RESUMEN
A portion of the antigen I/II (spaA, B, P1) gene of Streptococcus sobrinus 6715, containing the coding sequence for the amino terminal 684 amino acids of the protein, was cloned in bacteriophage lambda GT10. Selection was by immunological detection using a polyclonal antiserum to the antigen I/II from Strep. mutans. From the amino acid sequence, peptides were synthesized, 15 amino acids in length, that covered the entire sequence. In total, 260 synthetic peptides were synthesized and evaluated for their immunogenicity in Balb/C mice. Thirty-nine peptides were immunogenic, without carrier, and the antisera generated were tested for their ability to bind cells of Strep. mutans and Strep. sobrinus in a solid-phase assay. Antisera corresponding to peptides from five regions on the I/II molecule bound cells of both bacterial species. These peptides were then evaluated for their ability to stimulate in vitro murine lymphocyte proliferation, after in vivo immunization with Strep. sobrinus cells. Two of the peptides were capable of stimulating proliferation, as determined by incorporation of [3H]-thymidine into murine lymph node cells. The sequences of these 5 peptides were then compared to sequences found in the antigen I/II from Strep. mutans (Kelly et al., 1989). As expected, there was considerable homology between the cross-reactive peptides synthesized and the analogous region from Strep. mutans. This homology was not usually contiguous and suggests that the antibodies bind a face of antigen I/II that is in an alpha-helical conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Antígenos Bacterianos/genética , Proteínas Bacterianas/genética , Glicoproteínas de Membrana , Nucleótidos/genética , Streptococcus/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Antígenos de Superficie/genética , Antígenos de Superficie/inmunología , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos , Datos de Secuencia Molecular , Streptococcus/genética , Streptococcus mutans/genética , Streptococcus mutans/inmunología , Vacunas Sintéticas/síntesis química , Vacunas Sintéticas/inmunologíaRESUMEN
Monoclonal antibody MB3.6 (IgG3) recognizes disialoganglioside GD3, which represents a major surface marker on most human melanoma cells. We demonstrate that this antibody effectively lyses four human melanoma cell lines expressing significant levels of GD3 on their surface by either of two mechanisms: antibody-dependent cellular cytotoxicity (ADCC) or complement-mediated cytotoxicity. However, a melanoma cell line that expresses minimal levels of GD3 on 13% of the cells shows insignificant lysis by MB3.6 by either of these two mechanisms, suggesting that a threshold level of antigen expression may be required for effective in vitro cytolysis. In addition, monoclonal antibody (MAb) MB3.6 effectively inhibits establishment and growth of human melanoma tumors in the nude mouse when injected 24 hr after subcutaneous inoculation of tumor cells. Furthermore, MB3.6 produces specific regression of established melanoma tumors when injected 7 days after the subcutaneous inoculation of tumor cells. In contrast, tumor growth in animals injected with the melanoma cell line expressing minimal levels of GD3 was not affected by MAb MB3.6. These data indicate that once appropriate levels of the GD3 ganglioside are expressed on human melanoma cells, MAb MB3.6 can mediate tumor cell killing in vitro and in vivo and, thus, may prove useful for effective immunotherapy of human malignant melanoma.
Asunto(s)
Anticuerpos Antineoplásicos/uso terapéutico , Antígenos de Neoplasias/inmunología , Gangliósidos/inmunología , Melanoma/inmunología , Animales , Especificidad de Anticuerpos , Citotoxicidad Celular Dependiente de Anticuerpos , Proteínas del Sistema Complemento/inmunología , Citotoxicidad Inmunológica , Humanos , Melanoma/patología , Melanoma/terapia , Ratones , Ratones DesnudosRESUMEN
The simultaneous injection of monoclonal antibody 9.2.27, directed against a chondroitin sulfate proteoglycan preferentially expressed on human melanoma cells, and 2 X 10(7) mononuclear splenocytes, eradicated established, progressively growing human melanoma tumors in nude mice. Neither splenocytes nor antibody alone achieved significant tumor regression. The cells responsible for tumor elimination are most likely natural killer (NK) cells: they are present in splenocytes of T cell-deficient nude mice, and cloned cells with NK activity are able to suppress tumor growth. Moreover, splenocytes treated with anti-asialo GM1 and complement or harvested from NK-deficient C57BL/6 beige mice did not cause tumor rejection. Furthermore, treatment of BALB/c nude mice just before injection with anti-asialo GM1 antiserum, which is known to eliminate NK activity in vivo, resulted in better tumor growth. In addition, evidence is presented that cells with NK activity are probably the effectors responsible for melanoma target cell lysis in vitro: Antibody-dependent and -independent cell-mediated lysis of M21 melanoma cells was suppressed when splenocytes were preincubated with complement and antibodies specific for cell surface antigens of NK cells, i.e., anti-asialo GM1, anti-Qa5, and anti-NK1.1. Moreover, splenocytes of C57BL/6 beige mice were not able to lyse M21 cells in vitro. These results strongly support the conclusion that cells with NK activity are indeed responsible for the antibody-dependent destruction of M21 melanoma cells in vivo and in vitro.
Asunto(s)
Anticuerpos Antineoplásicos/inmunología , Gangliósido G(M1) , Células Asesinas Naturales/inmunología , Melanoma/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular , Glicoesfingolípidos/inmunología , Humanos , Inmunización Pasiva , Ratones , Ratones Desnudos , Bazo/inmunologíaRESUMEN
A murine monoclonal antibody (monoclonal antibody 126) produced against cultured human neuroblastoma cells (LAN-1) was found to be specifically directed to a disialoganglioside (GD2) antigen preferentially expressed on both cell lines and tissues derived from melanoma and neuroblastoma. In enzyme-linked immunosorbent assays, monoclonal antibody 126 failed to react with leukemic and lymphoblastoid cells as well as with a variety of carcinoma and sarcoma cell lines. Immunohistological analysis by the immunoperoxidase technique revealed strong reactivity of monoclonal antibody 126 with frozen and formaldehyde-fixed neuroblastoma and melanoma tissues. Tissues from patients with glioma or with small cell cancer of the lung showed faint staining, whereas those from individuals with sarcoma, lymphoma, and a variety of other neoplasms proved to be negative. Sera of neuroblastoma patients showed significantly elevated GD2 levels compared to normal children (p less than 0.001) and children with other tumors (p less than 0.001) as determined by a quantitative competitive enzyme-linked immunosorbent assay. Furthermore, the GD2 serum level of one neuroblastoma patient, when followed serially, was found to correlate with progression of disease, suggesting the potential usefulness of this assay for the diagnosis and monitoring of neuroblastoma.
Asunto(s)
Gangliósidos/análisis , Neuroblastoma/análisis , Anticuerpos Monoclonales , Línea Celular , Ensayo de Inmunoadsorción Enzimática , Gangliósidos/sangre , Humanos , Neoplasias/análisis , Neuroblastoma/sangre , Neuroblastoma/patologíaRESUMEN
Experiments were performed to analyze the modulation of macrophage Ia expression and biosynthesis by Salmonella minnesota-derived lipopolysaccharide (LPS) in vivo. The i.p. injection of LPS into LPS-responder mice caused a dramatic increase in the Ia expression of the peritoneal macrophage population harvested 1 wk after injection. As little as 1 ng of lipid-rich Re595 LPS per mouse caused a significant I-Ak increase, and 1 microgram was optimal; wild-type S. minnesota LPS was less active. No I-Ak induction by LPS was observed in the LPS-nonresponder strain C3H/HeJ. LPS-induced macrophages showed a 6- to 16-fold increase in I-Ak expression by radioimmunoassay (RIA), a 3- to 10-fold increase in the proportion of I-Ak-positive cells, and a 10- to 15-fold increase in I-Ak biosynthetic capacity. The magnitude of this induction by LPS was comparable to increases observed after injection of live Listeria monocytogenes. The kinetics of I-Ak induction by LPS and by L. monocytogenes were different: LPS caused an initial decrease in I-Ak expression 1 day after injection, and I-Ak induction by LPS occurred more slowly and maintained heightened expression longer. Several H-2 gene products (H-2Kk, I-Ak, and I-Ek) were augmented in LPS-induced macrophages. In keeping with increased I-A and I-E expression, LPS-induced macrophages were more effective than normal macrophages in presenting antigen to T lymphocytes. We suggest that the modulation of macrophage Ia expression is one important mechanism contributing to the immunoregulatory activity of LPS.