RESUMEN

Much is known about the structure function relationships of a large number of bacterial protein toxins, the nature of their cell surface receptors, and their enzymatic activities which lead to the inactivation of their respective cytosolic targets. Despite this wealth of knowledge a detailed understanding of the mechanisms which underlie translocation of the catalytic domain across the eukaryotic cell membrane to the cytosol, the penultimate event in the intoxication process, have been slow in developing. In the case of diphtheria toxin, two prominent hypotheses have been advanced to explain how the catalytic domain is translocated from the lumen of endocytic vesicles to the target cell cytosol. We discuss each of these hypotheses and provide an overview of recent observations that tend to favor a mechanism employing a Cytosolic Translocation Factor complex in the entry process. This facilitated mechanism of translocation appears to rely upon protein-protein interactions between conserved domains within the transmembrane domain of diphtheria toxin with host cell factors to effect delivery of the enzymatic moiety. We have recently identified a 10 amino acid motif in the transmembrane domain of diphtheria toxin that is conserved in anthrax Lethal and Edema Factors, as well as in botulinum neurotoxins A, C and D. Stable eukaryotic cell transfectants that express a peptide containing this motif become resistant to the toxin, and sensitivity is completely restored by co-expression of siRNA which inhibits peptide expression. Data obtained from use of the protein fusion toxin DAB(389)IL-2 in cytotoxicity assays using susceptible Hut 102/6TG and resistant transfectant Hut102/6TG-T1 cells, as well as pull down assays have led to the formulation of a working model of facilitated delivery of the diphtheria toxin catalytic domain to the cytosol of target cells which is discussed in detail.

Asunto(s)

Toxinas Bacterianas/metabolismo , Neurotoxinas/metabolismo , Animales , Toxinas Bacterianas/química , Humanos , Neurotoxinas/química , Transporte de Proteínas , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo

RESUMEN

The expression of neural regulatory molecules by immune cells that infiltrate the nervous system upon injury may be a mechanism for cross-regulation between the nervous system and the immune system. Several lines of evidence implicate nerve growth factor (NGF) signaling through its receptors (TrkA and p75(NGFR)) as a potential source of communication between the two systems. We observed changes in NGF mRNA expression and protein secretion by T lymphocytes polarized toward the Th2 phenotype. The presence of NGF did not affect T cell proliferation or cytokine production in vitro. Mice treated with NGF by i. p. injection following induction of experimental autoimmune encephalomyelitis, an inflammatory, demyelinating disease of the central nervous system, showed a delayed onset of disease and lower clinical scores during the course of disease. These data suggest a role for NGF signaling in the regulation of the immune response, possibly by enhancing sympathetic innervation of lymphoid tissues.

Asunto(s)

Encefalomielitis Autoinmune Experimental/etiología , Factor de Crecimiento Nervioso/fisiología , Animales , Citocinas/biosíntesis , Encefalomielitis Autoinmune Experimental/inmunología , Interleucina-4/farmacología , Activación de Linfocitos , Linfocitos/metabolismo , Ratones , Ratones Transgénicos , Factor de Crecimiento Nervioso/genética , ARN Mensajero/análisis

RESUMEN

Given the critical role of cytokines in the regulation of an inflammatory response, we investigated whether certain cytokines are expressed in the brains of normal mice during maturation that could contribute to the immune-privileged nature of the CNS or potentially influence an immune-mediated illness such as experimental allergic encephalomyelitis. The gene expression of IFN gamma (Th1 cytokine) and IL-4 (Th2 cytokine) was analyzed in the brain of several strains of mice. IFN gamma was not detectable. However, IL-4 was present in the brains of neonatal mice, but not adult mice. Resident CNS cells are believed to be the source of the IL-4, because mice deficient in T cells (SCID and RAG2-/-) expressed the IL-4 gene in the CNS. Further analysis indicated that the gene expression of the Th2 cytokine transcription factor, GATA-3, correlated with IL-4 and IL-10 expression in the brain. Since GATA-3-deficient mice have an abnormal CNS, brain-derived Th2 cytokines may play an important role in CNS development, as well as potentially contribute to the immune-privileged nature of the brain.

Asunto(s)

Química Encefálica/genética , Química Encefálica/inmunología , Regulación del Desarrollo de la Expresión Génica/inmunología , Interleucina-4/genética , Factores de Edad , Animales , Animales Recién Nacidos , Encéfalo/crecimiento & desarrollo , Encéfalo/inmunología , Cartilla de ADN , Proteínas de Unión al ADN/genética , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Factor de Transcripción GATA3 , Interferón gamma/genética , Interleucina-10/genética , Ratones , Ratones Endogámicos , Linfocitos T/inmunología , Transactivadores/genética

RESUMEN

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated, autoimmune disorder characterized by central nervous system (CNS) inflammation and demyelination, features reminiscent of the human disease, multiple sclerosis (MS). In addition to the signal the encephalitogenic T cell receives through the T cell receptor (TCR), a second signal, termed costimulation, is required for complete T cell activation. The B7 family of cell surface molecules expressed on antigen presenting cells (APC) is capable of providing this second signal to T cells via two receptors, CD28 and CTLA-4. Our studies have shown that costimulation provided by B7 molecules to its ligand CD28 is important in the initiation of the autoimmune response in EAE. Further, it appears the costimulation provided by B7-1 is important in disease development, while B7-2 may play an important regulatory role. We and others later showed that B7/CTLA-4 interaction plays a critical role in down-regulating the immune response. Previous work has shown that activated T cells and T cells of a memory phenotype are less dependent on costimulation than naive T cells. T cells reactive with myelin components that are involved in the pathogenesis of EAE and possibly MS would be expected to have been activated as part of the disease process. Building upon our prior work in the EAE model, we have tested the hypothesis that myelin-reactive T cells, which are relevant to the pathogenesis of CNS inflammatory demyelination, can be distinguished from naive myelin-reactive T cells by a lack of dependence upon costimulation for activation and that the costimulatory requirements of these myelin-reactive T cells change during the course of disease. Our studies in the EAE model have also addressed the mechanisms of extrathymic (peripheral) T cell tolerance following intravenous (i.v. ) administration of high dose antigen. It is believed that TCR signaling in the absence of costimulation is a vital component of peripheral tolerance mechanisms. However, recent evidence suggests that peripheral tolerance of antigen-specific T cells induced in vivo may require CTLA-4 engagement of the tolerized T cells. We have begun to examine the molecular mechanisms of tolerance induction following intravenous and intraperitoneal administration of myelin antigens in the EAE model and test the hypothesis that tolerance induction is dependent on the B7:CD28/CTLA-4 pathway. The results from our studies will enhance our understanding of the role that myelin-reactive T cells may play in the pathogenesis of MS. We have determined that MBP-reactive T cells in MS patients are less dependent upon CD28 costimulation than in normal controls, suggesting that these T cells were previously primed in vivo. Characterization of these CD28-independent myelin-specific T cells will have broad implications for a variety of immunologically based therapies in diseases such as MS.

Asunto(s)

Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/terapia , Inmunoterapia , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Animales , Humanos

RESUMEN

Recent evidence suggests that co-stimulation provided by B7 molecules through CTLA-4 is important in establishing peripheral tolerance. In the present study, we examined the kinetics of tolerance induction and T cell differentiation following i.p. administration of myelin basic protein (MBP) Ac1-11 in mice transgenic for a TCR V(beta)8.2 gene derived from an encephalitogenic T cell clone specific for MBP Ac1-11. Examination of the lymph node cell response after antigen administration demonstrated a dependence on CTLA-4 for i.p. tolerance induction. Examination of splenocyte responses suggested that i.p. antigen administration induced a T(h)2 response, which was potentiated by anti-CTLA-4 administration. Interestingly, i.p. tolerance was able to inhibit the induction of experimental autoimmune encephalomyelitis and anti-CTLA-4 administration did not alter this phenotype, suggesting that CTLA-4 blockade did not block tolerance induction. Thus, T cell differentiation and the dependence on CTLA-4 for tolerance induction following i.p. antigen administration differs between lymph node and spleen in a model of organ-specific autoimmunity.

Asunto(s)

Encefalomielitis Autoinmune Experimental/inmunología , Tolerancia Inmunológica , Inmunoconjugados , Proteína Básica de Mielina/administración & dosificación , Linfocitos T/fisiología , Abatacept , Animales , Antígenos CD , Antígenos de Diferenciación/fisiología , Antígeno CTLA-4 , Diferenciación Celular , Citocinas/biosíntesis , Inyecciones Intraperitoneales , Ratones , Ratones Transgénicos , Proteína Básica de Mielina/inmunología , Ovalbúmina/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología

RESUMEN

Interactions between B7 molecules on antigen-presenting cells and CTLA-4 on T cells have been shown to be important in establishing tolerance. In the present study, we examined the kinetics of tolerance induction following i.v. administration of myelin basic protein (MBP) Ac1-11 in mice transgenic for a TCR V(beta)8.2 gene derived from an encephalitogenic T cell clone specific for MBP Ac1-11. Examination of the lymph node cell (LNC) response 10 days after antigen administration demonstrated an accentuation of i.v. tolerance induction with anti-CTLA-4 blockade. Anergy was induced in splenocytes by i.v. antigen administration as shown by a decrease in MBP-specific proliferation and IL-2 production, and anti-CTLA-4 potentiated this effect. In addition, i.v. antigen plus anti-CTLA-4 and complete Freund's adjuvant was not encephalitogenic. Interestingly, i.v. tolerance (a single injection) did not inhibit experimental autoimmune encephalomyelitis (EAE) and anti-CTLA-4 administration did not alter this phenotype. These results suggest that while the majority of MBP-specific T cells are tolerized by i.v. antigen and that this process is potentiated by anti-CTLA-4 administration, a population of T cells remains that is quite efficient in mediating EAE.

Asunto(s)

Antígenos de Diferenciación/fisiología , Encefalomielitis Autoinmune Experimental/inmunología , Tolerancia Inmunológica , Inmunoconjugados , Proteína Básica de Mielina/administración & dosificación , Linfocitos T/fisiología , Abatacept , Animales , Antígenos CD , Antígenos de Diferenciación/inmunología , Antígeno CTLA-4 , Diferenciación Celular , Citocinas/biosíntesis , Inyecciones Intravenosas , Activación de Linfocitos , Ratones , Ratones Transgénicos , Proteína Básica de Mielina/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología

RESUMEN

Previous studies have shown complex roles for the B7 receptors in providing both positive and negative regulation of experimental autoimmune encephalomyelitis (EAE). B7 blockade can ameliorate clinical EAE by indirectly interfering with CD28 signaling. However, B7 blockade can also result in disease exacerbation, presumably by interfering with regulatory B7:CTLA-4 interactions. Therefore, we have directly targeted T cell CD28 with specific mAbs both during initial Ag priming and after the onset of clinical signs of EAE. We found that CD28 blockade ameliorated EAE during the efferent and afferent limbs of the immune response. Disease amelioration at disease onset was associated with suppression of TNF-alpha production. Finally, Ab blockade of T cell CD28 during the first disease episode resulted in significant attenuation of the subsequent disease course, with no significant relapses. In contrast to previous studies targeting APC B7 with CTLA4-Ig, reagents targeting CD28 can block ongoing disease. Therefore, the present results suggest a clinically relevant therapeutic scenario for human diseases, such as multiple sclerosis.

Asunto(s)

Antígenos CD28/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/prevención & control , Activación de Linfocitos , Subgrupos de Linfocitos T/inmunología , Animales , Anticuerpos Bloqueadores/metabolismo , Anticuerpos Bloqueadores/farmacología , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales/farmacología , Antígeno B7-1/inmunología , Antígeno B7-1/metabolismo , Sitios de Unión de Anticuerpos , Antígenos CD28/metabolismo , Progresión de la Enfermedad , Encefalomielitis Autoinmune Experimental/etiología , Femenino , Interleucina-2/antagonistas & inhibidores , Interleucina-2/biosíntesis , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Subgrupos de Linfocitos T/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

We present a physical and molecular genetic characterization of Drosophila melanogaster TFIIE (dTFIIE), a component of the basal RNA polymerase II transcription apparatus. We have purified dTFIIE to near homogeneity from nuclear extracts of Drosophila embryos and found that it is composed of two subunits with apparent molecular weights of 55 and 38 kDa. Peptide sequence information derived from the two subunits was used to isolate the corresponding cDNA clones, revealing that dTFIIE and human TFIIE share extensive amino acid similarity. Functional conservation was demonstrated by the ability of bacterially expressed dTFIIE to substitute for human TFIIE in an in vitro transcription assay reconstituted from purified components. Cytological mapping analysis shows that both subunits are encoded by single copy genes located on chromosome III.

Asunto(s)

Drosophila melanogaster/química , Factores de Transcripción TFII , Factores de Transcripción/aislamiento & purificación , Transcripción Genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , ARN Polimerasa II/metabolismo , Proteínas Recombinantes , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo