RESUMO
Invariant chain (Ii) contributes in a number of distinct ways to the proper functioning of major histocompatibility complex (MHC) class II molecules. These include promoting effective association and folding of newly synthesized MHC class II alpha and beta subunits, increasing transit of assembled heterodimers out of the endoplasmic reticulum (ER), inhibiting class II peptide binding, and facilitating class II movement to or accumulation in endosomes/lysosomes. Although the cytoplasmic tail of Ii makes a key contribution to the endocytic localization of class II, the relationship between the structure of Ii and its other diverse functions remains unknown. We show here that two thirds of the lumenal segment of Ii can be eliminated without affecting its contributions to the secretory pathway events of class II folding, ER to Golgi transport, or inhibition of peptide binding. These same experiments reveal that a short (25 residue) contiguous internal segment of Ii (the CLIP region), frequently found associated with purified MHC class II molecules, is critical for all three functions. Together with other recent findings, these results raise the possibility that the contributions of Ii to the early postsynthetic behavior of class II may depend on its interaction with the class II binding site. This would be consistent with the intracellular behavior of unoccupied MHC class I and class II molecules as incompletely folded proteins and imply a related structural basis for the similar contributions of Ii to class II and of short peptides to class I assembly and transport.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/fisiologia , Fragmentos de Peptídeos/fisiologia , Dobramento de Proteína , Sequência de Bases , Sítios de Ligação , Transporte Biológico , Células Cultivadas , Antígenos de Histocompatibilidade Classe II/metabolismo , Dados de Sequência MolecularRESUMO
Calnexin is a resident protein of the endoplasmic reticulum (ER) that associates with nascent protein chains. Among the newly synthesized integral membrane proteins known to bind to calnexin is invariant chain (Ii), and Ii release from calnexin coincides with proper assembly with major histocompatibility complex (MHC) class II heterodimers. Although calnexin association with several membrane glycoproteins depends on interactions involving N-linked glycans, we previously reported that a truncation mutant of mouse Ii (mIi1-107) lacking both N-glycosylation sites was highly effective in associating with MHC class II heterodimers and escorting these dimers through the secretory pathway. This could indicate that calnexin, despite binding to both Ii and class II, is not necessary for the proper interaction of these proteins, or that in contrast to most membrane glycoproteins, the N-linked glycans of Ii are not critical to its interaction with this chaperone. To examine this issue, we have directly explored the binding of calnexin to both Ii truncation mutants lacking the typical sites of N-glycosylation or Ii produced in cells treated with tunicamycin to prevent glycan addition. These experiments revealed that either method of eliminating N-linked carbohydrates on Ii also inhibited association with calnexin. A lumenally truncated form of Ii (mIi1-131) that still has N-linked carbohydrates showed a decreased affinity for calnexin compared with intact Ii, however, indicating that calnexin-Ii binding is not determined solely by the sugar moieties. All forms of Ii lacking N-linked sugars and showing defective association with calnexin also had enhanced rates of preendosomal degradation. Despite this effect on degradation rate, tunicamycin treatment did not inhibit the association of class II with glycan-free Ii. These data support the view that calnexin is not an absolute requirement for the proper assembly of class II-Ii nonamers, but rather acts primarily to retain Ii in the ER and to inhibit its degradation. These two properties of calnexin-Ii interaction may help ensure that sufficient intact Ii is available for efficient inactivation of the binding sites of newly synthesized class II molecules, while limiting the ability of excess free Ii to alter the transport properties of the early endocytic pathway.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Animais , Antígenos de Diferenciação de Linfócitos B/química , Sequência de Bases , Transporte Biológico , Calnexina , Células Cultivadas , Chlorocebus aethiops , Primers do DNA/química , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicosilação/efeitos dos fármacos , Glicosiltransferases/antagonistas & inibidores , Antígenos de Histocompatibilidade Classe II/química , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Processamento de Proteína Pós-Traducional , Tunicamicina/farmacologiaRESUMO
Leucine-based signals in the cytoplasmic tail of invariant chain (Ii) control targeting of newly synthesized major histocompatibility complex class II molecules to the endocytic pathway for acquisition of antigenic peptides. Some protein determinants, however, do not require Ii for effective class II presentation, although endocytic processing is still necessary. Here we demonstrate that a dileucine-based signal in the cytoplasmic tail of the class II beta chain is critical for this Ii-independent presentation. Elimination or mutation of this signal reduces the rate of re-entry of mature surface class II molecules into the endocytic pathway. Antigen presentation controlled by this signal does not require newly synthesized class II molecules and appears to involve determinants requiring only limited proteolysis for exposure, whereas the opposite is true for li-dependent determinants. This demonstrates that related leucine-based trafficking signals in li and class II control the functional presentation of protein determinants with distinct processing requirements, suggesting that the peptide binding sites of newly synthesized versus mature class II molecules are made available for antigen binding in distinct endocytic compartments under the control of these homologous cytoplasmic signals. This permits capture of protein fragments produced optimally under distinct conditions of pH and proteolytic activity.
Assuntos
Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Sequência de Aminoácidos , Animais , Citoplasma/metabolismo , Endopeptidases/fisiologia , Endossomos/metabolismo , Feminino , Leucina , Camundongos , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
Invariant chain (Ii) associates with newly synthesized class II molecules in the endoplasmic reticulum (ER), an interaction that has been shown to interfere with peptide binding to class II molecules. The class II-associated invariant chain peptide (CLIP) region (residues 81-104) of Ii is believed to mediate this inhibition by engaging the binding domain of class II like an antigenic peptide. Together, these findings have given rise to a model in which CLIP association with the class II groove acts to prevent inappropriate presentation of peptides imported into the ER for association with major histocompatibility complex class I molecules. However, the properties of class II molecules synthesized by cells lacking coexpressed Ii are at least superficially inconsistent with this paradigm in that they do not show clear evidence of peptide acquisition. At the same time, we have previously shown the shortest form of Ii still containing CLIP to play an essential role in regulation of early class II molecule assembly and transport in the secretory pathway. Using covalent peptide technology, we now show that occupancy of the class II binding site in the ER regulates class II trafficking to the Golgi complex, an event that is the locus of the major defect in cells of Ii-deficient mice. These data argue that CLIP occupies the class II binding site, not to prevent interaction with short peptides meant for class I, but rather to maintain the structural integrity of class II molecules that are labile without engaged binding regions, and that would also associate with intact proteins in the ER if left unoccupied. By these means, CLIP occupancy of the class II binding site promotes effective export of useful class II molecules for endocytic peptide acquisition.
Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Complexo Principal de Histocompatibilidade , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Diferenciação de Linfócitos B/metabolismo , Sequência de Bases , Transporte Biológico , Células COS , Variação Genética , Antígenos de Histocompatibilidade Classe II/genética , Camundongos , Dados de Sequência Molecular , Peptídeos/imunologia , Ligação ProteicaRESUMO
Invariant chain (Ii), which associates with major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum, contains a targeting signal for transport to intracellular vesicles in the endocytic pathway. The characteristics of the target vesicles and the relationship between Ii structure and class II localization in distinct endosomal subcompartments have not been well defined. We demonstrate here that in transiently transfected COS cells expressing high levels of the p31 or p41 forms of Ii, uncleaved Ii is transported to and accumulates in transferrin-accessible (early) endosomes. Coexpressed MHC class II is also found in this same compartment. These early endosomes show altered morphology and a slower rate of content movement to later parts of the endocytic pathway. At more moderate levels of Ii expression, or after removal of a highly conserved region in the cytoplasmic tail of Ii, coexpressed class II molecules are found primarily in vesicles with the characteristics of late endosomes/prelysosomes. The Ii chains in these late endocytic vesicles have undergone proteolytic cleavage in the lumenal region postulated to control MHC class II peptide binding. These data indicate that the association of class II with Ii results in initial movement to early endosomes. At high levels of Ii expression, egress to later endocytic compartments is delayed and class II-Ii complexes accumulate together with endocytosed material. At lower levels of Ii expression, class II-Ii complexes are found primarily in late endosomes/prelysosomes. These data provide evidence that the route of class II transport to the site of antigen processing and loading involves movement through early endosomes to late endosomes/prelysosomes. Our results also reveal an unexpected ability of intact Ii to modify the structure and function of the early endosomal compartment, which may play a role in regulating this processing pathway.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Endocitose/fisiologia , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/metabolismo , Complexo Principal de Histocompatibilidade/imunologia , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Retículo Endoplasmático/química , Retículo Endoplasmático/fisiologia , Retículo Endoplasmático/ultraestrutura , Imunofluorescência , Expressão Gênica/genética , Expressão Gênica/fisiologia , Genes MHC da Classe II/genética , Antígenos de Histocompatibilidade Classe II/química , Membranas Intracelulares/química , Membranas Intracelulares/fisiologia , Membranas Intracelulares/ultraestrutura , Lisossomos/química , Lisossomos/fisiologia , Lisossomos/ultraestrutura , TransfecçãoRESUMO
In mice double transgenic for functionally rearranged T cell receptor (TCR) V beta 2 and V beta 8.2 genes we found that most T lymphocytes express both TCR beta chains simultaneously. These T cells show no abnormality in thymic selection in vivo and their TCRs are capable of transducing activation signals in vitro. These results indicate that multispecific T cells may appear in the periphery if allelic exclusion of TCR beta genes is not established at the level of gene rearrangement.
Assuntos
Rearranjo Gênico da Cadeia beta dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Alelos , Animais , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Cruzamentos Genéticos , Citometria de Fluxo , Regulação da Expressão Gênica , Genótipo , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Transdução de Sinais , Timo/imunologiaRESUMO
BACKGROUND: CAT25 (T25mononucleotide repeat of the Caspase 2 gene), is a promising DNA marker for detecting microsatellite instability (MSI) in colorectal cancer. CAT25 has the potential to be incorporated into the Bethesda panel, a commonly used panel of DNA microsatellites, or replace it in its entirety. We aimed to develop and validate a high-resolution melting-PCR (HRM-PCR) method for CAT25 instability detection in clinical samples. METHODS: The instability of CAT25, BAT25 (a poly(A) tract occurring in c-kit) and BAT26 (a poly(A) tract localized in hMSH2) microsatellites were assessed in DNA from tumour and peripheral blood obtained from 110 patients with colorectal cancer using HRM-PCR and capillary electrophoresis. Immunohistochemistry (IHC) staining for MSH2, MSH6, MLH1, and PMS2 enzymes was performed on tumours with jigj MSI. Allelic size variation of CAT25 was analysed on peripheral blood DNA from 208 healthy volunteers. RESULTS: The HRM-PCR for CAT25 was validated in clinical samples. CAT25 showed a tight range of 64-66 base pairs. Of 110 tumours, 11 had High MSI, later confirmed by IHC. CAT25 defines MSI alone as well as when used together with BAT25 and BAT26. CAT25 results provided 100% predictive values and p < 0.0001 to classify a tumour as having high MSI. CONCLUSIONS: We developed and validated a new HRM-PCR assay to detect CAT25 instability. Our findings showed a limited allelic size variation of CAT25 and highlighted to CAT25 as a promising marker for MSI analysis.
Assuntos
Caspase 2/genética , Neoplasias Colorretais/genética , Marcadores Genéticos/genética , Repetições de Microssatélites/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodosRESUMO
Gold-specific T lymphocyte clones were isolated from a patient with rheumatoid arthritis who developed delayed type hypersensitivity reactions to gold. All of the isolated T cell clones required histocompatible antigen presenting cells as well as gold for induction of proliferation. Using a panel of HLA-homozygous Epstein Barr virus-transformed B (EBV-B) cells and anti-HLA antibodies, the clones were shown to recognize gold in the context of DR1 molecules. Gold recognition did not require active antigen processing since specific proliferation was not affected by glutaraldehyde fixation of the DR1 homozygous antigen presenting cells. Furthermore, we could show that gold salts inhibited peptide-induced responses of a peptide-specific T cell clone. In addition to providing evidence for gold-specific T cells in gold-treated RA patients exhibiting delayed type hypersensitivity responses, these data suggest that gold can alter MHC-peptide complexes. The latter observation may in part explain the mechanism/s responsible for both the therapeutic and the toxic effects of gold.
Assuntos
Artrite Reumatoide/imunologia , Ouro/imunologia , Hipersensibilidade Tardia/imunologia , Subpopulações de Linfócitos T/imunologia , Células Apresentadoras de Antígenos/imunologia , Artrite Reumatoide/tratamento farmacológico , Células Clonais/imunologia , Feminino , Glutaral , Ouro/farmacologia , Antígenos HLA/imunologia , Humanos , Ativação Linfocitária/imunologia , Cooperação Linfocítica/imunologia , Masculino , Desnaturação Proteica/imunologia , Preservação de TecidoRESUMO
Mucosal antigen-specific CD4 T-cell responses to intestinal pathogens remain incompletely understood. Here we examined the CD4 T-cell response after oral infection with an internalin A 'murinized' Listeria monocytogenes (Lm). Oral Lm infection induced a robust endogenous listeriolysin O (LLO)-specific CD4 T-cell response with distinct phenotypic and functional characteristics in the intestine. Circulating LLO-specific CD4 T cells transiently expressed the 'gut-homing' integrin α4ß7 and accumulated in the intestinal lamina propria and epithelium where they were maintained independent of interleukin (IL)-15. The majority of intestinal LLO-specific CD4 T cells were CD27- Ly6C- and CD69+ CD103- while the lymphoid LLO-specific CD4 T cells were heterogeneous based on CD27 and Ly6C expression and predominately CD69-. LLO-specific effector CD4 T cells transitioned into a long-lived memory population that phenotypically resembled their parent effectors and displayed hallmarks of residency. In addition, intestinal effector and memory CD4 T cells showed a predominant polyfunctional Th1 profile producing IFNγ, TNFα, and IL-2 at high levels with minimal but detectable levels of IL-17A. Depletion of CD4 T cells in immunized mice led to elevated bacterial burden after challenge infection highlighting a critical role for memory CD4 T cells in controlling intestinal intracellular pathogens.
Assuntos
Memória Imunológica , Mucosa Intestinal/metabolismo , Listeria monocytogenes/imunologia , Listeriose/imunologia , Células Th1/imunologia , Administração Oral , Animais , Antígenos CD/metabolismo , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/imunologia , Diferenciação Celular , Células Cultivadas , Citocinas/metabolismo , Proteínas de Choque Térmico/imunologia , Proteínas Hemolisinas/imunologia , Integrina alfa4/metabolismo , Cadeias beta de Integrinas/metabolismo , Mucosa Intestinal/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Retorno de Linfócitos/metabolismoRESUMO
HMG-box containing protein 1 (HBP1) is a member of the high mobility group (HMG) of chromosomal proteins. Since HBP1 exhibits tumor-suppressor activity in nonmyeloid tissues, we examined the effects of ectopic overexpression of HBP1 upon the growth and differentiation of myeloid cells. We prepared transient and stable transfectants of the myeloblast cell line K562, which overexpress HBP1 mRNA and protein. HBP1 transfectants displayed slower growth in cell culture and reduced colony formation in soft agar, retardation of S-phase progression, reduced expression of cyclin D1 and D3 mRNAs and increased expression of p21 mRNA. HBP1 transfectants also underwent increased apoptosis, as demonstrated by morphology and binding of Annexin V. Fas ligand mRNA levels were increased in HBP1 transfectants, suggesting involvement of the Fas/Fas ligand pathway. HBP1 overexpression enhanced differentiation of K562 cells towards erythroid and megakaryocyte lineages, as evidenced by increased hemoglobin and CD41a expression. Overexpression of HBP1 modulated mRNA levels for myeloid-specific transcription factors C/EBPalpha, c-Myb, c-Myc, and JunB, as well as lineage-specific transcription factors PU.1, GATA-1, and RUNX1. These findings suggest that in myeloid cells HBP1 may serve as a tumor suppressor and a general differentiation inducer and may synergize with chemical differentiating agents to enhance lineage-specific differentiation.
Assuntos
Diferenciação Celular , Proliferação de Células , Proteínas de Grupo de Alta Mobilidade/biossíntese , Proteínas de Grupo de Alta Mobilidade/fisiologia , Leucemia Mieloide/patologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/fisiologia , Transformação Celular Neoplásica , Perfilação da Expressão Gênica , Humanos , RNA Mensageiro/biossíntese , Fatores de Transcrição/fisiologia , Células Tumorais Cultivadas , Regulação para CimaRESUMO
We investigated whether variations in gene expression of enzymes associated with anaerobic resistance of laboratory-derived strains of Trichomonas vaginalis could be detected in a group of 28 clinical isolates with variations in metronidazole sensitivity. We compared isolates by real-time PCR because this method allows for highly sensitive quantification of mRNA and for evaluation of several genes simultaneously. We found that PFOR gene A mRNA levels were highly correlated with PFOR gene B levels, as well as the D subunit of malic enzyme and ferrodoxin. Ferrodoxin mRNA expression was also significantly correlated with that of malic enzyme and hydrogenase. However, when we evaluated relationships between these enzymes and resistance to metronidazole, we found no significant correlations between aerobic or anaerobic in vitro sensitivity to drug and mRNA levels of any of the enzymes tested. Similarly, using a Student's t-test, no significant differences in enzyme mRNA levels were observed between isolates separated by metronidazole resistance or susceptibility. The lack of correlation between gene expression and resistance or susceptibility could be the result of differences in expression at the protein level or because other biochemical pathways or genes are involved in the resistance observed in clinical settings.
Assuntos
Antiprotozoários/farmacologia , Resistência a Medicamentos/genética , Metronidazol/farmacologia , Vaginite por Trichomonas/tratamento farmacológico , Trichomonas vaginalis/genética , Animais , Células Cultivadas , Primers do DNA/química , Feminino , Expressão Gênica/genética , Genes de RNAr/genética , Humanos , Hidrogenase/genética , Reação em Cadeia da Polimerase/métodos , Piruvato Sintase/genética , RNA Mensageiro/análise , Estatística como Assunto , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/isolamento & purificaçãoRESUMO
The air quality of three different microenvironments (school, dwelling, and coffee bar) located in the city of Rome, Italy, was assessed. Indoor and outdoor concentrations of polycyclic aromatic hydrocarbons (PAHs) associated with PM2.5 particles were determined during an intensive 3-week sampling campaign conducted in March 2013. In interiors, total particulate PAHs ranged from 1.53 to 4.96 ng/m(3) while outdoor air contained from 2.75 to 3.48 ng/m(3). In addition, gaseous toxicants, i.e., NO2, NOx , SO2, O3, and BTEX (benzene, toluene, ethyl-benzene, and xylene isomers), were determined both in internal and external air. To solve the origin of indoor and outdoor PAHs, several source apportionment methods were applied. Multivariate analysis revealed that emissions from motor vehicles, biomass burning for heating purposes, and soil resuspension were the major sources of PAHs in the city. No linear correlation was established between indoor and outdoor values for PM2.5 and BTEX; the respective indoor/outdoor concentration ratios exceed unity except for PM2.5 in the no smoking home and benzene in all school floors. This suggests that important internal sources such as tobacco smoking, cleaning products, and resuspension dust contributed to indoor pollution. Using the monitoring stations of ARPA Lazio regional network as reference, the percentage within PAH group of benzo[a]pyrene, which is the WHO marker for the carcinogenic risk estimates, was ca. 50% higher in all locations investigated.
Assuntos
Poluentes Atmosféricos/análise , Poluição do Ar em Ambientes Fechados/análise , Monitoramento Ambiental/métodos , Substâncias Perigosas/análise , Material Particulado/análise , Habitação/normas , Hidrocarbonetos Policíclicos Aromáticos/análise , Cidade de Roma , Instituições Acadêmicas/normas , Compostos Orgânicos Voláteis/análise , Local de Trabalho/normasRESUMO
An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.
Assuntos
Apoptose/efeitos dos fármacos , Isquemia Encefálica/patologia , Inibidores Enzimáticos/farmacologia , Necrose , Neurônios/patologia , Fármacos Neuroprotetores/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Animais , Benzamidas/farmacologia , Isquemia Encefálica/enzimologia , Isquemia Encefálica/metabolismo , Caspase 3 , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/enzimologia , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Gerbillinae , Técnicas In Vitro , Isoquinolinas/farmacologia , Microscopia Eletrônica , Microscopia de Fluorescência , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Neurônios/metabolismo , Fenantrenos/farmacologia , Piperidinas/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Células Piramidais/efeitos dos fármacos , Células Piramidais/enzimologia , Células Piramidais/metabolismo , Células Piramidais/patologia , Ratos , Ratos WistarRESUMO
Membrane recycling in pancreatic acinar cells involves endocytic vesicle formation at the apical cell surface and rapid membrane traffic to the Golgi complex. During this process a small amount of extracellular content is taken up from the acinar lumen. In order to determine whether secretory proteins already released into the pancreatic acinar lumen are reinternalized during membrane retrieval, 3H-labeled amylase or 125I-labeled secretory proteins were reinfused through the pancreatic duct until the lumina were reached. Tissue samples from various time points were prepared for light and electron microscope autoradiography. The observations showed that [3H]amylase and, to a lesser extent, the 125I-labeled secretory proteins were internalized at the apical cell surface and rapidly (within 2-5 min) transferred to the Golgi cisternae and the condensing vacuoles; only a minor proportion of silver grains was observed over lysosomes. In addition, at later time points, mature secretion granules close to the Golgi complex became labeled. The results indicate that exocytosis in the rat exocrine pancreas does not operate at 100% efficiency; part of the exported amylase and part of the total secretion product are reinternalized concomitantly with the endocytic removal of plasma membrane and are copackaged together with newly synthesized secretory proteins.
Assuntos
Pâncreas/metabolismo , Proteínas/metabolismo , Animais , Autorradiografia , Membrana Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Endocitose , Exocitose , Complexo de Golgi/metabolismo , Masculino , Microscopia Eletrônica , Modelos Biológicos , Pâncreas/ultraestrutura , Ratos , Ratos Endogâmicos , alfa-Amilases/metabolismoRESUMO
The regulation of epidermal growth involves a number of ions, growth factors and cytokines and possibly additional but as yet unknown factors. Here we report on the potential role of the secretory N-terminal domain (sAPP) of the Alzheimer amyloid precursor protein (APP) in the regulation of keratinocyte proliferation. In human skin APP was detectable predominantly in the basal cell layer of the epidermis whereas the immunocytochemical signal in the underlying mesenchymal tissue was very low. Cultured normal human keratinocytes expressed the three APP isoforms 695, 751 and 770 with highest values for the isoforms 751 and 770. HaCaT cells, a spontaneously immortalized human keratinocyte cell line, exhibited almost identical patterns in the expression of the APP isoforms and in the release of endogenous sAPP. In HaCaT cells, recombinant sAPP (sAPPrec) was found to compete with endogenous sAPP for the same binding sites. Binding of sAPPrec was specific and occurred in microdomains of approximately 0.1 to approximately 0.3 microm in diameter. At 10 nM, sAPPrec binding induced a 2- to 4-fold increase in the rate of cell growth. sAPP concentrations in the conditioned media were found to reach 5-20 nM which is in the mitogenic range of sAPPrec. The proliferative effect of sAPP was inhibited by approximately 50% when antisense oligonucleotides directed against the APP mRNA were applied. The predominant expression of
Assuntos
Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/fisiologia , Queratinócitos/citologia , Precursor de Proteína beta-Amiloide/metabolismo , Divisão Celular , Células Cultivadas , Células Epidérmicas , Feminino , HumanosRESUMO
Acute, low-dose ultraviolet B (UVB) radiation impairs contact hypersensitivity induction in mice by a mechanism due at least in part to Langerhans cells alterations. To better define the effects of UVB on Langerhans cells, we have compared the action of this agent on the skin of intact mice and in skin explants incubated in vitro up to 24 h. Using immunofluorescence, we detected a reduction in the length of the dendrites of Langerhans cells and a significant reduction in the number of Ia-positive Langerhans cells per unit area within 2 h of UVB; these changes reversed within 24 h in vivo, but not in vitro. By electron microscopy, the number of dendritic cells per 100 basal keratinocytes increased in vivo, but decreased in vitro by 2 h after UVB, a discordance that was significant. On the contrary, the number of dendrite profiles per dendritic cell body decreased significantly 2 h after UVB, both in vivo and in vitro. Many epidermal dendritic cells, 2 h after UVB in vivo, were deficient in cytoplasmic organelles, whereas the few cells that remained after UVB in vitro retained their Birbeck granules, and displayed many, dilated cytoplasmic vesicles. We interpret these data to mean that low doses of UVB radiation destroy the functional and morphologic integrity of epidermal Langerhans cells, and that these cells are rapidly replaced by precursor cells that mature in situ into normal-appearing Langerhans cells.
Assuntos
Células Epidérmicas , Células de Langerhans/citologia , Células de Langerhans/efeitos da radiação , Monócitos/citologia , Raios Ultravioleta , Animais , Contagem de Células/efeitos da radiação , Movimento Celular , Técnicas In Vitro , Células de Langerhans/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Pele/citologiaRESUMO
In this study, we have investigated by light and electron microscopy the presence, distribution, and inner structure of CD36(OKM5)+ dendritic cells (DC) in the lamina propria and epithelium of the oral mucosa of HIV- and HIV+ subjects; in the latter, both clinically healthy areas and areas of hairy leukoplakia (HL) were studied. Perivascular CD36+ DC were present in the lamina propria of all the specimens studied. They were also found in small numbers in the epithelium of clinically healthy mucosa of HIV- and HIV+ subjects, but were practically absent from the epithelium of HL. CD36+ DC seemed to be regularly HLA-DR+ in HIV-subjects; this positivity was recognized only in some cells in the clinically healthy mucosa of HIV+ subjects, and practically never in HL. Because the only perivascular cells observed in the clinically healthy areas of HIV+ subjects were CD36+, we investigated the ultrastructure of perivascular DC in these same areas. These cells were characterized by the presence of a prominent Golgi apparatus, many lysosomes, and focal adhesions to the extracellular matrix. It may be concluded that 1) CD36+ DC are physiologic components of the oral mucosa, 2) they share some ultrastructural features with macrophages, 3) no differences in numbers were found between HIV+ and HIV- subjects, and 4) these cells are affected in their expression of HLA-DR antigens during HIV infection, particularly in areas of HL. This may be a hint that the antigen-presenting function of these cells in the oral mucosa is negatively affected during HIV infection.
Assuntos
Anticorpos Monoclonais/análise , Antígenos CD/imunologia , Células Dendríticas/imunologia , Soropositividade para HIV/imunologia , Mucosa Bucal/citologia , Adulto , Antígenos CD36 , Feminino , Corantes Fluorescentes , Antígenos HLA-DR/análise , Humanos , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Masculino , Microscopia Eletrônica , Pessoa de Meia-IdadeRESUMO
Cyclosporine (CsA), administered to rats at daily doses of 10 and 50 mg/kg body weight, for 21 days, influenced negatively the structures involved in the synthesis, storage and secretion of digestive enzymes in pancreatic acinar cells. A dose-related, significant reduction in basophilic cell regions, secretion granule content, and overall size of acinar cells was appreciable by light microscopy and morphometry. By electron microscopy, the acinar cells of the rats given 10 mg/kg/day CsA were similar to the controls, whereas with the higher dose most cells showed reduction in the size of nucleoli, increase in the number of lysosomes, and evidence of autophagy. In only a few cells was autophagy particularly severe and involved almost the entire cytoplasm. Nine weeks after withdrawal from CsA treatment, the structural recovery of acinal cells was complete, and features indicating enhanced protein synthesis and mitochondrial multiplication were observed by electron microscopy. In conclusion, prolonged administration of CsA to rats induces changes in the acinar cells indicating a depression of their activity, without substantial impairment in the viability of the most of them, even at high doses. This accounts for complete restoration of the acinar tissue upon withdrawal.
Assuntos
Ciclosporinas/efeitos adversos , Pâncreas/efeitos dos fármacos , Animais , Ciclosporinas/administração & dosagem , Relação Dose-Resposta a Droga , Masculino , Pâncreas/patologia , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
In the case of the malaria CS protein we have shown that there is at least one T cell determinant which is able to bind to and be recognized by most human MHC class II molecules, while for the 190L polypeptide, derived from a conserved region of the p190 merozoite surface protein, we have identified several epitopes recognized by T cell clones in association with different HLA-class II isotypes and alleles. In addition, binding analysis of these epitopes indicated that most of the peptides are able to bind to multiple allelic forms of class II molecules. Although there are important obstacles to malaria vaccine development we believe that, in the light of these results, unresponsiveness in humans, caused by MHC restriction, might not be a major constraint in development of a subunit vaccine.
Assuntos
Antígenos de Protozoários/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Malária/imunologia , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Antígenos H-2/imunologia , Antígenos HLA-D/imunologia , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas de Protozoários/imunologia , Vacinas Protozoárias/imunologiaRESUMO
BACKGROUND: Helicobacter pylori gastric infection has been associated with various digestive and extra-digestive diseases. The systemic influence of gastric H. pylori infection seems to be mediated by the release of various cytokines. In liver disease, bacterial infections have been associated with the impairment of liver metabolic function. AIMS: To evaluate the influence of H. pylori infection on liver function as assessed by means of the monoethylglycinexylidide test, which depends upon liver blood flow and cytochrome P-450 activity, and the 13C-galactose breath test, which depends on cytosolic enzymatic activity and is correlated with hepatic functional mass. Moreover, to evaluate whether H. pylori-associated modifications of liver function may be related to tumour necrosis factor-alpha serum levels. PATIENTS AND METHODS: Thirty-five patients with liver cirrhosis of various aetiologies, who underwent monoethylglycinexylidide and 13C-galactose breath tests, were retrospectively evaluated for H. pylori infection by means of anti-H. pylori immunoglobulin G. The main clinical, biochemical and functional characteristics of the patients as well as their tumour necrosis factor-alpha serum levels were then analysed on the basis of the presence of H. pylori infection. RESULTS: Twenty-one patients tested positive for H. pylori infection (60%), and 11 tested negative (31.4%). No clinical or biochemical differences were observed between H. pylori-infected and non-infected patients. H. pylori infection showed no difference in distribution according to Child-Pugh classes (A, 55%; B and C, 67%). The monoethylglycinexylidide test results were significantly lower at each sampling time in H. pylori-positive patients compared to H. pylori-negative patients (MEGX15, P=0.027; MEGX30, P=0.014; MEGX60, P=0.028), while 13C-galactose breath test showed no significant differences considering both cumulative percentage dose and percentage dose/h. The median tumour necrosis factor-alpha serum levels were no different between H. pylori-positive (16.1 pg/mL, 95% confidence interval, 8.7-28.7) and H. pylori-negative (12.3 pg/mL, 95% confidence interval, 8.7-23.4) patients. CONCLUSIONS: In cirrhotic patients, H. pylori infection seems to selectively affect cytochrome P-450 liver activity, while hepatic functional mass does not seem to be impaired. Tumour necrosis factor-alpha does not seem to be the mediator of this impairment. Further studies are needed to evaluate the impact of H. pylori eradication on parameters of liver function.