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1.
Pharm Res ; 33(7): 1736-51, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27037576

RESUMEN

PURPOSE: To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates. METHODS: IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines. RESULTS: IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs). CONCLUSIONS: Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.


Asunto(s)
Formación de Anticuerpos/inmunología , Inmunidad Innata/inmunología , Inmunoglobulinas Intravenosas/inmunología , Línea Celular , Citocinas/inmunología , Humanos , Leucocitos Mononucleares/inmunología , Monocitos/inmunología , Tamaño de la Partícula , Receptores de IgG/inmunología , Receptores Toll-Like/inmunología
2.
Am J Transplant ; 6(4): 724-35, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16539629

RESUMEN

Immunity to collagen V [col(V)] contributes to lung 'rejection.' We hypothesized that ischemia reperfusion injury (IRI) associated with lung transplantation unmasks antigenic col(V) such that fresh and well-healed lung grafts have differential susceptibility to anti-col(V)-mediated injury; and expression of the autoimmune cytokines, IL-17 and IL-23, are associated with this process. Adoptive transfer of col(V)-reactive lymphocytes to WKY rats induced grade 2 rejection in fresh isografts, but induced worse pathology (grade 3) when transferred to isograft recipients 30 days post-transplantation. Immunhistochemistry detected col(V) in fresh and well-healed isografts but not native lungs. Hen egg lysozyme-reactive lymphocytes (HEL, control) did not induce lung disease in any group. Col(V), but not HEL, immunization induced transcripts for IL-17 and IL-23 (p19) in the cells utilized for adoptive transfer. Transcripts for IL-17 were upregulated in fresh, but not well-healed isografts after transfer of col(V)-reactive cells. These data show that IRI predisposes to anti-col(V)-mediated pathology; col(V)-reactive lymphocytes express IL-17 and IL-23; and anti-col(V)-mediated lung disease is associated with local expression of IL-17. Finally, because of similar histologic patterns, the pathology of clinical rejection may reflect the activity of autoimmunity to col(V) and/or alloimmunity.


Asunto(s)
Colágeno Tipo V/inmunología , Rechazo de Injerto/patología , Interleucina-17/genética , Interleucinas/genética , Pulmón/patología , Linfocitos/inmunología , Daño por Reperfusión/inmunología , Animales , Autoinmunidad/genética , Autoinmunidad/inmunología , Rechazo de Injerto/inmunología , Interleucina-23 , Subunidad p19 de la Interleucina-23 , Pulmón/inmunología , Trasplante de Pulmón/inmunología , Ratas , Ratas Endogámicas , Transcripción Genética , Regulación hacia Arriba
3.
J Biol Regul Homeost Agents ; 19(3-4): 93-104, 2005.
Artículo en Inglés | MEDLINE | ID: mdl-16602623

RESUMEN

Current evidence suggests that MHC class II-restricted CD4+ T-cells play a crucial role in orchestrating host immune responses against cancer as well as autoimmune and infectious diseases. Antigens must be processed within endosomal and lysosomal compartments of antigen presenting cells (APC) before binding to MHC class II molecules for display to T-cells. Only a limited number of processed peptides termed immunodominant are selected for display by MHC class II molecules and prove capable of inducing strong T-cell responses. Thus processing reactions within APC are of central importance for the development of effective vaccines as they modulate the number of peptide: class II complexes by enhancing or disrupting epitope formation and display. Studies suggest that there are substantial gaps in our knowledge of how antigen processing and presentation by APC regulates epitope selection and immunodominance in disease situations. Here we describe new insights in antigen processing and epitope selection with relevance to immunotherapeutic strategies for cancer, autoimmunity and infectious diseases.


Asunto(s)
Presentación de Antígeno , Inmunoterapia/métodos , Animales , Células Presentadoras de Antígenos/inmunología , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/terapia , Linfocitos T CD4-Positivos/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Epítopos Inmunodominantes , Infecciones/inmunología , Infecciones/terapia , Neoplasias/inmunología , Neoplasias/terapia , Inmunología del Trasplante
4.
J Immunol ; 167(2): 632-5, 2001 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-11441064

RESUMEN

HLA-DM catalyzes the exchange and selection of ligands for MHC class II molecules within mature endosomal/lysosomal compartments. Here, evidence is provided that DM edits peptides in early endosomes, thus influencing presentation via recycling class II molecules. Maximal class II-restricted presentation of an albumin-derived peptide, dependent on endocytosis and recycling class II molecules, was observed in cells lacking HLA-DM. DM editing of this epitope was observed in early endocytic compartments as shown using inhibitors of early to late endosomal transport. Editing was tempered by coexpression of HLA-DO, suggesting that DM:DO ratio may be important in guiding epitope editing in early endosomal compartments. Thus, HLA-DM appears to interact with, and edit epitopes displayed by, recycling class II molecules.


Asunto(s)
Antígenos HLA-D/fisiología , Antígenos de Histocompatibilidad Clase II/metabolismo , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Edición de ARN/inmunología , Presentación de Antígeno/genética , Línea Celular , Endosomas/inmunología , Endosomas/metabolismo , Antígenos HLA-D/genética , Antígenos HLA-D/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Sustancias Macromoleculares , Fragmentos de Péptidos/genética , Transporte de Proteínas/genética , Transporte de Proteínas/inmunología , Transfección
5.
J Cell Biochem ; 82(1): 11-21, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11400159

RESUMEN

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non-osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20-17. However, human bone marrow stromal cells cultured with 1 microM dexamethasone for four days, or further stimulated to become osteoblast-like cells with 50 microg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 microM dexamethasone was reduced if the cells were not given 50 microg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells.


Asunto(s)
Adenoviridae/genética , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteínas Morfogenéticas Óseas/biosíntesis , Proteínas Morfogenéticas Óseas/genética , Factor de Crecimiento Transformador beta , Adenoviridae/patogenicidad , Animales , Ácido Ascórbico/farmacología , Proteína Morfogenética Ósea 2 , Diferenciación Celular/efectos de los fármacos , Transformación Celular Viral , Dexametasona/farmacología , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Ratones , Proteínas Recombinantes , Recombinación Genética/genética , Células del Estroma/citología , Células del Estroma/metabolismo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismo
6.
Arch Microbiol ; 175(3): 208-19, 2001 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-11357513

RESUMEN

We isolated an obligately anaerobic halophilic bacterium from the Dead Sea that grew by respiration of selenate. The isolate, designated strain DSSe-1, was a gram-negative, non-motile rod. It oxidized glycerol or glucose to acetate + CO2 with concomitant reduction of selenate to selenite plus elemental selenium. Other electron acceptors that supported anaerobic growth on glycerol were nitrate and trimethylamine-N-oxide; nitrite, arsenate, fumarate, dimethylsulfoxide, thiosulfate, elemental sulfur, sulfite or sulfate could not serve as electron acceptors. Growth on glycerol in the presence of nitrate occurred over a salinity range from 100 to 240 g/l, with an optimum at 210 g/l. Analysis of the 16S rRNA gene sequence suggests that strain DSSe-1 belongs to the order Halanaerobiales, an order of halophilic anaerobes with a fermentative or homoacetogenic metabolism, in which anaerobic respiratory metabolism has never been documented. The highest 16S rRNA sequence similarity (90%) was found with Acetohalobium arabaticum (X89077). On the basis of physiological properties as well as the relatively low homology of 16S rRNA from strain DSSe-1 with known genera, classification in a new genus within the order Halanaerobiales, family Halobacteroidaceae is warranted. We propose the name Selenihalanaerobacter shriftii. Type strain is strain DSSe-1 (ATCC accession number BAA-73).


Asunto(s)
Bacterias Anaerobias/metabolismo , Sedimentos Geológicos , Compuestos de Selenio/metabolismo , Filogenia , Ácido Selénico
7.
J Immunol ; 166(7): 4543-51, 2001 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-11254711

RESUMEN

Peptides bind cell surface MHC class II proteins to yield complexes capable of activating CD4(+) T cells. By contrast, protein Ags require internalization and processing by APC before functional presentation. Here, T cell recognition of a short peptide in the context of class II proteins occurred only after delivery of this ligand to mature endosomal/lysosomal compartments within APC. Functional and biochemical studies revealed that a central cysteine within the peptide was cysteinylated, perturbing T cell recognition of this epitope. Internalization and processing of the modified epitope by APC, was required to restore T cell recognition. Peptide cysteinylation and reduction could occur rapidly and reversibly before MHC binding. Cysteinylation did not disrupt peptide binding to class II molecules, rather the modified peptide displayed an enhanced affinity for MHC at neutral pH. However, once the peptide was bound to class II proteins, oxidation or reduction of cysteine residues was severely limited. Cysteinylation has been shown to radically influence T cell responses to MHC class I ligands. The ability of professional APC to reductively cleave this peptide modification presumably evolved to circumvent a similar problem in MHC class II ligand recognition.


Asunto(s)
Células Presentadoras de Antígenos/metabolismo , Cisteína/metabolismo , Endocitosis/inmunología , Antígenos HLA-D/metabolismo , Fragmentos de Péptidos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Alanina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/inmunología , Animales , Presentación de Antígeno/inmunología , Células Presentadoras de Antígenos/inmunología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Línea Celular , Técnicas de Cocultivo , Cisteína/química , Cistina/química , Cistina/metabolismo , Humanos , Hibridomas , Concentración de Iones de Hidrógeno , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Cadenas kappa de Inmunoglobulina/metabolismo , Ligandos , Ratones , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/inmunología , Unión Proteica/inmunología
8.
J Cell Biochem ; 80(4): 532-7, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11169737

RESUMEN

Differentiation of osteoprogenitor cells into osteoblasts is a pivotal step during the normal development and repair of bone. Upregulation of endogenous cellular alkaline phosphatase activity (AP) is a commonly used intracellular marker for the assessment of osteoprogenitor cell differentiation into the osteoblastic phenotype. Current methods for assaying AP involve colorimetric detection of the enzyme's activity using the synthetic substrate p-nitrophenol phosphate. In this paper, we explored an alternative method of detecting AP using the chemiluminescent substrate disodium 3-(4-methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.1(3,7)]decan]-4-yl) phenyl phosphate (CSPD) for enhanced AP sensitivity and a more simplified assay. Using calf intestinal alkaline phosphatase as a standardizing enzyme, we determined that the chemiluminescent detection system was four orders of magnitude more sensitive than the standard colorimetric method of detection. Moreover, the chemiluminescent assay was faster and markedly simpler to perform. To maximize the utility of this assay system, two osteoprogenitor cell lines were compared for their ability to generate alkaline phosphatases in vitro when exposed to recombinant human bone morphogenetic protein (rhBMP-2). The W20-17 cell line was substantially more sensitive to rhBMP-2 than the C3H10T1/2 cell line, where each cell line produced detectable increases in AP after exposure to rhBMP-2 levels of 5 and 25 ng/ml, respectively. The experimental design for AP responsiveness to rhBMP-2 was further optimized for chemiluminescent detection with the W20-17 cell line by comparing the effects of reporter cell seeding density and the day of assay. In summary, the data presented in this paper demonstrate a faster, simpler, and more sensitive chemiluminescent method to monitor changes in AP levels during osteodifferentiation.


Asunto(s)
Adamantano/análogos & derivados , Fosfatasa Alcalina/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Huesos/citología , Mediciones Luminiscentes , Osteoblastos/enzimología , Espectrofotometría/métodos , Factor de Crecimiento Transformador beta , Adamantano/farmacología , Animales , Proteína Morfogenética Ósea 2 , Bovinos , Diferenciación Celular , Línea Celular , Colorimetría , Relación Dosis-Respuesta a Droga , Humanos , Intestinos/enzimología , Ratones , Fenotipo , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Regulación hacia Arriba
11.
Curr Protoc Immunol ; Chapter 18: Unit 18.7, 2001 May.
Artículo en Inglés | MEDLINE | ID: mdl-18432749

RESUMEN

Cell surface receptors, such as transferrin receptors and MHC molecules, are internalized into the endocytic pathway and recycled to the plasma membrane. Previous assays used to measure endocytosis and recycling were cumbersome and often required radioactive reagents. This unit describes protocols that employ the combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules allowing for rapid and safe quantitation of cell surface protein expression, endocytosis, and recycling.


Asunto(s)
Biotinilación/métodos , Extractos Celulares/análisis , Membrana Celular/metabolismo , Endocitosis , Proteínas de la Membrana/análisis , Biotina/química , Biotina/metabolismo , Extractos Celulares/química , Membrana Celular/química , Clatrina/metabolismo , Endosomas/química , Endosomas/metabolismo , Complejo Mayor de Histocompatibilidad , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptores de Transferrina/metabolismo , Transferrina/metabolismo
12.
J Exp Med ; 191(9): 1513-24, 2000 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-10790426

RESUMEN

Biochemical and functional studies have demonstrated major histocompatibility complex (MHC) class II-restricted presentation of select epitopes derived from cytoplasmic antigens, with few insights into the processing reactions necessary for this alternate pathway. Efficient presentation of an immunodominant epitope derived from glutamate decarboxylase (GAD) was observed regardless of whether this antigen was delivered exogenously or via a cytoplasmic route into human histocompatibility leukocyte antigen class II-DR4(+) antigen-presenting cells. Presentation of exogenous as well as cytoplasmic GAD required the intersection of GAD peptides and newly synthesized class II proteins. By contrast, proteolytic processing of this antigen was highly dependent upon the route of antigen delivery. Exogenous GAD followed the classical pathway for antigen processing, with an absolute requirement for endosomal/lysosomal acidification as well as cysteine and aspartyl proteases resident within these organelles. Presentation of endogenous GAD was dependent upon the action of cytoplasmic proteases, including the proteasome and calpain. Thus, translocation of processed antigen from the cytoplasm into membrane organelles is necessary for class II-restricted presentation via this alternate pathway. Further trimming of these peptides after translocation was mediated by acidic proteases within endosomes/lysosomes, possibly after or before class II antigen binding. These studies suggest that processing of exogenous and cytoplasmic proteins occurs through divergent but overlapping pathways. Furthermore, two cytoplasmic proteases, the proteasome and calpain, appear to play important roles in MHC class II-restricted antigen presentation.


Asunto(s)
Presentación de Antígeno , Autoantígenos/inmunología , Citoplasma/metabolismo , Glutamato Descarboxilasa/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Procesamiento Proteico-Postraduccional , Ácido Aspártico Endopeptidasas/metabolismo , Autoantígenos/metabolismo , Transporte Biológico , Polaridad Celular , Cisteína Endopeptidasas/metabolismo , Endocitosis , Endosomas/metabolismo , Glutamato Descarboxilasa/metabolismo , Antígeno HLA-DR4/inmunología , Epítopos Inmunodominantes/inmunología , Lisosomas/metabolismo , Fragmentos de Péptidos/inmunología , Vesículas Sinápticas/inmunología
14.
Traffic ; 1(7): 561-9, 2000 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-11208144

RESUMEN

Exogenous antigenic peptides captured and presented in the context of major histocompatibility (MHC) class II molecules on APC, have been employed as potent vaccine reagents capable of activating cellular immune responses. Binding and presentation of select peptide via surface class II molecules has been reported. Here, a role for endocytosis and early endosomes in the presentation of exogenous peptides via MHC class II molecules is described. T cell recognition of a 14 amino acid human serum albumin-derived peptide in the context of HLA-DR4 was observed only with metabolically active APC. The delayed kinetics and temperature dependence of functional peptide presentation via APC, were consistent with a requirement for peptide internalization to early endosomal compartments prior to T cell recognition. Ablating endocytosis by exposing cells to inhibitors of ATP production completely blocked the display of functional peptide:class II complexes on the surface of the APC. Presentation of the peptide was also found to be sensitive to primaquine, a drug that perturbs the recycling of transport vesicles containing endocytic receptors and mature class II complexes. Functional presentation of the endocytosed peptide was dependent upon these mature class II complexes, as inhibitor studies ruled out a requirement for newly synthesized class II molecules. N-terminal processing of the endocytosed peptide was observed upon trafficking through endosomal compartments and linked to the formation of functional peptide:class II complexes. These findings establish a novel mechanism for regulating class II-restricted peptide presentation via the endocytic pathway.


Asunto(s)
Presentación de Antígeno/fisiología , Antígenos Virales/metabolismo , Endocitosis/fisiología , Antígenos HLA-DR/inmunología , Antígeno HLA-DR4/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Macrólidos , Fragmentos de Péptidos/metabolismo , Albúmina Sérica/metabolismo , Antibacterianos/farmacología , Antígenos Virales/inmunología , Biotinilación , Brefeldino A/farmacología , Línea Celular , Cloroquina/farmacología , Desoxiglucosa/farmacología , Endocitosis/efectos de los fármacos , Genes MHC Clase II , Antígenos HLA-DR/genética , Antígenos HLA-DR/metabolismo , Antígeno HLA-DR4/genética , Antígeno HLA-DR4/metabolismo , Cadenas HLA-DRB1 , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Humanos , Sustancias Macromoleculares , Fragmentos de Péptidos/inmunología , Transporte de Proteínas/efectos de los fármacos , Albúmina Sérica/inmunología , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patología , Azida Sódica/farmacología , Transfección
15.
J Immunol ; 163(12): 6413-23, 1999 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-10586031

RESUMEN

Following antigenic challenge, MHC-restricted T cell responses are directed against a few dominant antigenic epitopes. Here, evidence is provided demonstrating the importance of APC in modulating the hierarchy of MHC class II-restricted T cell responses. Biochemical analysis of class II:peptide complexes in B cells revealed the presentation of a hierarchy of peptides derived from the Ig self Ag. Functional studies of kappa peptide:class II complexes from these cells indicated that nearly 20-fold more of an immunodominant epitope derived from kappa L chains was bound to class II DR4 compared with a subdominant epitope from this same Ag. In vivo, T cell responses were preferentially directed against the dominant kappa epitope as shown using Ig-primed DR4 transgenic mice. The bias in kappa epitope presentation was not linked to differences in class II:kappa peptide-binding affinity or epitope editing by HLA-DM. Rather, changes in native Ag structure were found to disrupt presentation of the immunodominant but not the subdominant kappa epitope; Ag refolding restored kappa epitope presentation. Thus, Ag tertiary conformation along with processing reactions within APC contribute to the selective presentation of a hierarchy of epitopes by MHC class II molecules.


Asunto(s)
Células Presentadoras de Antígenos/inmunología , Epítopos de Linfocito T/metabolismo , Epítopos Inmunodominantes/metabolismo , Secuencia de Aminoácidos , Animales , Presentación de Antígeno , Células Presentadoras de Antígenos/metabolismo , Línea Celular , Epítopos de Linfocito T/inmunología , Antígenos HLA/química , Antígenos HLA/inmunología , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase II/química , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Inmunización Pasiva , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/inmunología , Cadenas kappa de Inmunoglobulina/inmunología , Cadenas kappa de Inmunoglobulina/metabolismo , Inmunoglobulinas/inmunología , Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Péptidos/inmunología , Péptidos/metabolismo , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Linfocitos T/inmunología , Linfocitos T/metabolismo
16.
Appl Environ Microbiol ; 65(10): 4385-92, 1999 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-10508064

RESUMEN

Washed-cell suspensions of Sulfurospirillum barnesii reduced selenate [Se(VI)] when cells were cultured with nitrate, thiosulfate, arsenate, or fumarate as the electron acceptor. When the concentration of the electron donor was limiting, Se(VI) reduction in whole cells was approximately fourfold greater in Se(VI)-grown cells than was observed in nitrate-grown cells; correspondingly, nitrate reduction was approximately 11-fold higher in nitrate-grown cells than in Se(VI)-grown cells. However, a simultaneous reduction of nitrate and Se(VI) was observed in both cases. At nonlimiting electron donor concentrations, nitrate-grown cells suspended with equimolar nitrate and selenate achieved a complete reductive removal of nitrogen and selenium oxyanions, with the bulk of nitrate reduction preceding that of selenate reduction. Chloramphenicol did not inhibit these reductions. The Se(VI)-respiring haloalkaliphile Bacillus arsenicoselenatis gave similar results, but its Se(VI) reductase was not constitutive in nitrate-grown cells. No reduction of Se(VI) was noted for Bacillus selenitireducens, which respires selenite. The results of kinetic experiments with cell membrane preparations of S. barnesii suggest the presence of constitutive selenate and nitrate reduction, as well as an inducible, high-affinity nitrate reductase in nitrate-grown cells which also has a low affinity for selenate. The simultaneous reduction of micromolar Se(VI) in the presence of millimolar nitrate indicates that these organisms may have a functional use in bioremediating nitrate-rich, seleniferous agricultural wastewaters. Results with (75)Se-selenate tracer show that these organisms can lower ambient Se(VI) concentrations to levels in compliance with new regulations proposed for release of selenium oxyanions into the environment.


Asunto(s)
Bacillus/metabolismo , Nitratos/metabolismo , Proteobacteria/metabolismo , Compuestos de Selenio/metabolismo , Selenio/metabolismo , Cinética , Ácido Selénico , Suspensiones
17.
Int J Syst Bacteriol ; 49 Pt 3: 1177-80, 1999 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-10425777

RESUMEN

Two strains of dissimilatory arsenate-reducing vibrio-shaped bacteria are assigned to the genus Sulfurospirillum. These two new species, Sulfurospirillum barnesii strain SES-3T and Sulfurospirillum arsenophilum strain MIT-13T, in addition to Sulfurospirillum sp. SM-5, two strains of Sulfurospirillum deleyianum, and Sulfurospirillum arcachonense, form a distinct clade within the epsilon subclass of the Proteobacteria based on 16S rRNA analysis.


Asunto(s)
Arseniatos/metabolismo , Bacterias Gramnegativas/clasificación , Compuestos de Selenio/metabolismo , Técnicas de Tipificación Bacteriana , ADN Ribosómico/química , ADN Ribosómico/genética , Genes de ARNr , Bacterias Gramnegativas/citología , Bacterias Gramnegativas/genética , Bacterias Gramnegativas/fisiología , Datos de Secuencia Molecular , Oxidación-Reducción , Filogenia , ARN Ribosómico 16S/genética , Ácido Selénico , Análisis de Secuencia de ADN
18.
J Leukoc Biol ; 64(1): 85-91, 1998 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-9665280

RESUMEN

The mannose receptor, present on the plasma membrane of macrophages, promotes the internalization of glycoproteins and glycoconjugates via both endocytic and phagocytic pathways. The expression of this receptor is tightly modulated during monocyte/Mphi differentiation and cellular activation. We isolated clonal populations from murine J774 macrophage tumor cells, which differ in their surface expression of functional mannose receptors. To examine the potential mechanisms regulating receptor function in these cell lines, the interaction of receptor with ligand as well as receptor synthesis and degradation was analyzed. J774 clones with both high and low levels of mannose receptor activity were found to synthesize significant amounts of receptor protein, suggesting that the protein may be regulated at the level of synthesis and degradation. In J774 clones expressing very low receptor activity and protein, the half-life of mannose receptor molecules was substantially decreased. The evolution of multiple mechanisms modulating mannose receptor function may be critical in fine-tuning the role of this receptor in antigen processing and in scavenger and host defense functions.


Asunto(s)
Lectinas Tipo C , Macrófagos/ultraestructura , Lectinas de Unión a Manosa , Receptores de Superficie Celular/biosíntesis , Animales , Células Cultivadas , Células Clonales , Cinética , Macrófagos/metabolismo , Receptor de Manosa , Ratones , Fenotipo , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/fisiología
19.
J Immunol Methods ; 212(1): 9-18, 1998 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-9671148

RESUMEN

Cell surface receptors and antigens, such as TfR and MHC molecules, are endocytosed and subsequently redisplayed on the plasma membrane. The internalization and recycling of MHC molecules is thought to play an important role in antigen presentation, but studying this process has been hindered due to the lack of a rapid and easily quantitated assay. The combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules of interest, allows for the quantitation of their cell surface expression, endocytosis and recycling. The endocytosis of TfR and MHC II molecules was readily quantitated in B cell lines using this procedure with results nearly identical to previously published data using more laborious radioactive methods. Evidence for the recycling of class II antigens and TfR back to the plasma membrane was obtained by monitoring the exit of these molecules from endosomes. Exposing cells to hypertonic media blocks clathrin-dependent endocytosis and was found to inhibit the internalization of MHC class II proteins on B cells. This flexible assay to capture and quantitate the cell surface expression and endocytosis of MHC molecules and other surface antigens offers a sensitive and non-radioactive alternative to study the intracellular trafficking of diverse membrane proteins.


Asunto(s)
Endocitosis , Ensayo de Inmunoadsorción Enzimática/métodos , Antígenos de Histocompatibilidad Clase II/metabolismo , Receptores de Superficie Celular/metabolismo , Presentación de Antígeno , Biotinilación , Endosomas/metabolismo , Complejo Mayor de Histocompatibilidad
20.
Hum Immunol ; 59(2): 77-86, 1998 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-9536430

RESUMEN

Peptide binding to HLA-DR molecules in intracellular compartments is facilitated by HLA-DM molecules, present in most types of antigen-presenting cells. Allorecognition of DR specificities represents a form of T cell recognition of the MHC-peptide complex which in some cases is influenced by peptide binding. DRA and DRB*0401 (Dw4) genes were introduced into different cell types including DM-negative and DM-restored mutant cells to analyze recognition of DR4 subtypes by alloreactive T cell clones and Dw4-specific monoclonal antibodies. Distinct patterns of T cell recognition were identified: (i) deficient response to Dw4 molecules in the absence of DM expression in which T cell responses were restored by transfecting DM into the Dw4-expressing cells; and (ii) equivalent recognition of Dw4 on DM- and DM+ cells. Using several mAb to Dw4 molecules, a similar distinction was observed: a shared epitope on Dw4 and Dw14 molecules was partially DM-independent while a Dw4-specific epitope was DM-dependent and cell type-specific. Thus, a subset of both T cell and mAb allodeterminants are influenced by a DM-dependent interaction of MHC molecules with peptides, while the formation of DM-independent allodeterminants may represent direct MHC epitope recognition by the T cell receptor or an alternative peptide loading mechanism distinct from the HLA-DM pathways.


Asunto(s)
Epítopos de Linfocito B/inmunología , Epítopos de Linfocito T/inmunología , Antígenos HLA-D/inmunología , Antígeno HLA-DR4/inmunología , Antígenos de Histocompatibilidad Clase II , Anticuerpos Monoclonales/inmunología , Línea Celular Transformada , Humanos , Péptidos/inmunología , Linfocitos T/inmunología
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