RESUMEN
This study identifies activation characteristics of PPD-responsive T-cells that emerge after treatment with anti-CD4 monoclonal antibody (Mab). PPD-stimulated T-cell proliferations, OX40 phenotype and protein tyrosine phosphorylations involving p56lck (pp56lck) were compared to Con A stimulations using T-cells isolated from spleen and draining lymph node of CFA/PPD-immunized rats either untreated or treated in vivo with anti-CD4 Mab. Splenocytes stimulated by concanavalin A (Con A) showed correlated increases in proliferation, levels of pp56lck, and OX40 expression; these parameters were not correlated in splenocytes after PPD-stimulations. T-cells isolated from lymph nodes draining the site of CFA/PPD immunization proliferated in response to stimulation by either PPD or Con A, but only PPD-responsive cells showed correlation to the OX40 activation phenotype and increased levels of pp56lck. CD4+ T-cells isolated from either tissue compartment after anti-CD4 Mab treatments showed higher background and PPD-stimulated proliferations, and expressed lower levels of OX40. In contrast, anti-CD4 Mab treatments reduced (60%) and abolished Con A-stimulated proliferations of splenocytes and lymph node T-cells, respectively. The effects of anti-CD4 Mab treatment on pp56lck levels correlated only to the changes observed for Con A stimulations of splenocytes. These results demonstrate that PPD antigen-specific T-cell populations recovered from different tissue compartments were resistant to in vivo anti-CD4 Mab treatments and did not show the activation changes characteristics of CD4+ T-cells after Con A stimulation.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antígenos CD4/inmunología , Epítopos/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Concanavalina A/inmunología , Relación Dosis-Respuesta Inmunológica , Adyuvante de Freund/inmunología , Proteínas Tirosina Quinasas/inmunología , Ratas , Ratas Endogámicas Lew , Linfocitos T/efectos de los fármacos , Tuberculina/inmunologíaRESUMEN
In vitro incubations of CD4+ T cells with anti-CD4 monoclonal antibody (Mab) demonstrated saturation binding and inhibited both PPD-stimulated proliferation and delayed-type hypersensitivity (DTH) after passive transfer of these cells into naive rats. In comparison, in vivo administration of anti-CD4 Mab into rats, 2 weeks after CFA immunization (PPD priming), saturated CD4 molecules expressed on T lymphocytes and depleted significant percentages of CD4+ T cells from blood, spleen, and lymph nodes, yet failed to cause significant inhibition of PPD-stimulated DTH ear swelling. Only repeated in vivo administration of anti-CD4 Mabs during the CFA/PPD antigen priming caused significant decreases (36 +/- 5%) in PPD-stimulated DTH ear swelling. T cells isolated from the blood and spleen showed insignificant relative decreases in PPD-stimulated proliferation after anti-CD4 treatments. In contrast, PPD-stimulated proliferation of T cells isolated from the draining lymph nodes after anti-CD4 treatments showed dramatic increases in PPD reactivity. PPD stimulation of T cells isolated from the draining lymph nodes of anti-CD4 Mab-treated rats produced relative phenotypic changes in CD4 coexpressions with CD45RC (inverse memory), CD49d (inflammatory adhesion integrin VLA-4), and OX40 (CD4+ blast), representative of memory CD4+ T cell blasts with reduced capacity for endothelial infiltration. These data demonstrate that depletion of CD4+ T cells by anti-CD4 Mab injections, administered either during or after antigen priming, selectively enhanced memory responses in T lymphocytes in the draining lymph nodes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD4/inmunología , Memoria Inmunológica/inmunología , Linfocitos T/inmunología , Tuberculina/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Línea Celular , Supresión Clonal/inmunología , Citometría de Flujo , Hipersensibilidad Tardía/inmunología , Inmunofenotipificación , Ganglios Linfáticos/citología , Activación de Linfocitos/inmunología , Ratas , Ratas Endogámicas LewRESUMEN
The nontraditional Pharm.D. program at the University of Arkansas for Medical Sciences is described. The nontraditional program, started in 1990, includes 21 credit hours of didactic work and 12 of clerkships. In the self-paced didactic courses, videotapes, manuals, and texts are used, along with faculty consultation in person or by telephone. Clerkships can be traditional or nontraditional. Nontraditional clerkships can be completed by self-study in the student's community after an orientation period at the preceptor's site. More than 400 pharmacists have enrolled in the program and more than 80 have entered nontraditional clerkships. Thirty-nine pharmacists have received the Pharm.D. degree. The program offers an accessible alternative for pharmacists who want to earn the Pharm.D. degree while maintaining full-time employment.
Asunto(s)
Educación de Postgrado en Farmacia/métodos , Arkansas , Curriculum , Humanos , Mentores , Farmacéuticos/normas , Facultades de FarmaciaRESUMEN
Surface expression of the CD4 glycoprotein molecule is postulated to facilitate antigen recognition through the T cell receptor (TCR) and is itself a receptor for human immunodeficiency virus (HIV)-gp120 glycoprotein. Both antigen-stimulated TCR activation and HIV infectivity can be blocked by whole anti-CD4 antibodies. Although selective modulation of CD4 from the surface by gangliosides (GM1) blocks HIV infectivity, it enhances associated TCR function. Enhanced TCR function has also been observed after intracellular delivery of synthetic CD4 mRNA-antisense oligodeoxynucleotides (ODN) that block de novo synthesis of CD4. These specific CD4 modulations were mechanistically different from one another yet they both selectively removed the CD4 molecule from the T cell surface and enhanced antigen-stimulated function through the TCR. The proposed role of CD4 during TCR function and HIV infectivity was developed, in part, according to decreases following CD4 antagonism by whole antibody or down-modulation of CD4 by phorbol-stimulated protein kinase C activity. Selective CD4 modulations have independently redefined the specific contributions of CD4 surface expression during T cell activation and may establish a role for CD4 receptor subtypes during HIV-1 infection of CD4+ cells.
Asunto(s)
Antígenos CD4/inmunología , Animales , Antígenos CD4/biosíntesis , Antígenos CD4/genética , Infecciones por VIH/inmunología , Humanos , Linfocitos T/enzimología , Linfocitos T/inmunologíaRESUMEN
Immunization with disease-associated TCR V region peptides is an effective treatment for experimental autoimmune encephalomyelitis. Myelin basic protein-specific T cells, which induce experimental autoimmune encephalomyelitis in many animal strains, may be important in the pathogenesis of multiple sclerosis. Myelin basic protein-specific T cell clones from some multiple sclerosis patients preferentially use TCR V genes from the V beta 5.2 and V beta 6.1 families. To assess the safety and immunogenicity of TCR V beta 5.2 and V beta 6.1 peptides, we injected 11 multiple sclerosis patients with varying doses of two synthetic peptides, TCR V beta 5.2(39-59) and V beta 6.1(39-59), encompassing the CDR2 region of these V gene families. Low doses (100 to 300 micrograms) of peptide induced T cell immunity in 7 of 11 patients to one or both peptides. Delayed type hypersensitivity skin responses to the peptides were observed in three of seven responders, and TCR peptide-specific Ab occurred in two of seven T cell responders. Low doses of TCR peptides produced no side effects and did not cause broad spectrum immunosuppression. Synthetic TCR V region peptides can induce T cell immunity safely in humans and may prove useful in treating human autoimmune diseases.
Asunto(s)
Esclerosis Múltiple/inmunología , Esclerosis Múltiple/terapia , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Adulto , Anciano , Secuencia de Aminoácidos , Epítopos/genética , Femenino , Humanos , Hipersensibilidad Tardía , Inmunización , Técnicas Inmunológicas , Inmunoterapia , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunologíaRESUMEN
The biased expression of V beta 5.2 and V beta 6.1 by T cells specific for myelin basic protein (BP) has led to our use of TCR peptides from these V gene sequences to induce anti-TCR immunity in patients with multiple sclerosis (MS). Injection of V beta 5.2-39-59 or V beta 6.1-39-59 peptides significantly increased the peptide specific T cell frequency in 7 of 11 MS patients, often with an accompanying delayed hypersensitivity reaction at the injection site. Here, we validate these cellular immune responses by characterizing TCR peptide specific T cells from an MS patient with biased V beta 5.2 expression in BP reactive T cells before treatment with TCR peptides, and from two MS patients in whom the frequencies of anti-TCR peptide specific T cells were significantly boosted after injection with low doses of TCR peptides. In both cases, T cell lines were established with relative ease, especially after boosting with the peptides. A V beta 5.2-39-59 reactive line responded selectively to the boosting peptide and was restricted by both MHC class I (HLA-B7) and MHC class II (HLA-DR2) molecules. Characterization of 22 clonal isolates revealed that the responding T cells were predominantly activated CD4+CD8lo, circulating memory cells restricted by either HLA-B7 or HLA-DR2, that utilized mainly V beta 4, V beta 6, V beta 12, and V beta 14, but not V beta 5.2 in their TCR. T cell isolates specific for V beta 6.1-39-59 possessed similar characteristics but contained specificities cross-reactive with an N-terminal sequence on V beta 5.2-39-59. Upon stimulation with peptide or Con A, the TCR peptide specific T cell lines had increased message production for IFN-gamma, GM-CSF, IL-4, IL-5, and to a lesser degree, IL-2. This lymphokine mRNA profile differed from a BP-specific T cell line that produced message for IFN-gamma and GM-CSF but low or absent levels of IL-4 and IL-5. The extensive parallels between human T cells specific for V beta 5.2 and V beta 6.1 CDR2 peptides and rat T cells specific for V beta 8.2 CDR2 peptide that are highly protective against experimental encephalomyelitis strengthen the rationale for the therapeutic use of TCR peptides in human autoimmunity.
Asunto(s)
Esclerosis Múltiple/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Adulto , Anciano , Secuencia de Aminoácidos , Secuencia de Bases , Línea Celular , Cartilla de ADN/genética , Femenino , Expresión Génica , Humanos , Inmunoterapia , Linfocinas/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Esclerosis Múltiple/terapia , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/uso terapéutico , ARN Mensajero/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunologíaRESUMEN
Because of its proximity to the central nervous system, the cerebrospinal fluid (CSF) represents an important source of T cells that potentially could mediate putative autoimmune diseases such as multiple sclerosis (MS). To overcome the low CSF cellularity, we evaluated culture conditions that could expand CSF T cells, with a focus on the expression of T-cell receptor V beta genes utilized by T cells specific for the potentially encephalitogenic autoantigen myelin basic protein (BP). Expansion of "activated" CSF cells with IL-2/IL-4 plus accessory cells optimally retained BP-responsive T cells that over-expressed V beta 1, V beta 2, V beta 5, or V beta 18, compared to expansion using supernatants from PHA-stimulated blood cells, or anti-CD3 antibody that led to different V gene bias and rare reactivity to BP. Sequential evaluation of paired CSF and blood samples from a relapsing remitting MS patient indicated that BP-reactive T cells were present in CSF during the period of clinical activity, and the pattern of BP recognition in CSF was partially reflected in blood, even after CSF reactivity had dissipated during remission. Over-expressed V beta genes were not always constant, however, since in three sequential evaluations of a chronic progressive MS patient, V beta genes over-expressed in the first BP-reactive CSF switched to a different V beta gene bias that was present in the second and third CSF samples. Blood samples reflected each pattern of CSF V beta gene bias, but retained the initial bias for at least 4 months after its disappearance from CSF. These data indicate that selective expansion of IL-2/IL-4-responsive CSF cells favors growth of the BP-reactive subpopulation, and, in a limited number of patients studied, reflected clinical disease activity. In comparison, blood T cells provided a partial but longer lasting reflection of the CSF BP reactivity and V beta gene bias.
Asunto(s)
Esclerosis Múltiple/genética , Proteína Básica de Mielina/metabolismo , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Adulto , Anciano , Secuencia de Bases , División Celular/fisiología , Enfermedad Crónica , Femenino , Regulación de la Expresión Génica , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Esclerosis Múltiple/sangre , Esclerosis Múltiple/líquido cefalorraquídeo , Fenotipo , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T alfa-beta/genética , RecurrenciaRESUMEN
T cells made CD4- by ganglioside (GM1) treatment were cultured in antisense oligodeoxynucleotides complementary to mRNA for CD4. Antisense treatments decreased CD4 levels by 45% but did not prolong the total CD4 modulation by GM1 pretreatment. Northern blot analysis demonstrated that CD4 message production did not change after ganglioside modulation and consequently that it was different from antisense CD4 modulation. However, modulation of CD4 from the cell surface by either GM1 or antisense resulted in greater proliferation and enhanced DTH when challenged with recall antigen. These results demonstrate that selective decrease of CD4 increased antigen-specific T cell responses.
Asunto(s)
Antígenos CD4/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Animales , Antígenos , Secuencia de Bases , Antígenos CD4/genética , Línea Celular , Gangliósido G(M1)/farmacología , Hipersensibilidad Tardía , Inmunoterapia Adoptiva , Técnicas In Vitro , Activación de Linfocitos , Ratones , Datos de Secuencia Molecular , Oligonucleótidos Antisentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas Lew , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Tuberculina/inmunologíaRESUMEN
Ganglioside (GM1) modulation of CD4 off the surface of T lymphocytes defined functions of the CD4 molecule during signal transduction through the T cell receptor (TCR)/CD3 complex. Antibody cross-linking of CD3 alone (3 x 3) stimulated phospholipase C (PLC) activity, rapid Ca2+ flux, and protein phosphorylations in freshly isolated human T lymphocytes. Antibody cross-linking of CD4 and CD3 (3 x 4) stimulated greater signaling than that caused by 3 x 3. Cross-linking CD4 alone did not stimulate these signaling processes. GM1-modulation of CD4 from the cell surface blocked all aspects of the augmented signaling imparted by CD4 co-modulation with CD3. In comparison, pretreatment with the protein tyrosine kinase inhibitor genistein inhibited 3 x 4-stimulated PLC activity and protein phosphorylation but not Ca2+ flux. Antibody cross-linking of the tyrosine phosphatase CD45 with 3 x 4 (3 x 4 x 45) also inhibited CD4-augmented phosphorylations and like genistein did not reduce Ca2+ levels. In conclusion, these data demonstrate that CD4 can augment signal transduction through the TCR/CD3 complex by its physical proximity to CD3. TCR/CD3-signaling augmentation by CD4 stimulated protein tyrosine kinases and PLC activities but stimulated intracellular Ca2+ flux through an independent mechanism(s).
Asunto(s)
Antígenos CD4/fisiología , Linfocitos T CD4-Positivos/inmunología , Gangliósido G(M1)/fisiología , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Transducción de Señal , Calcio/metabolismo , Activación Enzimática , Gangliósido G(M1)/farmacología , Genisteína , Isoflavonas/farmacología , Activación de Linfocitos/fisiología , Muromonab-CD3/metabolismo , Fosfatidilinositol Diacilglicerol-Liasa , Hidrolasas Diéster Fosfóricas/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/fisiologíaRESUMEN
The development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats is mediated by V beta 8.2+ T cells specific for myelin basic protein. One consequence of this biased expression of V beta 8.2 is the spontaneous development of regulatory T cells and antibodies against residues 39-59 of the V beta 8.2 sequence. Moreover, a synthetic V beta 8.2-39-59 peptide could induce protection against and speed recovery from EAE. T cells and antibodies specific for V beta 8.2-39-59 could transfer protection from EAE. Recently, we reported that the protective T cell epitope is subsumed within the V beta 8-44-54 sequence. We now report that protection induced by V beta 8-44-54 lasted at least 102 days and produced "split tolerance," enhancing anti-myelin basic protein antibody titers but reducing anti-myelin basic protein T cell frequency. The shorter V beta 8-44-54 peptide induced a distinct set of antibodies that did not cross-react with the longer V beta 8.2-39-59 peptide, although both specificities could stain V beta 8.2+ T cells and were equally protective against EAE. However, the V beta 8.2-39-59 peptide, but not the V beta 8-44-54 peptide, would appear to represent the natural idiotope: antibodies to V beta 8.2-39-59 that develop spontaneously during EAE could be boosted to higher titers only by the V beta 8.2-39-59, but not by other TCR peptides from the V beta 8.2 sequence, including V beta 8-44-54 that contains the functional T cell epitope. These results suggest that natural processing of the TCR V beta-chain favors the formation of a peptide that resembles the V beta 8.2-39-59 sequence. The B cell epitope present on the V beta 8-44-54 sequence was evident only in the absence of residues 39-43 and 55-59, suggesting that the two peptides possess distinct conformations. However, the V beta 8-44-54 B cell epitope is most likely expressed on the V beta 8.2+ T cells, either as a low affinity determinant on the intact TCR alpha/beta heterodimer or as a cryptic epitope bound in the cleft of surface MHC molecules.
Asunto(s)
Autoinmunidad , Encefalomielitis Autoinmune Experimental/inmunología , Idiotipos de Inmunoglobulinas/análisis , Fragmentos de Péptidos/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/inmunología , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Reacciones Cruzadas , Encefalomielitis Autoinmune Experimental/etiología , Inmunización , Masculino , Ratones , Datos de Secuencia Molecular , Ratas , Ratas Endogámicas LewRESUMEN
Transforming growth factor-beta (TGF-beta) had a profound effect on the in vitro phenotypic development of Ag-activated Th cells and enhanced the in vivo effector function of these cells upon adoptive transfer. Previous studies have shown that there are two types of Th cell populations found in unimmunized animals, naive helper cells, which are short-lived and express low levels of CD44 and high levels of CD45R and Mel-14, and memory helper cells, which have a long life span and express high levels of CD44 and low levels of CD45R and Mel-14. Culturing of Ag-specific murine Th cell lines and clones in the presence of TGF-beta greatly enhanced both the memory phenotype of the cultured cells and the effector function upon adoptive transfer in experimental autoimmune encephalomyelitis. Histologic evaluation of spinal cords from recipients receiving passively transferred murine T cell lines cultured with TGF-beta revealed large demyelinated plaques (multiple sclerosis-like) that were not present in animals receiving cells cultured with Ag alone. TGF-beta also enhanced the capability of myelin basic protein-specific Lewis rat T cell lines to transfer experimental autoimmune encephalomyelitis and potentiated a purified protein derivative-specific rat helper cell line to transfer delayed type hypersensitivity. Thus, the effects of TGF-beta did not appear to be limited by species specificity, Ag specificity, or in vivo T cell function. This is the first study showing that TGF-beta can potentiate the development and maintenance of the Th cell memory phenotype in vitro and enhance their in vivo effector function in an animal disease model.
Asunto(s)
Encefalomielitis Autoinmune Experimental/inmunología , Memoria Inmunológica/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacología , Animales , Antígenos CD/análisis , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/patología , Femenino , Antígenos de Histocompatibilidad/análisis , Hipersensibilidad Tardía , Inmunoterapia Adoptiva , Antígenos Comunes de Leucocito , Ratones , Fenotipo , Ratas , Ratas Endogámicas Lew , Médula Espinal/patología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
In previous studies (Morrison et al., 1990), we showed that ganglioside (GM1) modulation of CD4 was associated with activation of phospholipase C and increased production of inositol triphosphate, but not with activation of protein kinase C. These results demonstrated a unique signal transduction pathway related to GM1 modulation of CD4 on T cells and raised the question as to whether intracellular Ca2+ levels and related protein kinases would be affected by GM1-induced signalling. We now show that GM1 modulation of CD4 from human T cells corresponds to decreased cellular Ca2+ without significant changes in cellular protein phosphorylation. In the course of this study we discovered that T cells challenged with GM1 exhibited new proteins in their surrounding media. Fractionation of cellular and supernatant proteins show that cells treated with GM1 released proteins with an approximate molecular weight (Mr) of 49,000. This was exclusive of GM1 protein association and GM1-induced protein phosphorylation. Immunoblots demonstrated the presence of CD4 in GM1-treated cell supernatants. Western immunoblots using anti-CD4 antibodies detected a lower Mr form (49,000) of CD4 in the supernatants of GM1-treated cells. These studies further define the unique nature of GM1 signalling relating to modulation of CD4 and demonstrate that the fate of GM1 modulated CD4, in part, involves protein shedding.
Asunto(s)
Antígenos CD4/metabolismo , Gangliósido G(M1)/farmacología , Linfocitos T/efectos de los fármacos , Antígenos CD4/aislamiento & purificación , Calcio/metabolismo , Células Cultivadas , Humanos , Immunoblotting , Técnicas In Vitro , Peso Molecular , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
T cell lines selected from Lewis rats recovered from experimental autoimmune encephalomyelitis (EAE) respond not only to the immunodominant 72-89 epitope of basic protein (BP), but also to secondary epitopes including the I-A restricted 43-67 region of guinea pig (Gp) BP and the I-E restricted 87-99 sequence of rat (Rt) BP. The current study demonstrates at the clonal level the diversity of T cell responses to Gp- and Rt-BP in EAE-recovered rats. As predicted from the response pattern of BP-selected T cell lines, T cell clones from the lines responded to both the dominant and secondary epitopes of BP. In addition, a new majority clonal type was identified that responded to whole BP but not to epitopes represented on enzymatic cleavage fragments or synthetic peptides spanning the BP molecule. Clones representative of each of the three types of Gp-BP responses were characterized for phenotype, major histocompatibility complex restriction, and biologic activity in vivo. All of the clones were strongly CD4+ and co-expressed CD8 at modest levels as measured by both immunofluorescence and Northern blots. All three T cell specificities were I-A restricted. However, only the 72-89 responsive clone could transfer clinical EAE, due most likely to its unique ability to respond to Rt-BP. In contrast, the Gp-BP 43-67 reactive T cell clone transferred protection against EAE, whereas the whole Gp-BP reactive clone transferred delayed-type hypersensitivity response but was neither encephalitogenic nor protective. Thus, the recovery process from EAE is distinguished by an increased diversity of protective clones as well as innocuous clones that may be spawned as encephalitogenic T cells are regulated.
Asunto(s)
Enfermedades Autoinmunes/inmunología , Linfocitos T CD4-Positivos/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Proteína Básica de Mielina/inmunología , Ratas Endogámicas Lew/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD/análisis , Linfocitos T CD4-Positivos/fisiología , Células Clonales , Cobayas , Ratas , Linfocitos T/fisiología , Linfocitos T Reguladores/fisiologíaRESUMEN
The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti-idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope.
Asunto(s)
Encefalomielitis Autoinmune Experimental/prevención & control , Epítopos , Homeostasis , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Animales , Células Clonales , Reacciones Cruzadas , Encefalomielitis Autoinmune Experimental/patología , Femenino , Expresión Génica , Cobayas , Hipersensibilidad Tardía , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Ratas , Ratas Endogámicas Lew , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
Ganglioside (GM1) treatment of CD4+ human CEM lymphoma cells stimulated transient phosphoinositide (PI) breakdown, production of inositol phosphates (IP), protein phosphorylation and rapid decrease of CD4 surface expression. A comparison between the actions of GM1 and other agents that affect these signal transduction pathways demonstrated a distinct mechanism for GM1-induced decrease of CD4. GM1 stimulated both phospholipase C activity and protein phosphorylation but had no effect on either cellular cAMP levels or tyrosine kinase activity. Phorbol myristate acetate (PMA) stimulated protein phosphorylation and caused a significant decrease in surface display of CD4. Both of these processes were blocked by pretreating cells with the protein kinase C (PKC) inhibitor H7. These results demonstrate that GM1 stimulates PI turnover and induces a rapid decrease of CD4 surface expression by processes that do not activate adenylate cyclase or tyrosine kinase. They further demonstrate that the mechanism for GM1-induced decrease of CD4 is distinct from the CD4 internalization processes mediated by PKC activity.
Asunto(s)
Antígenos CD4/metabolismo , Gangliósido G(M1)/farmacología , Transducción de Señal/inmunología , Adenilil Ciclasas/metabolismo , Humanos , Hidrólisis , Fosfatos de Inositol/biosíntesis , Fosfatidilinositoles/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/inmunología , Células Tumorales Cultivadas/metabolismoRESUMEN
Recent studies from our laboratory have shown, for the first time, that a synthetic peptide from that TCR VB chain used preferentially by encephalitogenic T cells induced the formation of protective, MHC class I-restricted T cells and prevented the development of EAE in Lewis rats. In this report we 1) demonstrate that immunization with the TCR-VB8-39-59 peptide generated peptide-specific antibodies that protect against experimental autoimmune encephalomyelitis induced by either of the two distinct encephalitogenic epitopes of basic protein, and 2) characterize the production and biologic functions of rat and rabbit antibody responses to the TCR peptide. The antibodies in both species increased in titer over time, were highly specific for the immunogen by direct reaction and inhibition assays, stained only VB8+ T cells, and suppressed clinical signs and to lesser extent the number of histologic lesions of experimental autoimmune encephalomyelitis mediated by VB8+ T cells. Coupled with our previous work, these results indicate that both humoral and cellular responses to the TCR-VB8-39-59 peptide can contribute independent immunoregulatory effects on encephalitogenic T lymphocytes that use common V region genes in response to epitopes of myelin basic protein.
Asunto(s)
Encefalomielitis Autoinmune Experimental/prevención & control , Proteína Básica de Mielina/inmunología , Fragmentos de Péptidos/inmunología , Péptidos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , Encefalomielitis Autoinmune Experimental/inmunología , Datos de Secuencia Molecular , Péptidos/síntesis química , Ratas , Ratas Endogámicas Lew , Receptores de Antígenos de Linfocitos T alfa-betaRESUMEN
The objective of this study was to establish whether binding of platelet activating factor (PAF) to its receptor was integral to the stimulation of phosphoinositide-specific phospholipase C (PLC) in rabbit platelets. Saturation binding curves for [3H]PAF indicated that the PAF receptor has a dissociation constant (KD) of 28.72 nM. In comparison, PAF-stimulated PLC activity, as monitored by [3H]inositol triphosphate production, increased at lower concentrations and had an half-maximal effective concentration (EC50) value of 1.5 nM. Unlabeled PAF inhibited [3H]PAF binding competitively and demonstrated two binding sites, a high affinity site with an inhibitory constant (Ki) of 2.65 nM and a low affinity site with a Ki of 0.80 microM. The inhibitory effects of four PAF antagonists, CV-3988, CV-6209, SRI 63-441 and SRI 63-675 on the binding of [3H]PAF were compared to the effects of the antagonists on PAF-stimulated PLC activity. The four antagonists inhibited [3H]PAF binding almost completely whereas their ability to inhibit PAF-stimulated PLC activity varied. CV-3988, SRI 63-441 and SRI 63-675 had half-maximal inhibitory concentration (IC50) values of 0.28, 0.78 and 0.42 microM, respectively, whereas CV-6209 was more potent at inhibiting [3H]PAF binding (IC50 = 7.73 nM). The SRI 63-441 and SRI 63-675 inhibited PLC totally with an IC50 value of 0.78 and 1.27 microM, respectively. The CV-3988 and CV-6209 showed a maximal PLC inhibition of about 45% with "apparent IC50" values of 1.05 and 0.17 microM, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Plaquetas/fisiología , Fosfatidilinositoles/metabolismo , Factor de Activación Plaquetaria/fisiología , Glicoproteínas de Membrana Plaquetaria , Receptores de Superficie Celular/efectos de los fármacos , Receptores Acoplados a Proteínas G , Fosfolipasas de Tipo C/antagonistas & inhibidores , Animales , Plaquetas/enzimología , Activación Enzimática/efectos de los fármacos , Técnicas In Vitro , Éteres Fosfolípidos/farmacología , Compuestos de Piridinio/farmacología , Quinolinas/farmacología , Compuestos de Quinolinio/farmacología , ConejosRESUMEN
Human platelet concentrates were stored in polyolefin bags at 22 to 24 degrees C on a horizontal shaker for up to 8 days. At different intervals, aliquots of platelet-rich plasma (PRP) were removed aseptically and five variables, i.e., platelet counts, morphology, platelet-activating factor (PAF)-stimulated aggregation, phosphoinositide turnover, and [3H]PAF binding to platelet receptors, were studied. The number of platelets did not change during the 8 days of storage. Scanning electron microscopy of the platelets revealed a gradual morphologic change from biconcave flat discs to irregular, crenated forms. The PAF-induced aggregation of platelets declined with time of storage. A decrease to 50 percent of the Day 1 aggregatory response to PAF was evident on Day 2, and there was a further decline to about 20 percent by Day 6. Similarly, PAF receptor-coupled phosphoinositide turnover, as monitored by 32P incorporation into individual phosphoinositides, decreased dramatically with storage. After 2 to 3 days of storage, the phosphoinositide turnover was reduced to 50 percent of the original response, and it continued to decline to about 25 percent of original response by Day 5 or 6. The binding of [3H]PAF to washed human platelets indicated subtle changes between Days 2 and 4, which became more noticeable by Day 6. These results have raised the possibility of changes in the number of the receptors and/or their affinity for the ligand during storage. We conclude that although the number of platelets was maintained during storage for 8 days, a general deterioration of their responses to PAF occurred at the levels of cell surface receptor, transmembrane signaling (phosphoinositide turnover), and response (aggregation).
Asunto(s)
Plaquetas/fisiología , Conservación de la Sangre , Fosfatidilinositoles/sangre , Factor de Activación Plaquetaria/farmacología , Agregación Plaquetaria , Glicoproteínas de Membrana Plaquetaria , Receptores de Superficie Celular/análisis , Receptores Acoplados a Proteínas G , Unión Competitiva , Plaquetas/metabolismo , Plaquetas/ultraestructura , Conservación de la Sangre/métodos , Supervivencia Celular , Humanos , Microscopía Electrónica de Rastreo , Plasma , Factor de Activación Plaquetaria/metabolismo , Recuento de Plaquetas , TritioRESUMEN
Treatment of 32P-labeled rabbit platelets with platelet-activating factor (PAF) caused a time- and dose-dependent phosphorylation of several proteins including five major phosphorylated proteins with apparent molecular weights of 20,000, 35,000, 40,000, 65,000, and 150,000. Both PAF and thrombin caused a rapid increase followed by a decrease in phosphorylation of proteins, indicating the occurrence of a phosphorylation-dephosphorylation process. Four separate PAF receptor antagonists, CV-3988, CV-6209, SRI-63-441, and SRI-63-675 drastically reduced the PAF-stimulated protein phosphorylation. The order of potency was SRI-63675 greater than SRI-63441 greater than or equal to CV-6209 greater than CV-3988. These antagonists had no effect on thrombin-stimulated protein phosphorylation. Pretreatment of platelets with PAF (0.1 nM) completely abolished any further protein phosphorylation by the same concentration of PAF. PAF pretreatment shifted the dose response of protein phosphorylation by about 2 log units, to the right. When platelets were treated with PAF (10 nM) for 10 min, this abolished phosphorylation of proteins by any concentration of PAF. These studies indicated a homologous desensitization of protein phosphorylation. Interestingly, PAF-pretreated platelets still exhibited phosphorylation of proteins by thrombin. On the other hand, a lack of protein phosphorylation by PAF or thrombin was observed in platelets preexposed to thrombin and this demonstrated a heterologous desensitization. It is concluded that phosphorylation of proteins by PAF is a PAF receptor-coupled event and that this process is desensitized in platelets preexposed to PAF. The fact that both the activation of phosphoinositide-specific phospholipase C and the phosphorylation of proteins are desensitized in PAF-pretreated platelets suggests that a close "regulatory" intercommunication between these processes exists.