RESUMO
Strain A/J mice made secondary indirect plaque-forming cell (PFC) responses to azobenzenearsonate (ABA) conjugates of giant keyhole limpet hemocyanin (KLH), a thymic-dependent antigen, but not to conjugates of Ficoll, a T-independent antigen. ABA-Ficoll was also unable to elicit a response in animals primed with ABA-KLH, which have an expanded anti-ABA memory cell pool. On the other hand, ABA-Ficoll rendered mice unresponsive to ABA-KLH when administered before priming or boosting with the T-dependent immunogen. Hence, the T-independent antigen was able to tolerize but unable to trigger B-memory cells responsive to the T-dependent antigen. A/J mice immunized with dinitrophenyl conjugates of Ficoll or bovine IgG (BGG) made vigorous IgM and IgG PFC responses. PFC responses to ABA-KLH and 2,4-dinitrophenyl (DNP)-BGG were abrogated by depleting mice of C3 with cobra venom factor, whereas the IgM and IgG PFC responses to DNP-Ficoll were unaffected. B lymphocytes were fractionated on the basis of receptors for C3 and the subpopulations were assayed for in vitro PFC responses to DNP-Ficoll. Very little response was obtained from complement receptor lymphocyte [CRL(+)] B cells, whereas CRL(-) cells were more responsive than unfractionated B cells. Both populations responded to a polyclonal B-cell mitogen (lipopolysaccharide). On the other hand, the in vitro PFC response to a T-dependent antigen (sheep erythrocytes) correlated with the presence of CRL(+) B cells in the cultures. However, a minor component of this response, sensitive to anti-Thy-1 serum, was made by CRL(-) B cells, indicating the existence of subpopulations of T-dependent B cells with different signalling requirements. The results suggest that most B cells responsive to T-dependent antigens possess receptors for C3 and that C3 plays an obligatory role in the response of these cells. A distinct subpopulation of B cells which lack C3 receptors respond to T-independent antigens. The precursors of PFC for the ABA epitope reside largely or exclusively in the CRL(+) compartment in A/J mice, whereas precursors for the DNP determinant are found in both compartments.
Assuntos
Formação de Anticorpos , Compostos Azo/imunologia , Linfócitos B/imunologia , Complemento C3 , Proteínas do Sistema Complemento , Linfócitos T/imunologia , p-Azobenzenoarsonato/imunologia , Animais , Proteínas de Transporte/imunologia , Feminino , Ficoll/imunologia , Hemocianinas/imunologia , Memória Imunológica , Camundongos , Camundongos Endogâmicos A , Receptores de Droga , Baço/imunologiaRESUMO
Monoclonal antibody 17.13., derived from a fusion of splenocytes of a BALB/c mouse immunized with a surgically resected poorly differentiated human laryngeal recurrent squamous cell carcinoma (SCC) with mouse Sp2/0 cells, is an IgM-K which recognizes a cytoplasmic component of basal cells. Tissue sections of malignant and normal squamous epithelium, tumors of nonsquamous origin, and normal and malignant cytological specimens were tested with an immunoperoxidase assay. Seventy-nine of 81 (98%) SCC of the head and neck, 26 of 26 (100%) SCC of the cervical and female gynecological tract, 29 of 30 (97%) SCC of the lung, 19 of 19 (100%) SCC of the oral cavity, and 17 of 17 (100%) SCC-involved neck lymph nodes reacted strongly. Various carcinomas from breast, colon, ovary, and others were unreactive. In normal squamous epithelial tissues, monoclonal antibody 17.13. reacts only with basal cells but not the cells above the basal layers. Normal tissues from heart, liver, spleen, kidney, bladder, colon, ovary, stomach, pancreas, breast, lung, prostate, thyroid, and lymph nodes were unreactive with the exception of myoepithelial cells. Monoclonal antibody 17.13. may be useful in the diagnosis and management of SCC.
Assuntos
Anticorpos Monoclonais/imunologia , Carcinoma de Células Escamosas/imunologia , Animais , Carcinoma de Células Escamosas/diagnóstico , Citodiagnóstico , Epitélio/imunologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Neoplasias Laríngeas/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Células Tumorais CultivadasRESUMO
We have developed a sensitive hemolytic assay for mouse C3, utilizing readily available guinea pig complement components and standard techniques, which allows C3 to be detected in mouse serum diluted many thousand-fold. Mouse serum also contains a potent, relatively heat-stable C3-inactivator system, which is largely blocked by Suramin.
Assuntos
Complemento C3/isolamento & purificação , Técnica de Placa Hemolítica , Camundongos Endogâmicos/sangue , Animais , Venenos Elapídicos/farmacologia , Feminino , Temperatura Alta , Camundongos , Concentração Osmolar , Suramina/farmacologia , Fatores de TempoRESUMO
High-throughput screening of in-house compound libraries led to the discovery of a novel antibacterial agent, compound 1 (MIC: 12-25 microM against S. pyogenes). In an effort to improve the activity of this active compound, a series of 2-substituted quinazolines was synthesized and evaluated in several antibacterial assays. One such compound (22) displayed improved broad-spectrum antibacterial activity against a variety of bacterial strains. This molecule also inhibited transcription/translation of bacterial RNA, suggesting a mechanism for its antibiotic effects. Structure-activity relationship studies of 22 led to the synthesis of another 24 compounds. Although some of these molecules were found to be active in bacterial growth assays, none were as potent as 22. Compound 22 was tested for its ability to cure a systemic K. pneumonia infection in the mouse and displayed moderate effects compared with a control antibiotic, gentamycin.
Assuntos
Antibacterianos/síntese química , Benzoatos/síntese química , Quinazolinas/síntese química , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Benzoatos/química , Benzoatos/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Infecções por Klebsiella/tratamento farmacológico , Klebsiella pneumoniae/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Biossíntese de Proteínas/efeitos dos fármacos , Quinazolinas/química , Quinazolinas/farmacologia , RNA Bacteriano/genética , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacos , Relação Estrutura-Atividade , Transcrição Gênica/efeitos dos fármacosRESUMO
Monoclonal antibody 17.13.Cl.10 is a murine IgM kappa monoclonal antibody (Mab) that stains frozen section squamous cell carcinoma (SCC) homogeneously and intensely with a sensitivity greater than 98%, including 106/107 SCC specimens from the head and neck. It was produced using a human laryngeal SCC as immunogen and screened using frozen section human tissue. Monoclonal antibody 17.13.Cl.10 faintly stains the basal layer of normal squamous epithelium, does not stain normal organ tissue other than myoepithelial cells, and reacts with few non-SCC tumors. It, therefore, may be a useful adjunct to standard histopathologic criteria for the diagnosis of SCC. It may prove helpful in the investigation of tumor-associated antigens. Despite major technical and immunologic problems, monoclonal antibodies to functional tissue-specific tumor-associated antigens have the potential to play a major role in imaging and in treatment in the future.
Assuntos
Anticorpos Monoclonais/imunologia , Carcinoma de Células Escamosas/imunologia , Neoplasias Laríngeas/imunologia , Animais , Anticorpos Monoclonais/biossíntese , Carcinoma de Células Escamosas/diagnóstico , Feminino , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Coloração e RotulagemRESUMO
The capacity of the trinitrophenyl haptenic group coupled to a series of chemically dissimilar carriers to cross-stimulate putative T-dependent and T-independent B-cell subpopulations was determined by using an in vitro limiting dilution technique to generate primary IgM responses. TNP-Ficoll and TNP-dextran, two T-independent antigens with little or no polyclonal mitogenicity, stimulate the same population of anti-TNP precursors, which is distinct from the precursor population activated by TNP-LPS, a T-independent polyclonal mitogen, or by TNP-HRBC, a T-dependent antigen. TNP-LPS and TNP-HRBC activate the same precursor population, indicating that LPS can substitute for the T cell signal in T-dependent B-cell responses, whereas nonmitogenic T-independent antigens cannot. However, the cumulative evidence from this and other laboratories suggests that LPS and T-dependent antigens activate B cells by different mechanisms. TNP conjugates of Ficoll and dextran, which are relatively poor inducers of polyclonal B cell activation, induced larger anti-TNP clones than did TNP-LPS, a strong polyclonal mitogen. Macrophages are required for the anti-TNP-Ficoll/anti-TNP-dextran response, whereas, a similar requirement has not been shown for the anti-TNP-LPS response. Thus, macrophages may function as polyclonal B cell activators in T-independent responses. Experiments in which TNP was coupled directly onto the macrophage surface support this hypothesis. B-cell heterogenity in T-dependent responses is suggested by experiments using the C3 receptor as a marker for functional subpopulations of B cells. Murine T cells cooperate with B cells that carry a receptor for C3 and with at least some B cells which lack the C3 receptor in a primary in vitro antibody response. In vitro culture experiments using populations of B cells fractionated on the basis of the C3 receptor showed that CR+ cells were unable to make T-dependent antibody responses in the presence of anti-C3 antibody, whereas the response of CR- B cells was unaffected. Using irradiated, carrier-primed spleen cells from B10.A mice as a source of helper cells for B cells derived from various congenic strains in an in vitro primary IgM response to TNP-KLH, CR+ B cells cooperated across haplotype differences in the I region of the MHC, whereas CR- B cells did not. Preliminary mapping experiments for the genetic restriction of CR- B cells suggest complementation between the I-A and I-C subregions of the MHC. These findings tentatively suggest the existence of alternative cooperative pathways between T cells and B cell subpopulations.
Assuntos
Formação de Anticorpos , Antígenos , Linfócitos B/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Animais , Separação Celular , Células Clonais/imunologia , Haptenos , Macrófagos/imunologia , Camundongos , Polissacarídeos/imunologia , Baço/imunologia , Trinitrobenzenos/imunologiaRESUMO
IgM-sensitized sheep E bearing guinea pig C4 and C3b (EAC43bgp) react as strongly with mouse spleen cell C3b receptors as do optimally prepared EACmo. Yet, when the C3b is converted to C3d by incubation with heated serum at low ionic strength, reactivity with mouse spleen cells is completely lost whereas binding to guinea pig spleen cells remains high. It is concluded on the basis of this and other evidence that EAC43bgp are a sensitive and specific indicator for mouse lymphocyte C3b receptors. Degradation of the C3b on these cells will not cause misleading binding to C3d receptors, as could occur with cells bearing mouse C3b.
Assuntos
Complemento C3 , Linfócitos/imunologia , Animais , Sítios de Ligação , Complemento C3/metabolismo , Eritrócitos/imunologia , Cobaias , Humanos , Imunoglobulina M , Camundongos , Receptores de Antígenos de Linfócitos B , Formação de Roseta , Ovinos , Especificidade da Espécie , Baço/imunologiaRESUMO
We assessed the ability of monoclonal antibody (MAb) 17.13 to react with human oral squamous cell carcinoma (OSCC) and other oral tissues. MAb 17.13 was reacted with acetone-fixed, serial frozen sections of 21 OSCC, 7 fibromas, 2 squamous papillomas, 1 melanosis and 3 normal oral mucosa, by means of an avidin-biotin-immunoperoxidase assay. Twenty-one of 21 tumors (100% sensitivity) reacted strongly with MAb 17.13, with a homogeneous staining pattern. Individual tumor cells could be clearly seen, improving the detection of microinvasion or borderline lesions. The epithelium of benign lesions and normal oral mucosa showed staining of the basal cells only. We conclude MAb 17.13 could be used as an accurate diagnostic tool for OSCC.
Assuntos
Anticorpos Monoclonais , Carcinoma de Células Escamosas/patologia , Neoplasias Bucais/patologia , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/classificação , Especificidade de Anticorpos , Carcinoma de Células Escamosas/diagnóstico , Epitélio/patologia , Humanos , Técnicas Imunoenzimáticas , Cadeias Leves de Imunoglobulina , Imunoglobulina M , Cadeias kappa de Imunoglobulina , Neoplasias Bucais/diagnóstico , Coloração e RotulagemRESUMO
BDF1 mice treated with CoV had markedly reduced levels (less than 20%) of native serum C3 32 hr later, whereas the frequency of splenic CR+ cells was normal. CoV treatment before immunization reduced the IgM PFC response to a T-dependent antigen (TNP-SRBC) by more than 60%. Inclusion of highly specific anti-C3 antibody had no effect on the T-dependent IgM response of CR- B cells. The residual PFC responses in cultures of unfractionated spleen cells treated with anti-C3 could be largely or completely accounted for by CR- B cells in the cultures. The effect of anti-C3 antibody was not due to cytotoxicity. These data collectively indicate that the effect of CoV on T-dependent antibody responses is due to decreased C3 in serum rather than to interaction of C receptors directly with CoV or with C3 cleavage products. They suggest the existence of at least two distinct pathways of T-B cooperation, one in which C3 is an obligatory participant and another in which it may be uninvolved.
Assuntos
Linfócitos B/imunologia , Proteínas do Sistema Complemento , Linfócitos T/imunologia , Animais , Anticorpos , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos , Complemento C3/análise , Complemento C3/antagonistas & inibidores , Testes Imunológicos de Citotoxicidade , Eritrócitos/imunologia , Feminino , Técnica de Placa Hemolítica , Imunoglobulina M , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos , Venenos de Serpentes/farmacologia , Trinitrobenzenos/imunologiaRESUMO
When EAC43b were treated with heated serum in EDTA, reactivity with bovine conglutinin appeared rapidly, even at 0 degrees C, and almost simultaneously with the loss of C3b rosetting capacity. At the time conglutinability first appeared, there was no detectable decrease in I-A or hemolytic C3 activity, and no detectable C3 antigen release from the cells. With prolonged exposure to heated serum in EDTA, I-A (immune adherence) and hemolytic C3 activity were lost. If this exposure was at 37 degrees C, C3 antigen became strongly detectable in the supernatant fluid, and eventually conglutinability was markedly reduced or lost, whereas C3d rosettes were unaffected. We suggest that bovine conglutinin reacts with some early product of C3b degradation, rather than with C3d, and propose that this intermediate be designated C3k. We have developed a semi-quantitative assay for bovine conglutinin, utilizing a Coulter Counter to register the decrease in total particles due to red cell aggregation. By using this method, we have detected conglutination with mouse complement (C) as well as with that from man and the guinea pig.
Assuntos
Complemento C3 , Testes de Fixação de Complemento , Animais , Bovinos , Proteínas do Sistema Complemento , Ácido Edético , Cobaias , Hemólise , Temperatura Alta , Humanos , Reação de Imunoaderência , Cinética , Camundongos , Camundongos Endogâmicos A , Concentração Osmolar , Formação de Roseta , Temperatura , Fatores de TempoRESUMO
We studied the reactivity of monoclonal antibodies (MAbs) 17.13 and 63.12 with normal and diseased human oral mucosa by means of the immunoperoxidase technique. The specimens included: 22 normal oral tissues, 20 benign tumors, 17 lichen planus, 23 focal keratosis and epithelial hyperplasias, 18 proliferative verrucous leukoplakias, 20 dysplasias, and 21 squamous cell carcinomas. In most cases of normal mucosa and benign lesions, MAb 17.13 stained basal epithelial cells only, whereas MAb 63.12 stained all cell layers above the basal cells. In the premalignant and malignant lesions MAb 17.13 stained above the basal cells and MAb 63.12 either stained areas not stained by MAb 17.13 or the staining was absent. Based on the different staining patterns observed, there appears to be a potential value of these new reagents in diagnostic histopathology regarding specimens with equivocal cellular morphology.
Assuntos
Anticorpos Monoclonais , Doenças da Boca/patologia , Mucosa Bucal/citologia , Neoplasias Bucais/patologia , Lesões Pré-Cancerosas/patologia , Carcinoma de Células Escamosas/patologia , Epitélio/patologia , Fibroma/patologia , Gengiva/citologia , Humanos , Hiperplasia , Técnicas Imunoenzimáticas , Ceratose/patologia , Leucoplasia Oral/patologia , Líquen Plano/patologia , Linfócitos/patologia , Mucosa Bucal/patologia , Transtornos da Pigmentação/patologia , Coloração e Rotulagem , Língua/citologiaRESUMO
A large, O-linked glycoprotein, termed PAS-O, is a major differentiation antigen on the surface of normal lactating breast epithelia and is also found on the surface of many mammary tumors and mammary carcinoma cell lines. A characteristic feature of populations of tumor cells that express PAS-O is the cell-to-cell heterogeneity with respect to the presence or absence of the molecule. In this study, we used the human mammary carcinoma line 734B and a set of six monoclonal antibodies reactive with PAS-O to study the basis of this heterogeneity. Extensive Western blot analysis of antibody binding to PAS-O in milk fat globule membranes and in skim milk revealed that the antibodies all recognized different epitopes of PAS-O. Moreover, the epitopes were destroyed by periodic acid oxidation, demonstrating their oligosaccharide basis. All six monoclonal antibodies stained the 734B cells heterogeneously. In addition, five clones derived from the parent 734B population also exhibited heterogeneity in the expression of each of the epitopes. An analysis of staining of the 734B clones revealed that, in some cases, certain cells within the cloned population stained with one monoclonal antibody but not with another antibody. Significantly, though, when the 734B cells were treated with neuraminidase prior to antibody staining, most of the heterogeneity was eliminated, and all but one of the monoclonal antibodies stained 90-100% of the cells. This increase in cell staining was matched by an increase in PAS-O staining on Western blots. We conclude that heterogeneity in PAS-O expression on 734B cells is due partly to masking of epitopes by sialic acid and a variation (on a cell-to-cell basis) in the extent of PAS-O sialylation.
Assuntos
Neoplasias da Mama/metabolismo , Comunicação Celular , Epitopos/biossíntese , Glicoproteínas de Membrana/biossíntese , Mucinas/biossíntese , Anticorpos Monoclonais , Western Blotting , Linhagem Celular , Células Clonais , Feminino , Humanos , Microscopia Eletrônica , Microscopia de Contraste de Fase , Peso Molecular , Mucina-1 , Ácido N-Acetilneuramínico , Ácidos Siálicos/análise , Propriedades de SuperfícieRESUMO
The arterioles on the surface of the mouse brain (pial arterioles) were observed by in vivo microscopy. A focus of minor endothelial damage was produced in a single pial arteriole in each mouse by briefly exposing the site to a helium neon laser after an intravenous injection of Evans blue. Mice were injected 10 minutes before injury with a monoclonal antibody (MAb) to mouse CD31, also known as platelet endothelial cell adhesion molecule. This treatment doubled (P < .01) the time required for the laser to produce a recognizable platelet aggregate. In additional experiments, an MAb to mouse CD61 and an MAb to mouse intercellular adhesion molecule 1 had no effect. The data support previous observations indicating that platelet adhesion/aggregation in this model is induced by endothelial injury without exposure of basal lamina. The data are consistent with the hypothesis that the endothelial injury exposes or activates a platelet endothelial cell adhesion molecule on the endothelium which is blocked by the MAb directed against CD31. This may be the first demonstration of an effect of an anti-platelet endothelial cell adhesion molecule on platelet endothelial cell adhesion molecule on platelet adhesion/aggregation in vivo.