RESUMEN
Macrophages from mice treated with a novel antineoplastic agent, bisantrene, were shown previously to be highly active in inhibiting the proliferation of tumor cells in culture. These activated cells have now been found to protect mice from dying of progressive tumors when injected into animals. The effect was observed not only in a Winn-type tumor cell neutralization assay but also in a setting of therapeutic intervention. Multiple treatments with bisantrene-activated cells seemed more effective than a single treatment. Macrophages appeared to be the major effectors in this system, since treatment with carrageenan abolished the protective effect. Thus, present findings suggest that in addition to a direct cytotoxic effect of bisantrene, the activation of macrophages may contribute to the overall antitumor activity of the drug.
Asunto(s)
Linfoma/terapia , Macrófagos/inmunología , Animales , Antracenos/farmacología , Carragenina/farmacología , Inmunización Pasiva , Inmunoterapia , Activación de Macrófagos/efectos de los fármacos , Ratones , Trasplante de NeoplasiasRESUMEN
The immunorestorative characteristics of a novel synthetic immunomodulator, N-(4-[(4-fluorophenyl)sulfonyl]phenyl)acetamide (CL 259, 763), has been investigated in several experimental models. In one situation, the compound was shown to enhance the induction of a cytolytic T-lymphocyte response to the murine MBL-2 leukemia implanted in its syngeneic host in which only a minimal reactivity to the tumor is normally displayed. In a Vaccinia virus model, the compound similarly augmented the lytic activity of cytolytic T-lymphocyte to virus-infected targets in not only viral antigen-primed but also cyclosporin A-impaired mice. Likewise, the alloreactive cytolytic T-lymphocyte activity was recovered in animals immunocompromised by inoculation with murine plasmacytomas or cytoreductive anticancer drugs, such as cyclophosphamide and 5-fluorouracil. Thus, the present findings suggest that CL 259,763 is effective in potentiating the immune response to weak antigens as well as in restoring alloreactivity by sparing the immunotoxicity associated with the administration of cytotoxic drugs and the growth of neoplasms.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Sulfonas/farmacología , Linfocitos T Citotóxicos/efectos de los fármacos , Animales , Ciclofosfamida/farmacología , Ciclosporinas/farmacología , Fluorouracilo/farmacología , Ratones , Ratones Endogámicos , Neoplasias Experimentales/inmunología , Virosis/inmunologíaRESUMEN
An investigation was carried out to determine the potential for activating tumor-inhibitory macrophages with the cytotoxic antitumor agent, bisantrene. Macrophages prepared from peritoneal exudates of mice treated i.p. with bisantrene were extremely active in inhibiting the growth of tumor cells. The minimal effective in vivo dose of this drug appeared to be 25 mg/kg, with peak activation being achieved at doses of 50 to 100 mg/kg. Effector cells became detectable 2 days after treatment and persisted for at least 4 weeks. Incubation of effector and target cells for 48 hr seemed necessary to achieve the maximum inhibitory effect. Treatment with carrageenan in vitro and in vivo abolished tumor cytostasis, whereas treatment with anti-T-cell antibody plus complement had no effect, suggesting that macrophages rather than T-lymphocytes were responsible for the observed results. Culture supernatants of activated macrophages were found to have antiproliferative effects on tumor cells, indicating that a cytostatic factor(s) was produced by these macrophages. Hydrogen peroxide and neutral proteases apparently did not function as cytostatic mediators since activated macrophages were resistant to treatment with catalase, N-alpha-p-tosyl-L-lysine chloromethyl ketone, and aprotinin. The present findings suggest that, in addition to direct toxicity to tumor cells, bisantrene may act as a macrophage-activating immunopotentiator. This observation may be of potential clinical usefulness in the design of immunochemotherapeutic trials for certain types of cancer.
Asunto(s)
Antibióticos Antineoplásicos/toxicidad , Citotoxicidad Inmunológica , Activación de Macrófagos , Macrófagos/inmunología , Sarcoma de Mastocitos/inmunología , Animales , Antracenos/toxicidad , Línea Celular , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
GH specifically interacts with a soluble binding protein in serum. The GH-binding protein (GHBP) has been shown to contain the extracellular portion of the cell surface GH receptor (GHR). In rats and mice there is a unique mRNA that encodes the GHBP. This mRNA contains an alternatively spliced exon that replaces the transmembrane and intracellular domains of the receptor with a short hydrophilic carboxy-terminus of 17 and 25 amino acids, respectively, in rats and mice. In humans and other species no mRNAs encoding the GHBP have been identified, suggesting that the GHBP is in these cases a proteolytically processed GHR. In this study a monoclonal antibody (GHBP 4.3) was raised to the rat GHBP using as immunogen a synthetic peptide containing the unique C-terminal 17 amino acids that are not found in the rat GHR. As predicted, this antibody is specific to rat GHBP and does not cross-react with rat GHR. In combination with polyclonal and monoclonal antibodies that recognize both GHBP and GHR, this antibody was used to show that all, or most, of the GHBP in rat serum is indeed derived from the alternatively spliced GHBP mRNA and not from proteolytic processing of the GHR. In addition, endogenous rat serum GHBP was found to exist in two forms, with apparent mol wt of 52 and 44 kDa, arising from a single protein core of 32 kDa by extensive glycosylation. The concentrations of GHBP in male and female rat plasma were also estimated to be 300 and 575 ng/ml, respectively (measured in nonglycosylated GHBP equivalents).
Asunto(s)
Proteínas Portadoras/sangre , Secuencia de Aminoácidos , Animales , Anticuerpos/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Western Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Electroforesis en Gel de Poliacrilamida , Femenino , Técnicas de Inmunoadsorción , Masculino , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Empalme del ARN , ARN Mensajero/genética , Ratas , Ratas Endogámicas , Receptores de Somatotropina/inmunologíaRESUMEN
The potentiation of the biological activity of recombinant porcine growth hormone (pGH) by immunologic manipulation was investigated. An anti-pGH monoclonal antibody, designated PS-7.6, was generated and its effect on pGH was evaluated in hypophysectomized (hypox) rats. As expected, administration with pGH for 5 consecutive days promoted these animals to grow. The effect was further augmented when pGH was given together with PS-7.6 antibody and the enhancing ability of the antibody lasted beyond the treatment period. The growth profile of rats receiving antibody alone did not differ from that of untreated controls, indicating that PS-7.6 antibody by itself was not a growth stimulant. The possible mechanism of action of the antibody was investigated by analyzing blood and tissue samples of animals following injection with 125I-labeled pGH either in its free form or complexed with PS-7.6 antibody. As compared to the pGH levels in animals receiving free pGH, approximately a half pGH was released into circulation from the injection sites when it was given in a complex form. Furthermore, 2-4-fold increases in pGH deposition were also found in various tissues of animals treated with pGH-antibody complexes over that of respective tissues of animals receiving free pGH. Therefore, the present findings suggest that PS-7.6 antibody is capable of augmenting the somatogenesis of pGH and the effect is, at least in part, explainable by its ability in altering the pharmacokinetics and biodistribution of pGH in animals.
Asunto(s)
Anticuerpos Monoclonales , Hormona del Crecimiento/inmunología , Crecimiento/efectos de los fármacos , Animales , Sinergismo Farmacológico , Femenino , Hormona del Crecimiento/farmacocinética , Hormona del Crecimiento/farmacología , Hipofisectomía , Ratas , Ratas Endogámicas , Distribución Tisular , Aumento de Peso/efectos de los fármacosRESUMEN
Upon engagement with appropriate ligands, receptors can be activated to initiate various metabolic and morphological changes in living cells. An attempt was made in this study to generate monoclonal antibodies (mAb) specific to recombinant rat growth hormone receptor (GHR) and subsequently to investigate their ability to act as biologically active ligands. Three mAbs, designated 1A9, 1H2 and 2C3, were produced and all were highly reactive with GHR in an enzyme-linked immunosorbent assay. In contrast to 1H2, 1A9 and 2C3 competed with radioactive growth hormone (GH) tracer for the binding to GHR in a radioreceptor assay, suggesting that the GH-binding sites of GHR were identical, or very close to its epitopes recognized by 1A9 and 2C3. The molecular interaction evaluated by the BIAcore technology further demonstrated the separate GHR epitopes for 1A9 and 2C3. 2C3 apparently targeted the precise GH-binding sites of GHR, while the antigenic determinants for 1A9 were not at the site, but adjacent to it. Functional analysis showed that 2C3 promoted the growth of hypophysectomized rats, whereas others failed to do so. Therefore, findings from the present study suggest that these mAbs recognize distinct GHR epitopes and are useful for investigating the structure-function relationship of GHR. Furthermore, 2C3 may prove important as a biologically active agonist for better understanding of the process of GHR activation relevant to growth.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Receptores de Somatotropina/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Peso Corporal/inmunología , Epítopos , Crecimiento/inmunología , Ratones , Ratones Endogámicos BALB C , RatasRESUMEN
The properties of four independent lines of monoclonal antibodies (MAbs) specific to rat GH-binding protein (GHBP) were examined. Three MAbs, designated GHR-12, GHR-13 and GHR-16, were raised against the entire GHBP molecule. The fourth MAb, designated as GHBP4.3, was raised against the 17 amino acid residues at the C-terminal end of rat GHBP. The interaction of these antibodies with GHBP and their effect on GH binding to GHBP were analysed by conventional competition binding assays and surface plasmon resonance, i.e. with a Biospecific Interaction Analysis (BIAcore) instrument. The binding affinity of these MAbs to GHBP ranged from 29 nmol/l to 30.9 pmol/l. The pair-wise antibody binding to GHBP on BIAcore suggested that GHR-13 and GHR-16 recognized different antigenic determinants while part of the GHR-12 epitope might be shared with the other antibodies. The antibodies inhibited the interaction of GH with GHBP in the competition binding assay. However, in sequential binding on the BIAcore instrument, they were able to bind GHBP after its interaction with GH, indicating that the inhibition observed in the competition binding assay resulted from steric hindrance rather than direct interference with the GH-binding site of GHBP. The present findings, therefore, suggest that these antibodies are useful for investigating GHBP and its interaction with GH.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Complejo Antígeno-Anticuerpo/metabolismo , Proteínas Portadoras/metabolismo , Hormona del Crecimiento/metabolismo , Animales , Unión Competitiva , Proteínas Portadoras/inmunología , Epítopos/metabolismo , RatasRESUMEN
A monoclonal antibody (mAb), designated PS-7.6, was generated in mice by immunizing these animals with recombinant porcine growth hormone (pGH). This antibody has been previously shown to enhance the growth-promoting activity of pGH in an experimental rat model. An effort was made in this report to further characterize the immunologic properties of this antibody and its effect on the interaction between pGH and GH binding protein (GHBP). This antibody was classified as IgG1 isotype and found to be highly specific to pGH in a competition radioimmunoassay (RIA). It did not recognize several other GHs including those of bovine, chicken and human origins, nor several growth related factors including prolactin, insulin, somatostatin and growth hormone releasing factor. In Western analysis, PS-7.6 mAb identified not only the 22.5 kDa recombinant pGH, but also the native pGH in swine pituitary gland. An additional 45 kDa protein was also detected in the gland by the antibody, presumably a dimer form of pGH. The association and dissociation rate constants of the antibody as determined by biospecific interaction analysis (BIA) were 2.43 x 10(4) M-1 s-1 and 1.29 x 10(-3) s-1, respectively and its affinity (Kd) was 4.85 x 10(-8) M. Furthermore, PS-7.6 mAb prevented pGH from interacting with GHBP in RIA and BIA, indicating that the antibody epitope closely associated with the pGH binding region for GHBP. Therefore, present findings suggest that a possible mechanism of action of PS-7.6 mAb in enhancing animal growth is to prevent pGH from being bound to GHBP in circulation, thus making more pGH available to tissue receptors.
Asunto(s)
Anticuerpos Monoclonales , Proteínas Portadoras/metabolismo , Hormona del Crecimiento/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Sitios de Unión , Sitios de Unión de Anticuerpos , Unión Competitiva , Epítopos/metabolismo , Hormona del Crecimiento/metabolismo , Inmunoglobulina G/inmunología , Ratones , Ratones Endogámicos BALB C , Adenohipófisis/química , Unión Proteica/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Porcinos/inmunologíaRESUMEN
A monoclonal antibody (mAb), PS-7.6, to porcine somatotropin (pST) significantly enhanced the growth responses to pST injections in hypophysectomized (hypox) rats but could not be tested in pigs because of the large quantity of antibody required for a growth trial. Because pST inhibits the hypoglycemic effects of insulin, an insulin tolerance test procedure was established to measure pST activity in jugular-catheterized pigs. Doses of 0, 30, 100, and 300 micrograms/kg per day of pST were split and administered subcutaneously (sc) in equal portions twice daily for 2 d. After a 17-hr fast, plasma samples were obtained at 10-min intervals for 30 min before an intravenous injection of insulin (0.08 IU/kg) and then for an additional 50 min. Because pST increased fasting plasma glucose concentrations, preinsulin glucose values were used as a covariate to adjust the postinsulin concentrations. pST caused a dose-dependent increase in resistance to the insulin injection in these pigs. The areas under the curves (AUC), for plasma glucose were 22.1, 29.0, 39.0, and 47.2 mg/dl per min for the 0, 30, 100, and 300 micrograms/kg pST doses, respectively. Because different doses of pST could be detected, the PS-7.6 enhancement of pST treatment was evaluated. In the first experiment, five pigs/group each received sc injections of either vehicle, pST (75 micrograms/kg; approximately 3.0 mg/d), pST (75 micrograms/kg) + PS-7.6 at 3.75 mg/kg, or pST (75 micrograms/kg) + PS-7.6 at 15 mg/kg for 2 d before the insulin test. The pST and PS-7.6 were combined and incubated for at least 1 hr at room temperature before being injected. The injection of pST alone did not significantly change insulin tolerance activity (23.1 vs. 21.1, AUC), but insulin resistance was enhanced when this dose of pST also included PS-7.6 (27.4 and 29.5, AUC, respectively; P < 0.05). In a second experiment, the effects of PS-7.6 and PS-4.2, a mAb that did not potentiate the pST-stimulated growth of hypox rats, were compared. The five pigs/treatment received either vehicle, pST (75 micrograms/kg), pST (75 micrograms/kg) + PS-7.6 (3.75 mg/kg), or pST (75 micrograms/kg) + PS-4.2 (3.75 mg/kg) for 2 d. The administration of pST increased the resistance to insulin (26.7 vs. 18.8, AUC; P < 0.01), which was markedly potentiated by PS-7.6 (54.3, AUC, P < 0.001) but not affected by PS-4.2 (27.6 AUC). The injection of PS-7.6 at 7.5 mg/kg without exogenous pST did not alter the sensitivity to insulin. These results indicate that PS-7.6, but not PS-4.2, enhanced the insulin antagonistic activity of pST in swine, suggesting that an enhancement of pST-stimulated growth would also occur in PS-7.6-treated pigs.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Hormona del Crecimiento/farmacología , Antagonistas de Insulina/farmacología , Insulina/farmacología , Porcinos , Animales , Glucemia/metabolismo , Sinergismo Farmacológico , Hormona del Crecimiento/administración & dosificación , Hormona del Crecimiento/inmunología , Resistencia a la InsulinaRESUMEN
Murine ascites has been shown to contain a variety of growth promoting activities. In this study, we examined the effects of ascites fluid on the efficiency of hybridoma production and cell growth. SP2/0 mouse myeloma cells were injected into peritoneal cavities of mice and ascitic fluid was collected. Lymphocytes from mice immunized with porcine growth hormone (pGH) were fused with myeloma cells in a standard hybridization procedure. These cells were then dispensed in 96-well plates in medium containing either 2.5% ascites or 20% fetal calf serum (FCS) and cultured for few days. Supernatants from these cultures were collected and analyzed for anti-pGH antibodies. It was demonstrated that ascites-supplemented medium increased the efficiency in generating specific antibody-secreting hybridomas by 4-fold over FCS-supplemented medium. Furthermore, hybridoma cells were cultured in microtiter plate and found to proliferate in response to ascites in a dose dependent manner. This effect was abolished by prior digestion of ascites with trypsin, indicating its protein nature. B-lymphocyte related cytokines seemed less likely involved because antibodies to IL-4 and IL-6 failed to alter the stimulatory effect of ascites. Ascites was fractionated by FPLC using Superose 12 column and the active moiety was found to be a small m.w. peptide (< 1,000 dalton). Therefore, murine ascites is capable of substituting for conventional FCS in culture medium in the area of hybridoma technology.
Asunto(s)
Líquido Ascítico/química , Sustancias de Crecimiento/farmacología , Hibridomas/efectos de los fármacos , Animales , Formación de Anticuerpos/efectos de los fármacos , División Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Hormona del Crecimiento/inmunología , Hibridomas/citología , Hibridomas/inmunología , Inmunización , Ratones , Ratones Endogámicos BALB C/inmunología , Porcinos , Terpenos , Células Tumorales CultivadasRESUMEN
An effort was made to generate stable swine hybridomas capable of releasing monoclonal antibodies (MAb) with antigenic specificity. Crossbred pigs were immunized with recombinant porcine growth hormone (r-pGH) and the splenic cells were harvested from these animals. B lymphocytes enriched by gradient centrifugation and nylon wool adherence were briefly stimulated in vitro with r-pGH prior to hybridization with murine SP2/0 myeloma cells. The fused hybrids were screened for their ability to produce anti-pGH antibody and the positive ones were subcloned by a limiting dilution procedure. The stable cell lines were maintained by serial passages in cultures for further analysis. One such hybridoma, designated PM20/20, was found to secrete swine IgM. It recognized not only the immunizing r-pGH but also the native pGH extracted from the swine pituitary glands, as demonstrated by Western analysis. It also recognized two smaller fragments with m.w. of 10 kD and 5 kD of r-pGH following trypsin digestion. In addition to pGH, PM20/20 immunoreacted with several other GH species including bovine, chicken, and human origins, but not with ovine prolactin nor rat GH binding protein. The binding association rate constant and dissociation rate constant of PM20/20 to pGH were 5.3 x 10(4) M-1 s-1 and 1.0 x 10(-4) s-1, respectively, thus producing a dissociation constant of 1.9 x 10(-9) M. Therefore, stable swine-mouse heterohybridoma lines have been established and shown to continuously release swine mAb in cultures. These mAb may serve as useful alternatives to murine mAb in certain areas of research.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Hormona del Crecimiento/inmunología , Porcinos/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Western Blotting , Cromatografía de Afinidad , Ensayo de Inmunoadsorción Enzimática , Hormona del Crecimiento/análisis , Hibridomas/inmunología , Isotipos de Inmunoglobulinas/inmunología , Ratones , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/inmunologíaAsunto(s)
Anticuerpos Monoclonales/biosíntesis , Técnicas Inmunológicas , Porcinos/inmunología , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Fusión Celular , Hormona del Crecimiento/inmunología , Hibridomas/citología , Hibridomas/inmunología , Inmunoglobulina M/biosíntesis , Ratones , Mieloma Múltiple/inmunología , Especificidad de la EspecieRESUMEN
CL 246,738 is a low molecular weight, synthetic immunomodulator. The present study was done to determine the interaction among interferon (IFN), macrophages, and natural killer (NK) cells in mice following oral administration of CL 246,738. Splenic NK activity as evidenced by lysis of YAC-1 lymphoma cells in vitro was found to be augmented by the compound not only in normal mice, but also in immunodeficient beige and nude mice. Lytic activity remained elevated from one to seven days after a single treatment and the peak activation varied depending on the source of NK cells. NK cell activity associated with the peritoneal exudate cell population peaked at day 1 and returned to normal by day 2, whereas NK cell activity of peripheral blood lymphocytes peaked at day 3 and remained significantly elevated until day 7. Liver associated NK activity peaked at day 4 and remained significantly elevated at day 7 after treatment with CL 246,738. Lung associated NK activity was elevated by day 1 after treatment, peaked at day 4 and returned to normal by day 7 after drug administration. The drug was also effective in inducing IFN in all mouse strains tested. When these drug-treated mice were given antibody to IFN-(alpha + beta) but not to IFN-(beta), both IFN levels and NK cell activity decreased, suggesting the importance of IFN-(alpha) in this system. Furthermore, mice that had received carrageenan prior to, but not after CL 246,738 administration showed reduced serum IFN titers as well as decreased NK cell activity, indicating that macrophages played an intermediate role in immune enhancement by the drug.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Acridinas/farmacología , Células Asesinas Naturales/efectos de los fármacos , Adyuvantes Inmunológicos/farmacología , Envejecimiento/inmunología , Animales , Interferones/antagonistas & inhibidores , Interferones/inmunología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Bazo/citología , Bazo/efectos de los fármacos , Bazo/inmunologíaRESUMEN
3,6-bis(2-piperidinoethoxy)acridine trihydrochloride (CL 246,738) has been investigated for its immunomodulatory effect on murine macrophages. Incubation of macrophages harvested from the peritoneal cavities of normal mice with the compound for 48 to 72 hr rendered these cells inhibitory to the growth of tumor cells in vitro. Activation of tumor-inhibitory macrophages occurred over a range of concentrations (0.025 to 0.1 micrograms/ml) producing no direct inhibitory effects on tumor cells. Treatment of effector cells with carrageenan abrogated the effect, whereas treatment with anti-Thy-1.2 antibody and C did not, suggesting that the primary effectors were macrophages rather than T lymphocytes. These activated macrophages also manifested in vitro tumor cytolysis. In vivo studies indicated that peritoneal macrophages from mice treated with single oral doses of 100 to 400 mg/kg of the compound were also inhibitory to tumor cell growth in vitro. Effector macrophages became demonstrable in mice as early as 1 day after drug administration, reached peak activity at day 12, and disappeared by day 31, indicating a rapid onset but long-persisting effect. The tumor cytostatic activity of these macrophages was augmented by endotoxin at the dose of endotoxin that, in itself, had no effect. The addition of protease inhibitors, N-alpha-p-tosyl-L-lysine chloromethyl ketone and aprotinin, to cultures markedly diminished the cytostatic effect, suggesting that the release of neutral protease(s) could account for the inhibitory effects of the macrophages. On the other hand, hydrogen peroxide and arginase seemed excluded as the mechanism of action because the effect was not sensitive to treatment with catalase and exogenous arginine. The present findings indicate that CL 246,738 is an orally active immunopotentiator capable of inducing tumor-inhibitory macrophages both in vitro and in vivo.
Asunto(s)
Acridinas/farmacología , Adyuvantes Inmunológicos/farmacología , Antineoplásicos/farmacología , Leucemia Experimental/terapia , Activación de Macrófagos/efectos de los fármacos , Animales , Líquido Ascítico/inmunología , Citotoxicidad Inmunológica/efectos de los fármacos , Leucemia Experimental/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBARESUMEN
An effort was made to investigate the effects of a novel immunopotentiator, N-[4-[(4-fluorophenyl)sulfonyl]phenyl]acetamide (CL 259,763), on the generation of tumoricidal effector cells. It was demonstrated that a single oral dose of the compound (100-600 mg/kg) induced in mice a population of peritoneal macrophages capable of inhibiting the growth of tumor cells. These activated macrophages released proteases which seemed responsible for the tumor cell inhibition because the cytostatic activity was abrogated in the presence of protease inhibitors TLCK and aprotinin. On the other hand, addition of catalase and exogenous arginine to the culture failed to alter the effect, suggesting that hydrogen peroxide and arginase did not participate in this system. Although induction of cytolytic T-lymphocytes (CTL) reactive with syngeneic tumor cells was achievable in mice previously sensitized to the tumor, treatment with CL 259,763 rendered these animals even more responsive to tumor antigens resulting in a significant enhancement of tumor cell destruction. The compound was effective in augmenting the CTL response over a rather broad dose range of 25-200 mg/kg. In contrast to these stimulatory effects, the cytolytic activity of natural killer cells seemed not to be affected by the compound. Taken together, CL 259,763 is an orally active immunomodulator capable of inducing tumor inhibitory macrophages and potentiating CTL responses to syngeneic tumor cells and, therefore, may prove clinically useful in the treatment of neoplastic diseases.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Neoplasias Experimentales/inmunología , Sulfonas/farmacología , Animales , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Activación de Macrófagos/fisiología , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias Experimentales/patología , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of 111In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Neoplasias , Melanoma/inmunología , Anticuerpos Antineoplásicos , Reacciones Antígeno-Anticuerpo/efectos de la radiación , Antígenos de Superficie , División Celular , Membrana Celular/inmunología , Células Cultivadas , Endocitosis , HumanosRESUMEN
Previous reports have documented that mice bearing plasmacytomas (PC) are suppressed in their ability to mount a primary antibody response, whereas cellular immunity appears to be normal. Studies presented here provide evidence that T cell responsiveness is also depressed in BALB/c mice bearing the Lieberman plasmacytoma (Lpc-1). For instance, splenocytes from mice bearing large tumours were impaired in their in vitro ability to respond to the T cell mitogen, mount an appropriate alloreactive cytolytic T lymphocyte response, and produce interleukin-2 (IL-2). A population of suppressor cells was detected in the spleens 7 days after tumour implantation as evidenced by their ability to prevent normal splenocytes not only from responding to antigens in mixed lymphocyte culture, but also from producing IL-2. A similar inhibitory effect was observed with culture supernatants of these cells, indicating the existence of a soluble suppressive factor. Therefore, the present study demonstrates that cellular immune responses are impaired in mice bearing Lpc-1 tumours and that this effect may be due to the generation of suppressor cells and/or a suppressive factor.
Asunto(s)
Inmunidad Celular , Linfocitos/inmunología , Monocitos/fisiología , Neoplasias Experimentales/inmunología , Plasmacitoma/inmunología , Animales , Sustancias de Crecimiento/inmunología , Interleucina-2/inmunología , Isoantígenos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Neoplasias Experimentales/etiología , Plasmacitoma/etiología , Bazo/inmunología , Linfocitos T Reguladores/inmunología , Factores de TiempoRESUMEN
Two monoclonal antibodies (mAb), designated PS-7.6 and PS-11.2, were generated against recombinant porcine growth hormone (pGH) and shown to enhance the hormonal activity in promoting the growth of animals. The mAb were compared and their functional relationship investigated. It was demonstrated by radioimmunoassay that PS-11.2 did not compete, but rather enhanced binding of 125I-pGH tracer to PS-7.6, suggesting that both mAb recognized distinct epitopes and also were additive in their antigen bindings. Surface plasmon resonance analysis using optical BIAcore technology (Pharmacia Biosensor, Piscataway NJ, USA) provided additional data to support this idea because pGH, after being captured by PS-11.2, remained capable of interacting with PS-7.6. An anti-idiotypic mAb was employed and shown to interact with PS-7.6 but not PS-11.2, implying that the differences in the Fab variable regions of these two mAb might contribute to their epitope specificity. Binding kinetics were determined by the BIAcore and the individual affinities of PS-7.6 and PS-11.2 to pGH were 6.8 x 10(-8) and 1.2 x 10(-9) mol/L, respectively. When these mAb were sequentially subjected to the BIAcore, however, their affinities decreased by approximately 100-fold. Therefore, binding of pGH with one mAb significantly impaired a subsequent interaction with another mAb despite the fact that both mAb targeted different epitopes. Hypophysectomized rats were used for functional analysis and pGH was active in promoting growth of these GH-deficient animals. The hormonal effect was further improved by incubating pGH with either PS-7.6 or PS-11.2 prior to administration. However, enhancement by individual mAb was completely abolished when pGH was treated with both mAb together, indicating their unpredictable biological interference with each other. Therefore, the present findings clearly demonstrate that although PS-7.6 and PS-11.2 recognize separate epitopes, their individual interactions with pGH are closely interrelated both immunologically and biologically.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Epítopos/inmunología , Hormona del Crecimiento/inmunología , Animales , Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales/metabolismo , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo/fisiología , Técnicas Biosensibles , Epítopos/análisis , Femenino , Cinética , Radioinmunoensayo , Ratas , Ratas Sprague-Dawley , PorcinosRESUMEN
A clinically active anticancer agent, mitoxantrone (MX): 1, 4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9, 10-anthracenedione dihydrochloride, was studied for its potential inhibitory effect on alloreactivity induction. Addition of MX to mixed lymphocyte cultures (MLC) not only inhibited the proliferative response of lymphocytes to alloantigens but also prevented the generation of cytolytic T lymphocytes (CTL). MX showed a long-lasting effect in vitro and acted at the inductive rather than the effector phase of the CTL response as indicated by its failure to alter the activity of those CTL already generated in MLC. MX also inhibited CTL induction in mice. However, the precursors of CTL appeared to be spared in these animals as supported by limiting dilution analysis and also because CTL could be reactivated by exposure of splenocytes to the same or different alloantigens in MLC. The present findings demonstrate that MX is a potent immunosuppressive agent and as such might prove to be clinically useful in the treatment of autoimmune diseases or find utility in the organ transplantation field.