RESUMEN
Humanized anti-CD154 antibody, IDEC-131, had a slightly, but reproducibly, better binding affinity for CD154 (Kd = 5.6 nM), compared to the parent antibody 24-31 (Kd = 8.5 nM). Otherwise it was indistinguishable from the murine parent antibody in its ability to bind to CD154, block CD154 binding to CD40 and inhibit T cell-dependent B cell differentiation. The latter activity was independent of FcR binding as the Fab'1 fragment of IDEC-131 had an equivalent biological activity to that of the whole antibody. IDEC-131 blocked soluble CD154 from inducing proliferation of purified B cells, and blocked T cell dependent anti-tetanus toxoid specific antibody production by human B cells in vitro. IDEC-131, gamma1, kappa, had strong Fc gammaRI, Fc gammaRII and C1q binding, but was unable to induce complement dependent (CDC) or antibody dependent cell-cytotoxicity (ADCC) of activated peripheral blood T cells, which express relatively low levels of CD154. IDEC-131 antibody inhibited both primary and secondary antibody responses to ovalbumin in cynomolgus monkeys at a dose of 5 mg/kg. In non-immunized animals, treatment with IDEC-131 at 50 mg/kg weekly for 13 weeks induced no change in any of the measured lymphocyte subsets, including B cells, CD4+ and CD8+ T cells. Similarly, a safety study in chimpanzees showed no discernible safety related issues at 20 mg/kg, including B and T cell subsets. These results show that the humanized anti-CD154 antibody, IDEC-131, has retained the affinity and functional activity of its murine parent antibody, is unlikely to deplete CD154 positive lymphocytes in humans, and is safe and effective in blocking antibody production in monkeys. Based on its safety and efficacy profile, IDEC-131 is being developed for therapy of systemic lupus erythematosus.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/fisiología , Antígenos CD40/fisiología , Ligando de CD40/fisiología , Activación de Linfocitos , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Secuencia de Bases , Células CHO , Diferenciación Celular , Cricetinae , Femenino , Genes de Inmunoglobulinas , Humanos , Región Variable de Inmunoglobulina/genética , Macaca fascicularis , Masculino , Datos de Secuencia Molecular , Pan troglodytesRESUMEN
CD23, the low affinity receptor for IgE (FcvarepsilonRII), is involved in regulation of IgE synthesis by B-lymphocytes. Five monoclonal antibodies to human CD23 were generated from cynomolgus macaques immunized with purified soluble CD23 (sCD23). Four of the five primate antibodies blocked the binding of IgE complexes to CD23 positive cells and also inhibited the production of IgE in vitro by IL-4 induced human peripheral blood mononuclear cells (PBMC). The variable domains of several primate antibodies were utilized to construct chimeric macaque/human (PRIMATIZED((R))) monoclonal antibodies. PRIMATIZED((R)) p5E8G1, containing human gamma 1 constant region, inhibited IgE production in vitro as efficiently as the parent primate antibody, but the human gamma 4 constant version, PRIMATIZED((R)) p5E8G4, was not as effective in IgE inhibition. An F(ab')(2) of p5E8G1 did not inhibit IgE production but did interfere with IgE inhibition by the intact anti-CD23 antibody in a dose dependent fashion. The murine monoclonal antibody MHM6 recognizes human CD23 at a different epitope than primate antibody 5E8, and inhibits IgE production by IL-4 induced PBMC. As with the F(ab')(2) of p5E8G1, the F(ab')(2) of MHM6 also failed to inhibit IgE production. These data imply that the mechanism by which anti-CD23 antibodies inhibit IgE production requires cross-linking of CD23 to an IgG receptor. These data also imply that neither bivalent cross-linking of CD23 alone or inhibition of CD23 binding to its natural ligands is sufficient to inhibit IgE production.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Inmunoglobulina E/biosíntesis , Fragmentos Fc de Inmunoglobulinas/fisiología , Receptores de IgE/fisiología , Animales , Humanos , Macaca fascicularisRESUMEN
Two human monoclonal antibodies, RF-1 and RF-2, specifically recognize the fusion protein of the human respiratory syncytial virus (RSV). These were isolated from spontaneous tumors in SCID mice reconstituted with human splenocytes and boosted with fusion protein. The tumors consisted of Epstein-Barr virus-transformed human B cells in animals with antigen-specific antibody titers>105. The binding affinity of RF-1 and RF-2 to the fusion protein is 1010 and 109 M-1, respectively. The antibodies bind specifically to a conformational epitope of the fusion protein on RSV-infected HEp-2 cells. Both antibodies display virus-neutralizing properties in vitro at concentrations varying between 8 and 1000 ng/mL. Virus neutralization applies to a broad variety of wild and laboratory-adapted virus strains belonging to both virus types A and B. These antibodies are potential candidates for passive immunotherapy of severe RSV infections.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Proteína HN , Neoplasias Experimentales/inmunología , Virus Sincitiales Respiratorios/inmunología , Proteínas Virales de Fusión/inmunología , Proteínas Virales/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Anticuerpos Antivirales/aislamiento & purificación , Anticuerpos Antivirales/metabolismo , Especificidad de Anticuerpos , Linfocitos B/virología , Transformación Celular Viral , Ensayo de Inmunoadsorción Enzimática , Infecciones por Herpesviridae/inmunología , Infecciones por Herpesviridae/virología , Herpesvirus Humano 4/inmunología , Humanos , Immunoblotting , Ratones , Ratones SCID , Pruebas de Neutralización , Bazo/citología , Infecciones Tumorales por Virus/inmunología , Infecciones Tumorales por Virus/virología , Proteínas del Envoltorio ViralRESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) infection is a major problem in the newborn and aging populations. Fully human monoclonal antibodies with the ability to neutralize RSV could have a major impact on the immunotherapy of the disease. The generation of human antibodies has been difficult because there exists no general way to activate B cells against an antigen of choice in vitro. MATERIALS AND METHODS: Human spleen cells from individuals exposed to RSV were used to repopulate SCID mice. Hu-SCID mice were boosted with RSV fusion (F)-protein and subsequently developed B cell tumors. The tumors were removed and cultured and subcloned in vitro, using a feeder layer of CD154-expressing T cells. Two of these tumors produced the antibodies designated RF-1 and RF-2. VL genes were isolated by standard PCR techniques, however, it was necessary to use high-temperature reverse transcriptase to clone the VH genes. RESULTS: RF-1 and RF-2 VH genes were both found to be closely related members of the VH2 family. Vk genes originated from the VK III family. RF-1 and RF-2 recombinant antibodies expressed in CHO cells (cRF-1 and cRF-2) were found to have affinities for RSV F-protein of 0.1 nM and 0.07 nM, respectively, and both were able to neutralize several A and B subtypes of RSV. CONCLUSION: The technique of immortalizing human B lymphocytes, by passage in SCID mice and expression as recombinant antibodies in CHO cells, provides a method by which high-affinity human antibodies can be developed for immunotherapy of viral diseases.
Asunto(s)
Anticuerpos Monoclonales/genética , Anticuerpos Antivirales/genética , Virus Sincitial Respiratorio Humano/inmunología , Anciano , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Antivirales/biosíntesis , Secuencia de Bases , Células CHO , Transformación Celular Viral , Clonación Molecular , Cricetinae , Cartilla de ADN/genética , Expresión Génica , Genes de Inmunoglobulinas , Herpesvirus Humano 4 , Humanos , Inmunoterapia , Recién Nacido , Ratones , Ratones SCID , Datos de Secuencia Molecular , Pruebas de Neutralización , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Infecciones por Virus Sincitial Respiratorio/inmunología , Infecciones por Virus Sincitial Respiratorio/terapia , Células Tumorales CultivadasRESUMEN
We report here that immunization of human PBMC reconstituted SCID mice (hu-PBL-SCID mice) with in vitro cultured autologous dendritic cells (DC) pulsed with prostate specific antigen (PSA) complexed to a PSA-specific mouse IgG2a (PSA-IgG2a) consistently and reproducibly stimulates PSA-specific human IgG production. On day 0, female PBMC were used to reconstitute SCID mice and to generate DC in vitro. DC cultures were pulsed with PSA or PSA-IgG2a on day 6. The previously reconstituted hu-PBL-SCID mice were immunized with either PSA-pulsed DC and PSA, PSA-IgG2a-pulsed DC and PSA-IgG2a, or additional PBMC and PSA-IgG2a on day 7. Mice immunized with PSA-IgG2a-pulsed DC had, on the average, up to 31.5 times greater PSA-specific IgG serum concentrations than control mice. Competition ELISA confirmed the PSA specificity of serum IgG. Immunoblot analysis suggested that sera IgG preferentially recognized conformational epitopes on PSA. Therefore, our results represent a major step toward cloning human tumor-associated Ag-specific human mAbs from hu-PBL-SCID mice. In addition, flow cytometry showed that PSA-pulsed DC express significantly more B7.1, B7.2, CD40, and MHC class II surface molecules than mock-treated DC, but PSA-IgG2a-pulsed DC only had significantly enhanced B7.2 surface expression. Interestingly, PSA-specific IgG responses were reproducibly stimulated by DC expressing more B7.2, a molecule associated with Th2-type immune deviation, but not by those expressing more B7.1 and CD40, molecules associated with Th1-type immune deviation. Thus, our results show that stimulation with either Ag or Ag complexed to mAb yields DC with different phenotypes and APC effector functions.
Asunto(s)
Células Dendríticas/inmunología , Epítopos/inmunología , Inmunoglobulina G/biosíntesis , Antígeno Prostático Específico/inmunología , Adolescente , Adulto , Animales , Especificidad de Anticuerpos , Células Cultivadas , Medio de Cultivo Libre de Suero , Células Dendríticas/trasplante , Femenino , Humanos , Inmunización/métodos , Immunoblotting , Inmunoglobulina G/sangre , Isotipos de Inmunoglobulinas/sangre , Inmunofenotipificación , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos ICR , Ratones SCIDRESUMEN
High titers of Ag-specific human IgG were consistently achieved in SCID mice reconstituted with human splenocytes that had been primed with Ag in vitro and then boosted with Ag after engraftment into SCID mice. Specific human IgG titers in the hu-SPL-SCID mice reached approximately 1:4 x 10(5) when the mice were immunized with a neo-antigen, whereas titers reached 1:2 x 10(6) when recall responses were induced. Booster immunizations with Ag 21 days after the initial in vivo boost further enhanced this response, and specific human IgG titers of 1:17 x 10(6) were achieved. This represented an essentially monospecific IgG population. These responses were CD4+ T cell dependent. In addition, affinity maturation of the human Ab responses was observed. Spleens of hu-SPL-SCID mice with Ag-specific titers < or = 1:1 x 10(6) were often significantly enlarged and often displayed visible tumors. Fourteen of sixteen B cell tumors removed from spleens of five such hu-SPL-SCID mice, produced Abs that were specific for the immunizing Ags. From such tumor, cloned cell lines were established. One such mAb, MLN-7 (gamma1, kappa), was raised to tetanus toxoid and had no identified cross-reactivity.
Asunto(s)
Antígenos/administración & dosificación , Antígenos/inmunología , Epítopos/inmunología , Inmunización Secundaria , Inmunoglobulina G/biosíntesis , Bazo/inmunología , Adulto , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/aislamiento & purificación , Afinidad de Anticuerpos , Línea Celular , Células Cultivadas , Ferritinas/inmunología , Humanos , Inmunización/métodos , Inmunoglobulina G/sangre , Activación de Linfocitos , Transfusión de Linfocitos , Ratones , Ratones SCID , Persona de Mediana Edad , Bazo/citología , Bazo/trasplante , Linfocitos T/inmunología , Toxoide Tetánico/inmunologíaRESUMEN
Efficiency of immunization of splenocytes in vitro by three kinds of prostate antigen has been studied. Intact cells of the human prostate cell line LNCaP, a membrane preparation (PMA) of LNCaP cells, and prostate specific antigen (PSA) were used as antigens. Three different schemes of in vitro immunization were tested: male spleen cells vs female; single donor spleen cells vs. mixed lymphocyte culture (MLC); addition of exogenous IL-2 vs. no lymphokine addition. The supernatant antibodies were tested for reactivity to the immunizing antigen by ELISA. Subsequently hybridomas were generated by fusing the primed lymphocytes to a heteromyeloma cell line, K6H6/B5. Only antigen specific IgM responses could be detected. Intact LNCaP cells induced the highest responses from mixed lymphocyte cultures. PMA also induced the highest responses from mixed lymphocyte cultures of male origin, whereas both single donor and mixed donor spleen cell cultures of female origin responded to PMA. However, anti-PMA responses by mixed lymphocyte cultures of female cells were significantly augmented by addition of IL-2. PSA only induced specific responses from mixed female splenocyte cultures supported with IL-2. Several anti-PMA and -PSA antibodies were generated after somatic fusion of the in vitro primed cells. Two monoclonal IgG antibodies from LNCaP primed spleen cells could be competitively inhibited with tumor membrane antigen.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/inmunología , Antígenos de Neoplasias/inmunología , Linfocitos B/inmunología , Biomarcadores de Tumor/inmunología , Membrana Celular/inmunología , Antígeno Prostático Específico/inmunología , Próstata/inmunología , Anticuerpos Antineoplásicos/biosíntesis , Células Cultivadas , Femenino , Humanos , Hibridomas/inmunología , Masculino , Factores Sexuales , Bazo/citologíaRESUMEN
Induction of antigen-dependent IgM and IgG responses by human in vitro spleen cell cultures were supported by addition of defined lymphokines. Whereas exposure to IL-2 alone throughout both immunization and cultivation period supported the highest antigen-directed responses, antibodies of higher relative affinity to the immunizing antigen were secreted under conditions of shorter IL-2 exposure. Continuous presence of IL-2 supported the antigen-driven responses for up to 15 days, the later part of which was characterized by a very low relative affinity index value. IL-2 supported cultures produced up to 55 times and 36 times more IgM and IgG, respectively, than cultures without added IL-2. Addition of IL-2-neutralizing antibodies throughout the cultivation period abrogated all responses. Addition of IL-4 alone had negligible effect on the antigen-directed IgM responses but supported antigen-independent IgG responses. Neutralization of IL-4 alone had negligible effect on the IgM and IgG responses, whereas neutralization of IL-4 during IL-2 support, resulted in reduction of antigen dependency of responses. Delayed IL-4 neutralization (24 hours) restored some of the antigen sensitivity. IL-4 reduced IL-2-induced, antigen-directed immunoglobulin responses but supported increased antigen-induced, antigen-directed responses, resulting in maximal antigen-specific responses. IL-4 reduced the IL-2-induced immunoglobulin production. IL-2 supported cell division, whereas IL-4 supported prolonged survival. Flow cytometry tests showed that IL-2 primarily induced CD8+ cells (T suppressor/cytotoxic) and OKT-10+ cells (plasma cells) cells, whereas the number of B1+ cells (B cells) decreased. IL-4 induced slight increases in the amount of B1+ cells and CD4+ cells (T helper).
Asunto(s)
Antígenos/inmunología , Linfocitos B/inmunología , Interleucina-2/farmacología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Especificidad de Anticuerpos , Linfocitos B/efectos de los fármacos , Células Cultivadas , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Bazo/citologíaRESUMEN
A protocol by which a human spleen cell culture could be subjected to both a primary and a secondary type immunization in vitro has been developed. Primary immunized cultures required the addition of autologous fresh cells at the time of antigen re-exposure for generation of optimal antigen-specific IgG responses. Antigen re-exposure (secondary stimulation) was done 10 days after initial immunization. Subsequent responses were characterized by novel or greatly enhanced antigen-directed IgG responses compared to primary antigen stimulation. Re-exposure of antigen to 10-day old human spleen cell cultures, without addition of fresh cells, did not result in measurable responses. Lethal irradiation of the fresh cell component of a restimulation culture showed that the IgG responses were derived from the original culture and not the fresh cells. The presence of antigen during both the primary and the secondary immunization was a prerequisite for induction of antigen-directed IgG responses, as spleen cell preparations that had been cultivated without antigen, during the primary stimulation period or the secondary stimulation period or both, did not show antigen-dependent IgG responses after the secondary cultivation. Induction of IgG responses by restimulation was supported by IL-2, but only when IL-2 was added to both the primary and the secondary culture according to a strict protocol. IL-4 added during the second antigen exposure supported antigen-directed responses, whereas IL-4 added during the initial antigen exposure abrogated all antigen-dependent responses.
Asunto(s)
Antígenos/inmunología , Linfocitos B/inmunología , Activación de Linfocitos , Especificidad de Anticuerpos , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/efectos de la radiación , Linfocitos B/efectos de los fármacos , Linfocitos B/efectos de la radiación , Células Cultivadas , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina M/biosíntesis , Interleucina-2/farmacología , Interleucina-4/farmacología , Activación de Linfocitos/efectos de los fármacos , Bazo/citología , Factores de TiempoRESUMEN
We have developed culture conditions for human lymphocytes that support primary in vitro immune responses to protein Ag of either human or nonhuman origin. We now show that these primed B cells can be efficiently immortalized by fusion with a heterohybrid fusion partner to generate human, Ag-specific IgM or IgG antibody-producing heterohybridomas at a rate of 17 to 50 hybrids/10(6) lymphocytes fused. Approximately 50% of the Ig-secreting clones were stable with respect to Ig secretion. Levels of secretion attained with terminal cultures ranged from less than 1 to 100 micrograms/ml. Fusions of cells between 2 and 5 days after initiation of in vitro exposure to Ag produced more Ag-reactive and Ag-specific antibodies than fusions at 1 day or fusions performed after 5 days. Ag-reactive hybrids could be isolated at frequencies of 3 to 10%, depending on antigenicity of the immunogen. Foreign proteins, horse spleen ferritin, and a murine monoclonal Ig, induced higher percentages of Ag-reactive mAb than immunization with the human-derived ferritin. Ag-reactive IgG mAb were produced at relatively high frequency, depending on immunization conditions and the nature of the Ag. The strategy for identification of the best hybrids included early elimination of unstable hybridomas and of hybridomas producing broadly cross-reactive antibody, followed by evaluation of units of Ag reactivity/micrograms Ig. Ferritin-specific mAb selected according to these criteria showed immunocytochemical reactivity with ferritin-containing tissues and apparent affinities in the range of 10(7) to 10(8)/mol.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Hibridomas/citología , Bazo/citología , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/metabolismo , Afinidad de Anticuerpos , Células Cultivadas , Ferritinas/inmunología , Caballos , Humanos , Técnicas In Vitro , Especificidad de la Especie , Bazo/inmunologíaRESUMEN
The aim of this study was to examine whether the unresponsiveness of MHC class I-negative subclones of the EL4 thymoma to CD3 cross-linking can be restored by transfection of class I genes into the H-2-negative cells. Cell activation experiments with selected MHC class I-negative subclones and H-2b- and H-2Ld-positive transfectants showed that these cells are equally capable of secreting interleukin 2 (IL-2) after exposure to the phorbol ester phorbol 12-myristate 13-acetate (PMA) and ionomycin. In contrast, only the parental H-2-positive EL4 cells are capable of responding to treatment with immobilized anti-CD3 antibody with IL-2 secretion and IL-2 receptor expression. Measurements of intracellular free Ca2+ (Ca2+i) following anti-CD3 antibody-induced cross-linking of parental EL4 cells and H-2-negative and H-2b gene-transfected subclones showed that the parental cells and two of the class I transfectants, one H-2-positive and one H-2-negative, responded with a slow rise in Ca2+i, whereas one H-2-positive transfected cell clone was completely refractory to CD3 cross-linking. Modulation experiments using parental EL4 cells, H-2-negative subclones and H-2-positive transfectants demonstrated that the CD3 and class I molecules of these different cells are modulated to the same extent after exposure to specific antibodies. The present findings thus indicate that the unresponsiveness of H-2-negative EL4 subclone cells to CD3 cross-linking is not functionally associated with a lack of class I surface expression.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos H-2/genética , Antígenos de Histocompatibilidad Clase I/fisiología , Activación de Linfocitos , Linfoma/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Linfocitos T/inmunología , Transfección , Antígenos de Diferenciación de Linfocitos T/inmunología , Complejo CD3 , Calcio/metabolismo , Humanos , Interleucina-2/biosíntesis , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Interleucina-2/biosíntesisRESUMEN
This paper examines the possibility of a functional linkage between class I MHC molecules and the T-cell receptor complex for antigen (T3-Ti). A newly developed anti-CD3 antibody (500A2) was used as an activation signal for EL4 lymphoma cells and allospecific cytotoxic T-cell clones (CTL), and the production of IL-2/IL-2 receptor in EL4 cells and serine esterase in CTL was determined. Anti-CD3 antibody-induced activation of both EL4 and CTL cells was enhanced in the presence of immunologically cross-linked and immobilized anti-H-2 (class I) antibody reactive against the H-2 haplotype of the responding T cells. A number of H-2-negative and H-2-positive EL4 subclones were generated and tested for anti-CD3 antibody-induced IL-2/IL-2 receptor production. Although both H-2-positive and -negative subclones expressed CD3 antigen and produced IL-2 after activation with the phorbol ester TPA, only the H-2-positive cell clones produced IL-2 and expressed IL-2 receptor after anti-CD3 antibody induction. Our results are compatible with the existence of a functional linkage between the class I and the CD3 molecules on the surface of T cells.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Complejo CD3 , Células Clonales , Interleucina-2/biosíntesis , Receptores de Interleucina-2/biosíntesis , Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
A method to efficiently antigen-prime B-lymphocytes with low amounts (less than 1 microgram/10(8) cells) of either immunogenic or non-immunogenic molecules is described. Keyhole limpet hemocyanin (KLH) and histone were used as prototypes for strongly immunogenic and for phylogenetically conserved non-immunogenic epitopes, respectively. Several modifications of previously reported methods were applied to the system and resulted in the requirement of antigen amounts sufficiently low to be obtainable by elution of proteins from electrophoretic gels. Antigen priming against highly purified antigen preparations is thereby feasible even when purified material cannot be obtained by conventional biochemical procedures. The amount of T- and B-lymphocytes and interleukin-2 production was estimated under various conditions during the priming procedure, and those optimal for generation of a high number of antigen-specific B-lymphocytes determined. In vitro antigen-primed B-lymphocytes were immortalized by conventional hybridoma technology. By fusion of lymphoid cells with myeloma cells at each day during the antigen-priming period, the optimal day of fusion to generate antigen-specific hybridomas was determined. Further, in 12 experiments with different antigens, 11 monoclonal antibodies to histones H3 and H4, two to the murine glucose transporter, 17 to trinitrophenyl-sheep red blood cells, and 20 to KLH were obtained. All specific hybridomas produced IgMs, as the antigen-priming period could not be extended for more than 9-10 days, whereafter a rapid decay in B-lymphocytes occurred.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Linfocitos B/inmunología , Hibridomas/metabolismo , Animales , Formación de Anticuerpos , Línea Celular , Células Cultivadas , Epítopos/análisis , Epítopos/inmunología , Eritrocitos/inmunología , Hemocianinas/inmunología , Histonas/inmunología , Hibridomas/inmunología , Inmunización , Masculino , Ratones , Ratones Endogámicos BALB C , Ovinos , Trinitrobencenos/inmunología , Trinitrobencenos/farmacologíaRESUMEN
Treatment of the eukaryotic organism Tetrahymena with various types of DNA-damaging agents has been reported to cause a 35-fold induction of a mitochondrial DNA polymerase. We here report that the enzyme can be induced in large-scale cultures by exposure of the cells to thymine starvation and/or intercalating agents. The induced DNA polymerase has been purified to near homogeneity, with a specific activity of approx. 300,000 units/mg protein. The relative molecular mass of the active form of the enzyme is approx. 100,000, as determined by glycerol gradient sedimentation. The subunit structure has been analysed by SDS polyacrylamide gel electrophoresis of the highly purified preparation and by immunoprecipitation with a monoclonal antibody directed to the DNA polymerase. A polypeptide of Mr 47,000 has been observed to be a subunit of the enzyme. This corresponds to the size of the subunits suggested for mitochondrial DNA polymerase from chicken embryos and mouse myeloma cells.
Asunto(s)
ADN Polimerasa Dirigida por ADN/aislamiento & purificación , Mitocondrias/enzimología , Tetrahymena/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Inducción Enzimática , Mitocondrias/metabolismo , Mapeo Peptídico , Conformación Proteica , Tetrahymena/genética , Tetrahymena/metabolismoRESUMEN
A content analysis of primary school readers in Sierra Leone revealed that the particular modern and traditional normative aspirations expressed in the National Development Plan for 1974/5-1978/9 were also generally reflected in the children's readers. Compared to the 1964 readers, the 1st indigenous readers developed circa 1977 contained markedly greater emphasis on traditional norms, though modernity norms continued to dominate, and substantially less emphasis on Efficacy (a central aspect of modernity) and on Nonparochial Affiliation. This closely corresponded with the intent of the National Plan to continue on a modernizing course employing the traditional norms of Manual Labor and Social Cohesion in a grassroots effort to develop the agricultural sector, with nationalism in a less important role. Apart from lesser emphasis on Efficacy and underemphasis on the Value of Education, both discordant with national goals, the 1977 readers seem to provide children and teachers with a fairly accurate image of the national ethos. This may help to account for the generally positive relationship that has been found between schooling and economic development.