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1.
Proc Natl Acad Sci U S A ; 98(26): 15137-42, 2001 Dec 18.
Artículo en Inglés | MEDLINE | ID: mdl-11752460

RESUMEN

Differentiation of naive CD4(+) T cells into IFN-gamma-producing T helper 1 (T(H)1) cells is pivotal for protective immune responses against intracellular pathogens. T-bet, a recently discovered member of the T-box transcription factor family, has been reported to play a critical role in this process, promoting IFN-gamma production. Although terminal T(H)1 differentiation occurs over days, we now show that challenge of mice with a prototypical T(H)1-inducing stimulus, Toxoplasma gondii soluble extract, rapidly induced IFN-gamma and T-bet; T-bet induction was substantially lower in IFN-gamma-deficient mice. Naive T cells expressed little T-bet, but this transcription factor was induced markedly by the combination of IFN-gamma and cognate antigen. Human myeloid antigen-presenting cells showed T-bet induction after IFN-gamma stimulation alone, and this induction was antagonized by IL-4 and granulocyte/macrophage colony-stimulating factor. Although T-bet was induced rapidly and directly by IFN-gamma, it was not induced by IFN-alpha, lipopolysaccharide, or IL-1, indicating that this action of IFN-gamma was specific. Moreover, T-bet induction was dependent on Stat1 but not Stat4. These data argue for a model in which IFN-gamma gene regulation involves an autocrine loop, whereby the cytokine regulates a transcription factor that promotes its own production. These findings substantially alter the current view of T-bet in IFN-gamma regulation and promotion of cell-mediated immune responses.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Células Dendríticas/metabolismo , Interferón gamma/fisiología , Macrófagos/metabolismo , Factores de Transcripción/biosíntesis , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular/fisiología , Células Cultivadas , Cartilla de ADN , Células Dendríticas/inmunología , Femenino , Humanos , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Proteínas de Dominio T Box , Factores de Transcripción/fisiología , Regulación hacia Arriba
2.
Trends Immunol ; 22(10): 556-60, 2001 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-11574279

RESUMEN

The suggestion that antigen-presenting cells (APCs) produce interferon gamma (IFN-gamma) is controversial because it conflicts with the initial paradigm in which the production of IFN-gamma was restricted to lymphoid cells. However, some answers to this skepticism have been provided by recent findings of high-level production and intracellular expression of IFN-gamma by interleukin-12 (IL-12)-stimulated macrophages and dendritic cells. New data are now emerging to explain the mechanism of production of IFN-gamma vby APCs. As in lymphoid cells, IL-12-induced IFN-gamma production in APCs requires signal transducer and activator of transcription 4 (STAT4), although the precise molecular events that govern the transcription of the gene encoding IFN-gamma are enigmatic still. Understanding these processes in lymphoid, and now nonlymphoid, cells remains an important challenge.


Asunto(s)
Células Presentadoras de Antígenos/inmunología , Interferón gamma/biosíntesis , Animales , Proteínas de Unión al ADN/inmunología , Humanos , Modelos Inmunológicos , Factor de Transcripción STAT4 , Transactivadores/inmunología
3.
Clin Immunol ; 100(3): 270-6, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11513540

RESUMEN

The convergence of innate and adaptive immunity is critical for host defense, allowing for early protection and the generation of specific responses. STAT-4 is at that point of convergence, unifying the IFNalpha and IL-12 pathways. Activation of STAT-4 is crucial to T cell polarization, B cell and NK cell activation, and the control of intracellular pathogens. However, techniques to detect phosphorylated STAT-4 are cumbersome and require many cells. We have developed a flow cytometric detection technique to investigate IL-12 signaling in human peripheral blood mononuclear cells. Using different polyclonal antibodies that recognize either total STAT-4 protein or tyrosine-phosphorylated STAT-4, we can easily detect IL-12 and IFNalpha signaling in PHA/IL-2 blasts derived from peripheral blood lymphocytes. This technique not only allows us to evaluate IL-12 signaling, but it is also less time consuming and labor intensive than alternative methods. Using this flow cytometry-based method, we should be able to detect patients with defects in IL-12 receptor signal transduction, who typically present with disseminated nontuberculous mycobacterial infections.


Asunto(s)
Proteínas de Unión al ADN/análisis , Transactivadores/análisis , Animales , Proteínas de Unión al ADN/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Interferón-alfa/farmacología , Interleucina-12/farmacología , Ratones , Ratones Endogámicos BALB C , Fosforilación , Factor de Transcripción STAT4 , Transactivadores/metabolismo
4.
Blood ; 98(3): 851-9, 2001 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-11468188

RESUMEN

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and intense inflammation. FMF attacks are unique in their sensitivity to the microtubule inhibitor colchicine, contrasted with their refractoriness to the anti-inflammatory effects of glucocorticoids. The FMF gene, MEFV, was recently identified by positional cloning; it is expressed at high levels in granulocytes and monocytes. The present study investigated the subcellular localization of the normal gene product, pyrin. These experiments did not support previously proposed nuclear or Golgi localizations. Instead fluorescence microscopy demonstrated colocalization of full-length GFP- and epitope-tagged pyrin with microtubules; this was markedly accentuated in paclitaxel-treated cells. Moreover, immunoblot analysis of precipitates of stabilized microtubules with recombinant pyrin demonstrated a direct interaction in vitro. Pyrin expression did not affect the stability of microtubules. Deletion constructs showed that the unique N-terminal domain of pyrin is necessary and sufficient for colocalization, whereas disease-associated mutations in the C-terminal B30.2 (rfp) domain did not disrupt this interaction. By phalloidin staining, a colocalization of pyrin with actin was also observed in perinuclear filaments and in peripheral lamellar ruffles. The proposal is made that pyrin regulates inflammatory responses at the level of leukocyte cytoskeletal organization and that the unique therapeutic effect of colchicine in FMF may be dependent on this interaction. (Blood. 2001;98:851-859)


Asunto(s)
Citoesqueleto de Actina/metabolismo , Microtúbulos/metabolismo , Proteínas/metabolismo , Animales , Células COS , Proteínas del Citoesqueleto , Citoesqueleto/metabolismo , Estabilidad de Medicamentos , Fiebre Mediterránea Familiar/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Mutación , Proteínas/genética , Pirina , Proteínas Recombinantes , Transfección
5.
J Immunol ; 166(7): 4446-55, 2001 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-11254700

RESUMEN

Autocrine activation of APC by IL-12 has recently been revealed; we demonstrate here that inducible expression of Stat4 in APC is central to this process. Stat4 is induced in dendritic cells (DC) in a maturation-dependent manner and in macrophages in an activation-dependent manner. Stat4 levels directly correlate with IL-12-dependent IFN-gamma production by APC as well as IFN-gamma production by DC during Ag presentation. The Th2 cytokines IL-4 and IL-10 suppress Stat4 induction in DC and macrophages when present during maturation and activation, respectively, diminishing IFN-gamma production. In contrast, IL-4 has no effect on Stat4 levels in mature DC and actually augments IFN-gamma production by DC during Ag presentation, indicating that IL-4 acts differently in a spatiotemporal manner. The functional importance of Stat4 is evident in Stat4(-/-) DC and macrophages, which fail to produce IFN-gamma. Furthermore, Stat4(-/-) macrophages are defective in NO production in response to IL-12 and are susceptible to TOXOPLASMA: Autocrine IL-12 signaling is required for high-level IFN-gamma production by APC at critical stages in both innate and adaptive immunity, and the control of Stat4 expression is likely an important regulator of this process.


Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/fisiología , Células Dendríticas/metabolismo , Macrófagos/metabolismo , Transactivadores/biosíntesis , Transactivadores/fisiología , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Comunicación Autocrina/genética , Comunicación Autocrina/inmunología , Antígenos CD8/biosíntesis , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Citocinas/fisiología , Proteínas de Unión al ADN/genética , Células Dendríticas/citología , Células Dendríticas/inmunología , Inmunidad Celular/genética , Inmunidad Innata/genética , Inyecciones Intravenosas , Interferón gamma/biosíntesis , Interleucina-12/fisiología , Lipopolisacáridos/administración & dosificación , Activación de Macrófagos/genética , Macrófagos/inmunología , Macrófagos/parasitología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor de Transcripción STAT4 , Transducción de Señal/genética , Transducción de Señal/inmunología , Células Th2/inmunología , Células Th2/metabolismo , Toxoplasma/inmunología , Toxoplasma/patogenicidad , Transactivadores/genética , Regulación hacia Arriba/genética , Regulación hacia Arriba/inmunología
6.
Genes Immun ; 2(8): 422-32, 2001 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-11781709

RESUMEN

Mutations of the Janus kinase 3 (JAK3) have been previously described to cause an autosomal recessive variant of severe combined immunodeficiency (SCID) usually characterized by the near absence of T and NK cells, but preserved numbers of B lymphocytes (T-B+SCID). We now report a family whose JAK3 mutations are associated with the persistence of circulating T cells, resulting in previously undescribed clinical presentations, ranging from a nearly unaffected 18-year-old subject to an 8-year-old sibling with a severe lymphoproliferative disorder. Both siblings were found to be compound heterozygotes for the same deleterious JAK3 mutations: an A96G initiation start site mutation, resulting in a dysfunctional, truncated protein product and a G2775(+3)C mutation in the splice donor site sequence of intron 18, resulting in a splicing defect and a predicted premature stop. These mutations were compatible with minimal amounts of functional JAK3 expression, leading to defective cytokine-dependent signaling. Activated T cells in these patients failed to express Fas ligand (FasL) in response to IL-2, which may explain the accumulation of T cells with an activated phenotype and a skewed T cell receptor (TcR) Vbeta family distribution. We speculate that residual JAK3 activity accounted for the maturation of thymocytes, but was insufficient to sustain IL-2-mediated homeostasis of peripheral T cells via Fas/FasL interactions. These data demonstrate that the clinical spectrum of JAK3 deficiency is quite broad and includes immunodeficient patients with accumulation of activated T cells, and indicate an essential role for JAK3 in the homeostasis of peripheral T cells in humans.


Asunto(s)
Proteínas Tirosina Quinasas/deficiencia , Adolescente , Secuencia de Aminoácidos , Linfocitos B/metabolismo , Línea Celular Transformada , Niño , ADN Complementario , Proteína Ligando Fas , Femenino , Expresión Génica , Humanos , Inmunofenotipificación , Interleucina-2/metabolismo , Janus Quinasa 3 , Masculino , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Linaje , Proteínas Tirosina Quinasas/genética , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Interleucina-2/metabolismo , Análisis de Secuencia de ADN , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Inmunodeficiencia Combinada Grave/patología , Transducción de Señal , Linfocitos T , Regulación hacia Arriba
7.
Arthritis Res ; 2(1): 16-32, 2000.
Artículo en Inglés | MEDLINE | ID: mdl-11094415

RESUMEN

Cytokines play a critical role in the normal development and function of the immune system. On the other hand, many rheumatologic diseases are characterized by poorly controlled responses to or dysregulated production of these mediators. Over the past decade tremendous strides have been made in clarifying how cytokines transmit signals via pathways using the Janus kinase (Jak) protein tyrosine kinases and the Signal transducer and activator of transcription (Stat) proteins. More recently, research has focused on several distinct proteins responsible for inhibiting these pathways. It is hoped that further elucidation of cytokine signaling through these pathways will not only allow for a better comprehension of the etiopathogenesis of rheumatologic illnesses, but may also direct future treatment options.


Asunto(s)
Adyuvantes Inmunológicos/fisiología , Citocinas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Animales , Humanos
8.
Mol Cell Biol ; 20(12): 4371-80, 2000 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-10825200

RESUMEN

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Interleucina-2/fisiología , Proteínas de la Leche , Proteínas Tirosina Quinasas/fisiología , Transducción de Señal , Transactivadores/fisiología , Animales , Línea Celular , Activación Enzimática , Precursores Enzimáticos/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Janus Quinasa 3 , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/fisiología , Fosforilación , Receptores de Interleucina-2/fisiología , Factor de Transcripción STAT5 , Quinasa Syk , Proteínas Supresoras de Tumor
9.
Blood ; 95(10): 3223-31, 2000 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-10807793

RESUMEN

Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever and neutrophil-mediated serosal inflammation. We recently identified the gene causing FMF, designated MEFV, and found it to be expressed in mature neutrophils, suggesting that it functions as an inflammatory regulator. To facilitate our understanding of the normal function of MEFV, we extended our previous studies. MEFV messenger RNA was detected by reverse transcriptase-polymerase chain reaction in bone marrow leukocytes, with differential expression observed among cells by in situ hybridization. CD34 hematopoietic stem-cell cultures induced toward the granulocytic lineage expressed MEFV at the myelocyte stage, concurrently with lineage commitment. The prepromyelocytic cell line HL60 expressed MEFV only at granulocytic and monocytic differentiation. MEFV was also expressed in the monocytic cell lines U937 and THP-1. Among peripheral blood leukocytes, MEFV expression was detected in neutrophils, eosinophils, and to varying degrees, monocytes. Consistent with the tissue specificity of expression, complete sequencing and analysis of upstream regulatory regions of MEFV revealed homology to myeloid-specific promoters and to more broadly expressed inflammatory promoter elements. In vitro stimulation of monocytes with the proinflammatory agents interferon (IFN) gamma, tumor necrosis factor, and lipopolysaccharide induced MEFV expression, whereas the antiinflammatory cytokines interleukin (IL) 4, IL-10, and transforming growth factor beta inhibited such expression. Induction by IFN-gamma occurred rapidly and was resistant to cycloheximide. IFN-alpha also induced MEFV expression. In granulocytes, MEFV was up-regulated by IFN-gamma and the combination of IFN-alpha and colchicine. These results refine understanding of MEFV by placing the gene in the myelomonocytic-specific proinflammatory pathway and identifying it as an IFN-gamma immediate early gene.


Asunto(s)
Fiebre Mediterránea Familiar/genética , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/farmacología , Leucocitos/metabolismo , Proteínas/genética , Diferenciación Celular/genética , Proteínas del Citoesqueleto , Fiebre Mediterránea Familiar/sangre , Humanos , Inflamación , Leucocitos/patología , Biosíntesis de Proteínas , Pirina , Células U937
10.
J Immunol ; 164(9): 4659-64, 2000 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-10779770

RESUMEN

Stat4 is a key transcription factor involved in promoting cell-mediated immunity, whose expression in mature cells has been reported to be restricted to T and NK cells. We demonstrate here, however, that Stat4 expression is not restricted to lymphoid cells. In their basal state, monocytes do not express Stat4. Upon activation, however, IFN-gamma- and LPS-treated monocytes and dendritic cells express high levels of Stat4. Monocyte-expressed Stat4 in humans is phosphorylated in response to IFN-alpha, but not IL-12. In contrast, the Th2 cytokines, IL-4 and IL-10, specifically down-regulate Stat4 expression in activated monocytes, while having little effect on Stat6 expression. Moreover, macrophages in synovial tissue obtained from patients with rheumatoid arthritis express Stat4 in vivo, suggesting a potential role in a prototypical Th1-mediated human disease. IFN-alpha-induced Stat4 activation in human monocytes represents a previously unrecognized signaling pathway at sites of Th1 inflammation.


Asunto(s)
Artritis Reumatoide/patología , Proteínas de Unión al ADN/biosíntesis , Células Dendríticas/metabolismo , Activación de Macrófagos , Macrófagos/metabolismo , Monocitos/metabolismo , Células TH1/inmunología , Transactivadores/biosíntesis , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Citocinas/fisiología , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/sangre , Proteínas de Unión al ADN/genética , Células Dendríticas/inmunología , Regulación hacia Abajo/inmunología , Humanos , Interferón-alfa/farmacología , Macrófagos/inmunología , Macrófagos/patología , Monocitos/inmunología , Fosforilación , ARN Mensajero/biosíntesis , ARN Mensajero/sangre , Factor de Transcripción STAT4 , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Células TH1/metabolismo , Células Th2/inmunología , Transactivadores/antagonistas & inhibidores , Transactivadores/sangre , Transactivadores/genética
11.
J Immunol ; 164(4): 1768-74, 2000 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-10657623

RESUMEN

Glucocorticoids are widely used in the therapy of inflammatory, autoimmune, and allergic diseases. As the end-effectors of the hypothalamic-pituitary-adrenal axis, endogenous glucocorticoids also play an important role in suppressing innate and cellular immune responses. Previous studies have indicated that glucocorticoids inhibit Th1 and enhance Th2 cytokine secretion. IL-12 promotes Th1 cell-mediated immunity, while IL-4 stimulates Th2 humoral-mediated immunity. Here, we examined the regulatory effect of glucocorticoids on key elements of IL-12 and IL-4 signaling. We first investigated the effect of dexamethasone on IL-12-inducible genes and showed that dexamethasone inhibited IL-12-induced IFN-gamma secretion and IFN regulatory factor-1 expression in both NK and T cells. This occurred even though the level of expression of IL-12 receptors and IL-12-induced Janus kinase phosphorylation remained unaltered. However, dexamethasone markedly inhibited IL-12-induced phosphorylation of Stat4 without altering its expression. This was specific, as IL-4-induced Stat6 phosphorylation was not affected, and mediated by the glucocorticoid receptor, as it was antagonized by the glucocorticoid receptor antagonist RU486. Moreover, transfection experiments showed that dexamethasone reduced responsiveness to IL-12 through the inhibition of Stat4-dependent IFN regulatory factor-1 promoter activity. We conclude that blocking IL-12-induced Stat4 phosphorylation, without altering IL-4-induced Stat6 phosphorylation, appears to be a new suppressive action of glucocorticoids on the Th1 cellular immune response and may help explain the glucocorticoid-induced shift toward the Th2 humoral immune response.


Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Dexametasona/farmacología , Inmunosupresores/farmacología , Interleucina-12/fisiología , Proteínas Proto-Oncogénicas , Linfocitos T/efectos de los fármacos , Células TH1/efectos de los fármacos , Transactivadores/antagonistas & inhibidores , Células 3T3 , Animales , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Factor 1 Regulador del Interferón , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-12/antagonistas & inhibidores , Interleucina-12/metabolismo , Janus Quinasa 2 , Ratones , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/biosíntesis , Fosfoproteínas/genética , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/inmunología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12 , Factor de Transcripción STAT4 , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Linfocitos T/metabolismo , TYK2 Quinasa , Células TH1/inmunología , Transactivadores/metabolismo , Transfección
12.
Clin Immunol ; 91(2): 234-41, 1999 May.
Artículo en Inglés | MEDLINE | ID: mdl-10227816

RESUMEN

We have described previously a family with an apparent genetic susceptibility to disseminated Mycobacterium avium complex infection and an underlying defect in IL-12 regulation leading to abnormally low interferon-gamma production. Their T cells appear to act normally when in the presence of normal accessory cells. Cell-to-cell contact was necessary for normal monocytes to complement the familial patient monocyte defect, suggesting the familial defect in interferon-gamma costimulation involves pathways requiring cell surface molecule interactions. In an effort to better characterize the abnormality in these patients, we examined the role of known costimulatory molecules in residual costimulation by patient PBMC compared to normals. Whereas normals utilized CD40/CD40L interactions and IL-12 production for optimal interferon-gamma costimulation in PHA-stimulated cocultures, familial patient interferon-gamma production was low and unaffected by their blockade. CD86 blockade caused a greater than 50% reduction in both normal and familial patient interferon-gamma production, implying that a majority of residual familial patient costimulation required this pathway. Furthermore, selected myelomonocytic cell lines (K562 and THP1) acted as potent accessory cells for interferon-gamma production by familial patient and normal T cells, largely independent of IL-12 production. However, CD86 blockade of K562 cell/familial cell cocultures resulted in less than a 20% reduction in interferon-gamma production, indicating that familial patient cells respond to IL-12- and CD86-independent costimulatory signals for interferon-gamma as well. Thus, we demonstrate that the familial defect also involves interferon-gamma costimulation pathways requiring both CD40/CD40L interaction and IL-12 production, while residual pathways remain that allow low-level interferon-gamma production. Familial Mycobacterium avium patient monocytes and certain myelomonocytic cell lines can be exploited to investigate IL-12-independent costimulation for interferon-gamma production.


Asunto(s)
Interferón gamma/biosíntesis , Interleucina-12/metabolismo , Infección por Mycobacterium avium-intracellulare/metabolismo , Transducción de Señal , Antígenos CD/metabolismo , Antígeno B7-2 , Antígenos CD40/metabolismo , Ligando de CD40 , Línea Celular , Humanos , Células K562 , Leucocitos Mononucleares/metabolismo , Glicoproteínas de Membrana/metabolismo , Monocitos , Complejo Mycobacterium avium
13.
Cell ; 97(1): 133-44, 1999 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-10199409

RESUMEN

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.


Asunto(s)
Antígenos CD/genética , Fiebre Mediterránea Familiar/genética , Mutación de Línea Germinal/genética , Receptores del Factor de Necrosis Tumoral/genética , Secuencia de Aminoácidos , Antígenos CD/biosíntesis , Antígenos CD/sangre , Antígenos CD/metabolismo , Análisis Mutacional de ADN/métodos , Femenino , Genes Dominantes/genética , Humanos , Leucocitos/metabolismo , Masculino , Datos de Secuencia Molecular , Linaje , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/sangre , Receptores del Factor de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral , Síndrome
14.
J Infect Dis ; 178(4): 1095-104, 1998 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-9806040

RESUMEN

Mycobacterial infections are critically controlled by interferon-gamma (IFN-gamma) and the cellular responses it elaborates, as shown by patients with mutations in the IFN-gamma receptor ligand-binding chain (IFN-gamma R1) who have disseminated nontuberculous mycobacterial infections. The immunologic sequelae of IFN-gamma R1 deficiency were characterized in 2 unrelated patients from the Indian subcontinent with novel homozygous recessive IFN-gamma R1 mutations. In vitro, these patients' peripheral blood mononuclear cells produced 10% of normal IFN-gamma and interleukin-12 (IL-12) in response to phytohemagglutinin (PHA) but normal amounts of IFN-gamma in response to PHA plus IL-12. Tumor necrosis factor-alpha (TNF-alpha) production was normal in response to endotoxin and to PHA but was not augmented by the addition of IFN-gamma. An abnormal phenotype was not found in heterozygous patient relatives. These patients demonstrate the critical role that the IFN-gamma receptor plays in the regulation of IFN-gamma, IL-12, and TNF-alpha.


Asunto(s)
Interferón gamma/inmunología , Interleucina-12/inmunología , Complejo Mycobacterium avium/inmunología , Receptores de Interferón/genética , Factor de Necrosis Tumoral alfa/inmunología , Secuencia de Aminoácidos , Preescolar , Genes Recesivos , Heterocigoto , Humanos , Leucocitos Mononucleares/inmunología , Masculino , Datos de Secuencia Molecular , Mutación , Pakistán/etnología , Linaje , Receptores de Interferón/deficiencia , Receptor de Interferón gamma
15.
J Immunol ; 157(1): 411-6, 1996 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-8683146

RESUMEN

We have described previously a family in which several members have disseminated Mycobacterium avium complex infection. PBMC from affected members produced abnormally low amounts of IFN-gamma upon stimulation with PHA. Using PHA-stimulated allogeneic cocultures of highly purified monocytes and T cells from familial patients and normal subjects, we have now demonstrated that familial patient monocytes are defective in accessory cell function for IFN-gamma production. Familial patient monocytes did not inhibit IFN-gamma production by normal cells, nor did inhibition of PG synthesis restore normal IFN-gamma production by familial patient cells. Familial patient cells responded to the addition of exogenous IL-12 by increasing IFN-gamma production, while addition of exogenous anti-IL-12 had an insignificant effect on their IFN-gamma production. IL-12 was undetectable in PHA-stimulated cocultures of familial patient monocytes with familial or normal T cells. In addition, IL-12 production by adherent cells from patients and their unaffected mothers was abnormally low following stimulation with fixed Staphylococcus aureus Cowan I strain. However, normal amounts of IL-12 were detected when adherent familial patient cells were stimulated with S. aureus Cowan I strain and IFN-gamma, suggesting abnormal regulation of IL-12 production by familial monocytes. This is the first report of defective IL-12 production associated with increased susceptibility to an infectious disease, a finding that supports the critical role of this cytokine in host defense.


Asunto(s)
Interferón gamma/biosíntesis , Interferón gamma/deficiencia , Interleucina-12/biosíntesis , Interleucina-12/deficiencia , Monocitos/inmunología , Monocitos/metabolismo , Infección por Mycobacterium avium-intracellulare/genética , Infección por Mycobacterium avium-intracellulare/inmunología , Niño , Femenino , Heterocigoto , Humanos , Interferón gamma/farmacología , Interleucina-12/farmacología , Masculino , Monocitos/efectos de los fármacos , Madres , Linaje , Fitohemaglutininas/farmacología , Staphylococcus aureus/inmunología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología
16.
Clin Infect Dis ; 20(5): 1292-7, 1995 May.
Artículo en Inglés | MEDLINE | ID: mdl-7620013

RESUMEN

The control of leishmaniasis depends on a knowledge of the magnitude of the disease and of exposure to it. Delayed-type hypersensitivity testing can detect prior exposure to the parasite, but there is little agreement regarding the choice of an antigen for such testing. New and Old World leishmanins were tested in a study of patients with confirmed prior cutaneous leishmaniasis (CL), patients with confirmed prior American visceral leishmaniasis (AVL), and controls from areas in Espírito Santo, Brazil, where leishmaniasis is not endemic. Biobrás antigen (a suspended mixture of Leishmania braziliensis guyanensis, Leishmania mexicana amazonensis, and Leishmania mexicana promastigotes) detected 100% of prior CL infections and thus was superior to the Old World antigen, Leishmania major, which detected only 19% of these infections (P < .00001). Soluble New World antigens of Leishmania chagasi evoked a response in 96% of cases of prior AVL, whereas the Old World counterpart, Leishmania infantum, evoked a response in 71% of cases (P < .042). Testing of family members of patients with prior AVL showed greater utility of the New World leishmanins and suggested subclinical exposure of a large number of healthy people in the hyperendemic region. New World skin-test antigens should be used in future epidemiological studies in the Americas to more accurately determine the extent of exposure.


Asunto(s)
Antígenos de Protozoos/inmunología , Leishmania/inmunología , Leishmaniasis/diagnóstico , Adolescente , Adulto , Anciano , Animales , Niño , Preescolar , Humanos , Lactante , Persona de Mediana Edad , Pruebas Cutáneas
17.
Virology ; 184(1): 319-29, 1991 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-1871974

RESUMEN

Intracellular transport and processing of the human immunodeficiency virus type 1 (HIV-1) envelope precursor polyprotein, gp160, proceeds via the endoplasmic reticulum (ER) and Golgi complex. We examined gp160 processing during the production of HIV-1 virions in transfected HeLa cells using wild-type and env mutant proviral molecular clones. Results from pulse-chase analyses indicated that a single amino acid substitution within a highly conserved domain of the env gene impaired gp160 export from the ER, leading to an increase in oligomeric forms of gp160 and a decrease in gp120 production. In contrast, gp160 which contained a mutated cleavage site was able to traverse the ER/Golgi complex, even in the absence of proteolytic processing, and become incorporated into budding virions. These findings indicate that export from the ER is a point in the intracellular trafficking of gp160 that is crucial to the production of the mature envelope components.


Asunto(s)
Productos del Gen env/genética , VIH-1/genética , Mutagénesis Sitio-Dirigida , Precursores de Proteínas/genética , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/metabolismo , Productos del Gen env/biosíntesis , Productos del Gen env/aislamiento & purificación , Glicósido Hidrolasas , Aparato de Golgi/metabolismo , Proteínas gp160 de Envoltorio del VIH , Células HeLa/fisiología , Humanos , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Sondas de Oligonucleótidos , Precursores de Proteínas/biosíntesis , Precursores de Proteínas/aislamiento & purificación , Transfección , Proteínas del Envoltorio Viral/biosíntesis , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/aislamiento & purificación , Virión/genética
18.
AIDS Res Hum Retroviruses ; 7(9): 729-33, 1991 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-1742079

RESUMEN

Previously described FVB/N mice harboring a human immunodeficiency virus (HIV) long terminal repeat (LTR)/chloramphenicol acetyl transferase (CAT) transgene were treated with varying amounts of 254 nm UV-C radiation or 312 nm UV-B radiation. At optimal exposure periods, a 20-fold increase in HIV-LTR-directed expression was observed in ear specimens collected 24 h following UV-C exposure; a fourfold increase in expression was induced by UV-B exposure. Investigation of the kinetics of UV-C induction in vivo revealed that LTR-directed gene expression began to increase 2 hours after exposure and reached a maximum on Day 3 following exposure (greater than 30-fold induction). In experiments examining the kinetics of UV-B activation, the maximum level of CAT activity in the ears of irradiated transgenic animals was fivefold above levels in unirradiated transgenic controls (Day 5). Furthermore, CAT activity was not induced in fur-bearing skin following UV exposure; however, a fourfold increase in HIV-LTR-directed expression could be elicited when hair was removed by shaving prior to UV-B treatment.


Asunto(s)
Duplicado del Terminal Largo de VIH/efectos de la radiación , Animales , Cloranfenicol O-Acetiltransferasa/genética , Expresión Génica/efectos de la radiación , Ratones , Ratones Transgénicos , Rayos Ultravioleta
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