RESUMEN
NZB mice manifest a defect in tolerance induction by deaggregated heterologous gamma globulins. We have used an adoptive transfer system to study the defect. Thymectomized, intact, or thymectomized recipients given thymic epithelial grafts were studied after lethal irradiation and reconstitution with NZB, DBA/2, or (NZB x DBA(F1 marrow depleted of mature T cells. NZB thymocytes were responsible for the tolerance defect of NZB mice. The information for the defect was present in the NZB marrow prethymocyte. That defect could only be expressed when there was further maturation in association with a thymus. However, the normal DBA/2 thymic epithelium served as well as the abnormal NZB thymic epithelium. These studies resolve existing conflicts as to whether the NZB marrow or thymus is responsible for the loss of tolerance in association with autoimmunity.
Asunto(s)
Tolerancia Inmunológica , Linfocitos T/citología , Animales , Médula Ósea/inmunología , Células de la Médula Ósea , Diferenciación Celular , Células Epiteliales , Epitelio/inmunología , Femenino , Células Madre Hematopoyéticas/inmunología , Masculino , Ratones , Ratones Endogámicos DBA , Ratones Endogámicos NZB , Linfocitos T/inmunología , Timo/citología , Timo/inmunologíaRESUMEN
A linkage map of distal mouse chromosome 1 was generated using restriction fragment length polymorphism (RFLP) analysis of DNA prepared from 95 [C3H-gld/gld X Mus spretus)F1 X C3H-gld/gld] backcross mice. The gene order was: (centromere) C4bp, Ren-1,2, Ly-5, [At-3/gld], Apoa-2/Ly-17, Spna-1 (telomere). All mice expressing the phenotype of gld homozygotes were homozygous for the At-3 RFLP characteristic of C3H mice and none of the mice heterozygous for At-3 RFLPs had characteristics of gld homozygotes, demonstrating close linkage between these genes. The identification of an RFLP closely linked to the gld gene provides a starting point for the identification of a genetic defect that results in abnormal T cells and autoimmune disease.
Asunto(s)
Enfermedades Autoinmunes/genética , Ligamiento Genético , Trastornos Linfoproliferativos/genética , Mutación , Polimorfismo Genético , Polimorfismo de Longitud del Fragmento de Restricción , Animales , Mapeo Cromosómico , Cruzamientos Genéticos , Enzimas de Restricción del ADN , Femenino , Genes Recesivos , Masculino , Ratones , Ratones Endogámicos C3H , Fenotipo , Recombinación GenéticaRESUMEN
Sarcolemmal membrane vesicles isolated from canine ventricular tissue accumulate calcium through the sodium-calcium exchange system when an outwardly directed sodium gradient is generated across the vesicle membrane. Moreover, calcium uptake under these conditions is accompanied by the transient accumulation of the lipophilic cation tetraphenylphosphonium. Since the distribution of tetraphenylphosphonium across biological membranes reflects the magnitude and direction of transmembrane potential differences and the characteristics of the transient accumulation of this cation closely resemble those of sodium-calcium exchange activity, it is concluded that a membrane potential, interior negative, is produced during calcium accumulation through the exchange system. Thus, the operation of the sodium-calcium exchange system generates a current in cardiac membrane vesicles, suggesting that three or more sodium ions exchange for each calcium ion.
Asunto(s)
Calcio/metabolismo , Membrana Celular/metabolismo , Corazón/fisiología , Miocardio/metabolismo , Compuestos Organofosforados , Sodio/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Membrana Celular/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Compuestos Onio/metabolismo , Valinomicina/farmacologíaRESUMEN
A possible direct effect of guanine nucleotide binding (G) proteins on calcium channels was examined in membrane patches excised from guinea pig cardiac myocytes and bovine cardiac sarcolemmal vesicles incorporated into planar lipid bilayers. The guanosine triphosphate analog, GTP gamma S, prolonged the survival of excised calcium channels independently of the presence of adenosine 3',5'-monophosphate (cAMP), adenosine triphosphate, cAMP-activated protein kinase, and the protein kinase C activator tetradecanoyl phorbol acetate. A specific G protein, activated Gs, or its alpha subunit, purified from the plasma membranes of human erythrocytes, prolonged the survival of excised channels and stimulated the activity of incorporated channels. Thus, in addition to regulating calcium channels indirectly through activation of cytoplasmic kinases, G proteins can regulate calcium channels directly. Since they also directly regulate a subset of potassium channels, G proteins are now known to directly gate two classes of membrane ion channels.
Asunto(s)
Proteínas de Unión al GTP/fisiología , Corazón/fisiología , Canales Iónicos/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico/farmacología , Animales , Calcio/metabolismo , Colforsina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato) , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacología , Cobayas , Canales Iónicos/efectos de los fármacos , Isoproterenol/farmacología , Leupeptinas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Fosforilación , Tionucleótidos/farmacología , Función VentricularRESUMEN
Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics.
Asunto(s)
Calcio/fisiología , Canales Iónicos/fisiología , Ácido 3-piridinacarboxílico, 1,4-dihidro-2,6-dimetil-5-nitro-4-(2-(trifluorometil)fenil)-, Éster Metílico , Animales , Bovinos , Conductividad Eléctrica , Corazón/fisiología , Técnicas In Vitro , Membrana Dobles de Lípidos , Ácidos Nicotínicos/farmacología , Nifedipino/análogos & derivados , Nifedipino/farmacología , Nimodipina , Potasio/fisiología , Sarcolema , Sodio/fisiologíaRESUMEN
Patients with systemic lupus erythematosus (SLE) produce excessive amounts of autoantibodies. It has also been demonstrated in several systems that such patients have a relative loss of suppressor thymus-derived (T) cells that inhibit the immune response. This loss of suppressor cells has been suggested as one of the causes of the excessive production of antibodies in patients with SLE. In the present report we have tested the hypothesis that anti-T-cell antibodies found in the plasma of some patients with SLE preferentially kill suppressor cells. T cells from normal individuals can be activated by concanavalin A to develop suppressor cell activity. We therefore cultured normal T cells together with concanavalin A in the presence of plasma or plasma fractions from patients with SLE. We found that plasma from patients with active SLE, in which anti-T-cell antibodies were present, inhibited the development of suppressor activity in such cultures. In contrast, plasma from other active patients and patients with inactive SLE, in which no anti-T-cell antibodies could be detected, failed to block the development of such suppressor activity. Absorption of the plasma that contained anti-T-cell antibodies with T cell, but not non-T cells, could eliminate the suppressor-inhibiting activity of the SLE plasma that contained anti-T-cell antibodies. The immunoglobulin (Ig)M, but not the IgG, fraction of the plasma was shown to possess the inhibiting property and complement was found to be necessary for the effect of such anti-T-cell antibodies. We also demonstrated that exposure of normal T cells to such anti-T-cell antibodies and complement did not affect another population of T cells that could proliferate in response to mitogens.Thus, certain patients with SLE have in their plasma an antibody of the IgM class that can selectively eliminate a population of T cells capable of developing suppressor function. The loss of suppressor T cells in patients with SLE may be the result of the effects of such antibody activity in vivo.
Asunto(s)
Autoanticuerpos/inmunología , Lupus Eritematoso Sistémico/inmunología , Linfocitos T/inmunología , Adulto , Proteínas del Sistema Complemento/inmunología , Concanavalina A/farmacología , Humanos , Inmunoglobulina M/inmunología , Lupus Eritematoso Sistémico/tratamiento farmacológico , Activación de Linfocitos/efectos de los fármacosRESUMEN
Antibodies to T cells present in the plasma of patients with active systemic lupus erythematosus (SLE) plus complement are able to eliminate concanavalin A-induced suppressor function for the proliferative responses of T cells to allogeneic lymphocytes (MLR) and of B cells to pokeweed mitogen (PWM). Such antibodies were found to be effective in eliminating suppressor function only when T cells were treated before activation; there was no effect when treatment was performed after activation. These studies indicate that the antibodies preferentially interact with a T cell necessary for the generation of suppressor cells, rather than with mature, activated suppressor cells. Studies of individual SLE patients indicate that the same defects observed in SLE T cells were induced in normal T cells by plasma from that patient. Such observations suggest that many T-cell defects associated with active SLE may not be intrinsic T-cell abnormalities, but, rather, secondary effects of anti-T-cell antibodies. Studies of the T-cell subpopulations responsible for suppression of the MLR and PWM responses indicate that only T gamma cells (T cells bearing receptors for the Fc portion of immunoglobulin [Ig]G) acted as precursors of suppressor cells for the MLR, whereas both T gamma and T non-gamma cells (T cells not bearing receptors for the Fc portion of IgG) could be activated to suppress the PWM response. Consistent with this observation, SLE anti-T-cell antibodies that preferentially killed T gamma cells preferentially eliminated suppressor cells for the MLR.
Asunto(s)
Autoanticuerpos/inmunología , Enfermedades Autoinmunes/inmunología , Lupus Eritematoso Sistémico/inmunología , Linfocitos T Reguladores/inmunología , Células Cultivadas , Concanavalina A/farmacología , Humanos , Inmunoglobulina G/inmunología , Mitógenos de Phytolacca americana/farmacología , Receptores de Concanavalina A/inmunología , Receptores Fc/inmunología , Receptores Mitogénicos/inmunologíaRESUMEN
Regulation of cytosolic Ca2+ and cytosolic Na+ is critical for lymphocyte cation homeostasis and function. To examine the influence of cytosolic Na+ on Ca2+ regulation in human peripheral blood lymphocytes, Ca2+ entry and cytosolic Ca2+ (measured with fura-2) were monitored in cells in which cytosolic Na+ was increased and/or the Na+ gradient was decreased by reduction of external Na+ concentration. Ouabain-treated cells (0.1 mM for 30 min at 37 degrees C), suspended in Na(+)-free medium, showed a 30-65% increase in Ca2+ uptake compared to cells in 140 mM Na+ medium. Enhanced Ca2+ influx was entirely dependent on ouabain pretreatment and reversal of the Na+ gradient. Na pump inhibition or Na ionophore addition and subsequent exposure to Na(+)-free medium resulted in a sustained elevation of cytosolic Ca2+. As preincubation of cells in Ca(2+)-free medium further enhanced the ouabain-dependent increase in cytosolic Ca2+, the effects of the microsomal Ca(2+)-ATPase inhibitor thapsigargin on Ca2+ influx and cytosolic Ca2+ were studied. Thapsigargin stimulated Ca2+ entry following ouabain pretreatment and reversal of the Na+ gradient; the effects of thapsigargin were retained in the presence of LaCl3, a potent inhibitor of store-dependent calcium influx pathways. These results show lymphocytes demonstrate Na+/Ca2+ exchange activity and suggest the Na+/Ca2+ exchanger modulates cytosolic Ca2+ following intracellular Ca2+ store depletion.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/fisiología , Linfocitos/metabolismo , Sodio/metabolismo , Humanos , Ouabaína/farmacología , Intercambiador de Sodio-Calcio , Terpenos/farmacología , TapsigarginaRESUMEN
The Na+-Ca2+ exchange system of the sarcolemma of rat mesenteric artery was solubilized and reconstituted in soybean phospholipid vesicles. In the reconstituted system, the exchange process showed about 4-fold higher specific activity compared to that of native vesicles. The inhibitory effect of monensin and the stimulatory effect of valinomycin in the presence of K+ on Na+ gradient-dependent Ca2+ uptake were preserved and were pronounced in the reconstituted system. The stimulation by valinomycin indicates that the exchange process is electrogenic. Thus, the stoichiometry, the characteristics and the mechanism of action which were difficult to study in the native vesicles can now be determined conveniently using the reconstituted system. Also, solubilization and reconstitution of the exchange system confirms its existence in vascular smooth muscle.
Asunto(s)
Calcio/metabolismo , Músculo Liso Vascular/metabolismo , Sarcolema/metabolismo , Sodio/metabolismo , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cinética , Liposomas/metabolismo , Arterias Mesentéricas/metabolismo , Monensina/farmacología , Músculo Liso Vascular/efectos de los fármacos , Potasio/farmacología , Ratas , Solubilidad , Valinomicina/farmacologíaRESUMEN
Plasma membrane vesicles were purified from rat aortic myocytes by centrifugation in a discontinuous sucrose gradient. Vesicles were prepared in the presence or absence of five proteinase inhibitors (aprotinin, benzamidine, leupeptin, pepstatin A and phenylmethylsulfonyl fluoride). The proteinase inhibitors decreased the Vmax by 3.4-fold and had no effect on the Km for Ca2+ of Na+ gradient-dependent 45Ca2+ influx. The proteinase inhibitors had no direct effect on exchange activity, and they had no effect on membrane purity as indicated by 5'-nucleotidase activity. Removing the proteinase inhibitors or adding trypsin or chymotrypsin increased exchange activity approx. 2-fold. The Vmax of exchange activity in intact aortic myocytes is approx. 10-fold higher than the Vmax in plasma membrane vesicles prepared in the presence of proteinase inhibitors. Exchange activity in plasma membrane vesicles is only a sixtieth of the expected value, because the vesicles have approx. 7-fold higher 5'-nucleotidase activity and approx. 6-fold higher specific exchange activity than the crude homogenate. The large loss of exchange activity may be caused by a change in a regulatory domain of the exchanger because endogenous proteolysis restores some of the activity lost during vesicle preparation.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Músculo Liso Vascular/metabolismo , 5'-Nucleotidasa/metabolismo , Animales , Aorta , Membrana Celular/enzimología , Células Cultivadas/metabolismo , Cinética , Músculo Liso Vascular/enzimología , Inhibidores de Proteasas/farmacología , Ratas , Intercambiador de Sodio-Calcio , Tripsina/farmacologíaRESUMEN
The P2U purinergic agonist ATP (0.3 mM) elicited an increase in [Ca2+]i due to Ca2+ release from intracellular stores in transfected Chinese hamster ovary cells that express the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). The following observations indicate that ATP-evoked Ca2+ release was accompanied by a Ca(2+)-dependent regulatory activation of Na+/Ca2+ exchange activity: Addition of extracellular Ca2+ (0.7 mM) 0-1 min after ATP evoked a dramatic rise in [Ca2+]i in Na(+)-free media (Li+ substitution) compared to Na(+)-containing media; no differences between Na(+)- and Li(+)-based media were observed with vector-transfected cells. In the presence of physiological concentrations of extracellular Na+ and Ca2+, the ATP-evoked rise in [Ca2+]i declined more rapidly in CK1.4 cells compared to control cells, but then attained a long-lived plateau of elevated [Ca2+]i which eventually came to exceed the declining [Ca2+]i values in control cells. ATP elicited a transient acceleration of exchange-mediated Ba2+ influx, consistent with regulatory activation of the Na+/Ca2+ exchanger. The acceleration of Ba2+ influx was not observed in vector-transfected control cells, or in CK1.4 cells in the absence of intracellular Na+ or when the Ca2+ content of the intracellular stores had been reduced by prior treatment with ionomycin. The protein kinase C activator phorbol 12-myristate 13-acetate attenuated the exchange-mediated rise in [Ca2+]i under Na(+)-free conditions, but did not inhibit the ATP-evoked stimulation of Ba2+ influx. The effects of PMA are therefore not due to inhibition of exchange activity, but probably reflect the influence of protein kinase C on other Ca2+ homeostatic mechanisms. We conclude that exchange activity is accelerated during ATP-evoked Ca2+ release from intracellular stores through regulatory activation by increased [Ca2+]i. In the absence of extracellular Ca2+, the stimulation of exchange activity is short-lived and follows the time course of the [Ca2+]i transient; in the presence of extracellular Ca2+, we suggest that the exchanger remains activated for a longer period of time, thereby stabilizing and prolonging the plateau phase of store-dependent Ca2+ entry.
Asunto(s)
Adenosina Trifosfato/farmacología , Células CHO/metabolismo , Células CHO/fisiología , Calcio/metabolismo , Proteínas Portadoras/metabolismo , Transfección , Animales , Bario/metabolismo , Células CHO/efectos de los fármacos , Proteínas Portadoras/genética , Bovinos , Cricetinae , Membranas Intracelulares/metabolismo , Concentración Osmolar , Proteína Quinasa C/metabolismo , Sodio/farmacología , Intercambiador de Sodio-CalcioRESUMEN
We examined Ba2+ influx using isotopic and fura-2 techniques in transfected Chinese hamster ovary cells expressing the bovine cardiac Na+/Ca2+ exchanger (CK1.4 cells). Ba2+ competitively inhibited exchange-mediated 45Ca2+ uptake with a Ki approximately 3 mM. Ba2+ uptake was stimulated by pretreating the cells with ouabain and by removing extracellular Na+, as expected for Na+/Ba2+ exchange activity. The maximal velocity of Ba2+ accumulation was estimated to be 50% of that for Ca2+. When the monovalent cation ionophore gramicidin was used to equilibrate internal and external concentrations of Na+, Ba2+ influx was negligible in the absence of Na+ and increased to a maximum at 20-40 mM Na+. At higher Na+ concentrations, Ba2+ influx declined, presumably due to the competition between Na+ and Ba2+ for transport sites on the exchanger. Unlike Ca2+, Ba2+ did not appear to be taken up by intracellular organelles. Thus, 133Ba2+ uptake in ouabain-treated cells was not reduced by mitochondrial inhibitors such as-Cl-CCP or oligomycin-rotenone. Moreover, intracellular Ca2+ stores that had been depleted of Ca2+ by pretreatment of the cells with ionomycin (a Ca2+ ionophore) remained empty during a subsequent period of Ba2+ influx. Ca2+ uptake or release by intracellular organelles secondarily regulated exchange activity through alterations in [Ca2+]i. Exchange-mediated Ba2+ influx was inhibited when cytosolic [Ca2+] was reduced to 20 nM or less and was accelerated at cytosolic Ca2+ concentrations of 25-50 nM We conclude that (a) Ba2+ substitutes for Ca2+ as a transport substrate for the exchanger, (b) cytosolic Ba2+ does not appear to be sequestered by intracellular organelles, and (c) exchange-mediated Ba2+ influx is accelerated by low concentrations of cytosolic Ca2+.
Asunto(s)
Bario/metabolismo , Células CHO/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Miocardio/metabolismo , Transfección , Animales , Células CHO/fisiología , Bovinos , Cricetinae , Membranas Intracelulares/metabolismo , Orgánulos/metabolismo , Concentración Osmolar , Sodio/metabolismo , Intercambiador de Sodio-CalcioRESUMEN
Recombinant proteins have been proposed as subunit vaccines for many viral, bacterial and parasitic diseases, to reduce adverse side effects associated with inactivated or attenuated vaccines. Yet little is known about the comparative immunogenicity of recombinant proteins vs native forms present in cells or on organisms, and little is known about comparisons of the specificities of such immune responses. In another observation about differing forms of an antigen, about 10% of AIDS patients have anti-CD4 autoantibodies recognizing sites seen in recombinant CD4 (rCD4) but not present on cell surface CD4. We have analyzed antibody responses of mice to human CD4 when presented in recombinant or in cellular form. The response to the whole molecule was examined, as well as the responses to two sites within the molecule. In addition, any effect of immune response genes in the responding animal was sought, which might potentially restrict or modify any response to CD4. Mice immunized with rCD4 generated a large response to rCD4, but a lower response to the cell surface form, implying that additional sites are recognized on the recombinant form that are not recognized in the cellular form. Mice immunized with cells containing surface CD4 had high titers of antibody reactive with whole cells, of which only a small portion was reactive with rCD4. Titers on rCD4 are much lower for these mice than in rCD4-immunized mice. Both forms of CD4 induced antibodies to the gp120 binding site with comparable efficiency. For another site in domain 3 or 4 of CD4, cellular CD4 induced antibodies more frequently than the recombinant form. Immune response gene differences did not play a detectable role in the anti-CD4 response.
Asunto(s)
Antígenos CD4/inmunología , Proteínas Recombinantes/inmunología , Animales , Formación de Anticuerpos , Especificidad de Anticuerpos , Antígenos de Superficie/inmunología , Antígenos CD4/química , Epítopos , Femenino , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Ratones , Ratones Endogámicos ARESUMEN
The objective of this study was to assess the contribution of Na+-Ca2+ exchange activity to Ca2+ efflux at various cytosolic Ca2+ concentrations ([Ca2+]i) in transfected Chinese hamster cells expressing the bovine cardiac Na+-Ca2+ exchanger. Ionomycin was added to fura-2 loaded cells and the resulting [Ca2+]i transient was monitored in Ca2+-free media with or without extracellular Na+. The presence of Na+ reduced both the amplitude and duration of the [Ca2+]i transient. Na+ had similar effects when the peak of the [Ca2+]i transient was buffered to 100 nM by cytosolic EGTA, or when Ca2+ was slowly released from internal stores with thapsigargin. Ca2+ efflux following ionomycin addition was directly measured with extracellular fura-2 and followed a biphasic time course (t(1/2) approximately = 10 s and 90s). The proportion of total efflux owing to the rapid phase was increased by Na+ and reduced by EGTA-loading. Na+ accelerated the initial rate of Ca2+ efflux by 65% in unloaded cells but only by 16% in EGTA-loaded cells. In both cases, the stimulation by Na+ was less than expected, given the pronounced effects of Na+ on the [Ca2+]i transient. We conclude that the exchanger contributes importantly to Ca2+ efflux activity at all [Ca2+]i values above 40 nM. We also suggest that Ca2+ efflux pathways may involve non-cytosolic or local routes of Ca2+ traffic.
Asunto(s)
Calcio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Animales , Transporte Biológico , Células CHO/efectos de los fármacos , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Bovinos , Cricetinae , Cricetulus , Ácido Egtácico/metabolismo , Inhibidores Enzimáticos/farmacología , Colorantes Fluorescentes/metabolismo , Fura-2/metabolismo , Ionomicina/farmacología , Ionóforos/farmacología , Sodio/metabolismo , Intercambiador de Sodio-Calcio/efectos de los fármacos , Intercambiador de Sodio-Calcio/genética , Tapsigargina/farmacología , TransfecciónRESUMEN
Differences in cation transport have been observed between African Americans and whites. These differences may underlie the increased predisposition of African Americans to essential hypertension. To further explore these racial differences, we used platelets as a cellular model for calcium regulation. We measured 45Ca fluxes in platelets from 21 African American and 25 white men. Additionally, using fura 2, we measured cytosolic free calcium levels in resting platelets and platelets treated with ouabain and thrombin. Platelet 45Ca uptake was described by two exchangeable pools: a small, rapidly exchangeable pool and a larger, slowly exchangeable pool. Both pools were larger in platelets from African Americans than from whites (263 versus 185 pmol per 1 x 10(8) platelets for the rapidly exchangeable pool, P < .05; 744 versus 532 pmol per 1 x 10(8) platelets for the slowly exchangeable pool, P < .01). 45Ca washout was described by a rapidly exchangeable pool and a static pool. The former was also higher in platelets from African Americans than from whites (246 versus 202 pmol per 1 x 10(8) platelets, P < .01). The cytosolic free calcium concentrations in resting platelets were lower in African Americans than in whites. After treatment with ouabain and thrombin, the sustained posttransient levels of cytosolic free calcium increased to a greater extent in platelets from African Americans (46.7 nmol/L) than from whites (34.5 nmol/L, P = .033). Platelets from African Americans demonstrate higher intracellular calcium stores than platelets from whites. This racial difference could explain the sensitivity of African Americans to vasoactive agents acting through calcium mobilization from intracellular stores and cytosolic calcium.
Asunto(s)
Población Negra , Plaquetas/metabolismo , Calcio/sangre , Población Blanca , Adulto , Transporte Biológico , Citosol/metabolismo , Fura-2 , Homeostasis , Humanos , Masculino , Ouabaína/farmacología , Trombina/farmacologíaRESUMEN
Immunoregulatory T-cell subsets as defined by differentiation antigens were studied in 32 patients with systemic lupus erythematosus (SLE) and 16 healthy persons using the monoclonal antibodies OKT 3 or anti-Leu 4 (T cells), anti-Leu 2a (suppressor/cytotoxic cells) and anti-Leu 3a (helper/inducer cells). Compared with the 95 percent confidence limits in control subjects, decreases or increases of Leu 3a+ cells were observed in 23 patients, whereas abnormal percentages of Leu 2a+ cells were observed in only 10 patients (p less than 0.002). The ratio of Leu 3a+ to Leu 2a+ cells varied over a much broader range (0.31 to 4.14) in patients with SLE than in control subjects (95 percent confidence limit 1.04 to 2.20). Furthermore, the helper:suppressor ratio correlated significantly (p less than 0.001) with a numerical clinical characterization of the patients. A low helper: suppressor ratio was observed in patients with severe renal disease, thrombocytopenia and onset of SLE by 20 years of age. Patients with a high helper:suppressor ratio had multisystem disease including lymphadenopathy, but only rarely SLE renal disease. Patients with a normal helper:suppressor ratio had the most widespread multisystem disease, often involving the kidneys and the central nervous system. The ratio was not correlated with duration of illness, disease activity or corticosteroid dosage in the patients examined. The study suggests that SLE is not one disease entity, but rather a symptom complex with different immunoregulatory abnormalities and associated manifestations.
Asunto(s)
Lupus Eritematoso Sistémico/inmunología , Linfocitos T/inmunología , Adulto , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Femenino , Humanos , Recuento de Leucocitos , Masculino , Persona de Mediana Edad , Linfocitos T Reguladores/inmunologíaRESUMEN
Development of a vaccine for acquired immunodeficiency syndrome (AIDS) has proven difficult, and so alternative approaches such as idiotypic manipulation have been suggested. As applied to AIDS, this approach could involve immunizing with an anti-CD4 antibody resembling gp120, to induce anti-idiotypic antibodies which would bind to gp120. The CD4 binding site on gp120 is conserved, and so, such an immune response should protect against all variants. Induction of anti-human immunodeficiency virus (HIV) immunity has been reported using anti-Leu3a, and this result has led to testing in humans. Negative results obtained by others have been attributed to differences in immunization protocols. Because of the importance of this question, we reinvestigated the potential of anti-Leu3a to induce anti-HIV antibodies, compared with control immunizations with OKT4A (another anti-CD4 antibody) and the irrelevant Ig MOPC-21. Responses to anti-Leu3a showed induction of high-titer anti-idiotypic activity, and included combining-site-related activity. Yet sera showed no binding to gp160 above controls and no detectable neutralizing activity in a sensitive HIV plaque assay, so the anti-idiotypes induced were not internal images of CD4. We conclude that the pronounced anti-HIV responses reported with anti-Leu3a cannot be generalized, and thus that anti-Leu3a does not offer promise as an HIV vaccine. However, these results do not negate the promise of the idiotypic approach, and a vaccine for AIDS based on idiotype manipulation remains a possibility.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Antígenos CD4/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Animales , Anticuerpos Antiidiotipos/biosíntesis , Anticuerpos Monoclonales/inmunología , Antígenos CD4/metabolismo , Femenino , Productos del Gen env/inmunología , Anticuerpos Anti-VIH/biosíntesis , Proteína gp120 de Envoltorio del VIH/inmunología , Proteínas gp160 de Envoltorio del VIH , Humanos , Cinética , Ratones , Ratones Endogámicos BALB C , Pruebas de Neutralización , Precursores de Proteínas/inmunologíaRESUMEN
Our experiments with transfected cells provide new insights into the role of Na-Ca exchange activity in Ca homeostasis and emphasize the role of local interactions in determining exchanger function. Thus, the effects of ATP depletion and cytochalasin D highlight the influence of the actin cytoskeleton in regulating exchange activity. Cytoskeletal interactions could provide a mechanism for modulating exchange activity by mechanical stretch and might constitute a novel feedback mechanism for regulating contractile activity in the heart. The effects of Na on Ca entry during SDCI in the transfected cells suggest that local gradients of [Ca]i are important determinants of exchanger function. The surface distribution of exchanger proteins in relation to that of Ca channels therefore represents another area in which interactions with the cytoskeleton may be a central element in understanding the physiological function(s) of the exchange activity. At present, it seems likely that the exchanger's central hydrophilic domain mediates the connection between the exchanger and the cytoskeleton. This provides a rationale for understanding the importance of tissue-specific alterations in the exchanger's hydrophilic domain, which appear to have little affect on the kinetic behavior of the exchanger. Future work in our laboratory will be directed toward clarifying the role of cytoskeletal interactions in exchanger function.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Homeostasis , Transfección , Secuencia de Aminoácidos , Animales , Células CHO , Proteínas Portadoras/química , Cricetinae , Expresión Génica , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sodio/metabolismo , Intercambiador de Sodio-CalcioRESUMEN
Third ventricular injections of vasoactive intestinal polypeptide (VIP) result in increased circulating levels of luteinizing hormone (LH) in conscious, freely moving, ovariectomized (OVX) rats. This effect of VIP has been hypothesized to be mediated via stimulation of luteinizing hormone-releasing hormone (LH-RH) secretion from hypothalamic neurons since VIP is incapable of stimulating LH release from rat pituitaries in vitro. To test this hypothesis, crude synaptosomes were prepared from OVX rat median eminence (ME) tissue. Release of LH-RH from these preparations displayed time and temperature dependencies. Additionally, depolarization-induced (elevated K+) LH-RH release was demonstrated to be Ca2+-dependent. VIP, in doses ranging from 1.5 x 10(-7) to 1.5 x 10(-9) M, was capable of stimulating significantly greater LH-RH release from ME synaptosomes than that from control preparations. VIP's close structural homolog, glucagon, was incapable at the same doses of stimulating increased LH-RH release. These findings offer an explanation for the effect of third ventricularly injected VIP on LH release and suggest a modulatory role for VIP in the hypothalamic control of LH secretion.
Asunto(s)
Hormonas Gastrointestinales/farmacología , Hormona Liberadora de Gonadotropina/metabolismo , Sistema Hipotálamo-Hipofisario/metabolismo , Eminencia Media/metabolismo , Sinaptosomas/metabolismo , Péptido Intestinal Vasoactivo/farmacología , Animales , Cloruro de Calcio/farmacología , Castración , Dopamina/farmacología , Femenino , Glucagón/farmacología , Eminencia Media/efectos de los fármacos , Cloruro de Potasio/farmacología , Ratas , TemperaturaRESUMEN
Mouse hybridomas were isolated that produce monoclonal antibodies (MAbs) to several regions of the HIV envelope, gp120, which may be used to map immunogenic regions in animal and human immunity. One series of MAbs was generated by repeated immunization with recombinant gp120 (rgp120) from a single isolate, and a second series by sequential immunization with rgp120 from three isolates. All MAbs bound to rgp120IIIB, but only one bound well to cell surface-expressed gp160. Synthetic peptides spanning much of the length of gp120 were used to map MAb reactivity. Two MAbs were mapped to the C1 region, one to the C2 region, and three to the C5 region by this approach. Six distinct epitopes were detected by competitive binding analysis. One MAb binding the C1 region blocks binding of CD4 to rgp120IIIB, binds by ELISA to rgp120MN (an isolate not used during immunization), and binds to cell surface-expressed gp160. These hybridomas will be made available to the scientific community through a hybridoma culture facility.