen processing impair class II specific allostimulation, however, suggests that the endogenous pathway is important for class II as well as class I alloreactivity. We attempted to establish the importance of endogenous peptides for alloreactivity by identifying common sequences of peptides bound by DR molecules of an HLA-DRB1*0401 homozygous B cell line. Peptides corresponding to three of these (calreticulin, HLA class I and an unidentified molecule) were used to restimulate established allospecific HLA-Dw4 reactive T cell clones, as well as to sensitize allogeneic T cells de novo in vitro. Xenogeneic chinese hamster ovary (CHO) cells coexpressing the relevant DR allele together with CD80 were used as antigen presenting cells. The role of CD80 could be determined on these cells because (1) they are xenogeneic and (2) they do not express B7 family members bound by CTLA-4Ig.(ABSTRACT TRUNCATED AT 250 WORDS)