RESUMO
Glioblastomas (GBM) are the most frequent and aggressive brain tumors due to their recurrence and resistance to current therapies. These characteristics are associated with the presence of glioma stem cells (GSCs), mainly identified by the detection of the membrane antigens CD133 and CD15. The main source of GSCs has been biopsies of tumors. However, alternatives are sought from cell lines because more homogeneous populations can be obtained with high yields. This chapter describes a method for the enrichment and characterization of GSCs from cell lines derived from human GBM by selective culture with serum-free neural stem cell medium and growth factors. The technique offers alternatives for the enrichment and characterization of GSCs, that could contribute to a better understanding of the biology of GBMs.
Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica/genética , Glioblastoma/patologia , Células-Tronco Neoplásicas/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Antígeno AC133/análise , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Citometria de Fluxo , Glioblastoma/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Antígenos CD15/análise , Células-Tronco Neoplásicas/fisiologia , Células-Tronco Neurais/citologiaRESUMO
Peri-implantitis is an infectious disease characterized by inflammation of the tissues surrounding the implant, bleeding on probing with or without suppuration, and bone loss. Peri-implant lesions contain a leukocyte infiltrate of plasma cells, lymphocytes, macrophages and neutrophils. A survey of the literature did not show any studies reporting an association between hypoxia and peri-implantitis. The aim of the present cross-sectional study was to evaluate histological changes and immunostaining for CD15, CD57 and HIF-1α in the peri-implant mucosa of patients with and without peri-implantitis. Mucosal biopsies were obtained from 18 patients with peri-implantitis and 10 control subjects without peri-implantitis at a private health care center between 2010 and 2012. The sections were fixed in 10% buffered formalin, processed and embedded in paraffin for histopathological and immunohistochemical study. Acanthosis, spongiosis and exocytosis were observed in both groups, with no significant difference between them. The peri-implantitis group showed increased immunostaining for CD15, a neutrophil marker, and HIF-1α, a tissue hypoxia marker, but no significant difference in immunostaining for CD57, a Natural Killer cell marker. The increase in neutrophil (CD15) and hypoxia (HIF-1α) markers in patients with peri-implantitis suggests an active participation of neutrophils and hypoxia in the pathogenesis of this disease. Since the present study was the first to evaluate the expression of CD15, CD57 and HIF-1α in peri-implant tissues, further studies should be performed to better understand the role of these molecules in peri-implantitis.
Assuntos
Implantes Dentários/efeitos adversos , Peri-Implantite/imunologia , Estomatite/imunologia , Idoso , Biomarcadores/análise , Biópsia , Antígenos CD57/análise , Antígenos CD57/biossíntese , Estudos Transversais , Feminino , Fucosiltransferases/análise , Fucosiltransferases/biossíntese , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/análise , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Antígenos CD15/análise , Antígenos CD15/biossíntese , Masculino , Pessoa de Meia-IdadeRESUMO
The aim was to compare the expression of MUC1 and carbohydrate antigens in 124 tissue samples; 42 fibroadenoma (FA), 23 nonproliferative benign diseases (NPF), 25 usual epithelial hyperplasia (UEH), 7 atypical ductal hyperplasia (ADH), and 27 breast normal tissues. An immunohistochemical approach was adopted, using the following antibodies: reactive with MUC1 variable number of tandem repeats (C595, HMFG2, and SM3 monoclonal antibodies), anti-MUC1-cytoplasmic tail polyclonal antibody (CT33), and anti-carbohydrate antigens (sialyl Lewis x, Lewis x, Lewis y, Tn, and Thomsen-Friedenreich epitopes). Positive area of reaction, intensity, and pattern of expression were considered. A reactivity index was calculated as intensity (I) x 100+percentage of positive area (A). Statistical analysis comprised frequency analysis, P < 0.05, analysis of variance, and multiple correlation with principal component analysis. All samples expressed MUC1, detected by at least one anti-MUC1 antibody whereas Lewis x was the carbohydrate antigen most frequently found in all groups whereas variable number of tandem repeats MUC1 and Lewis x showed the highest correlation: 93% of normal samples, 62.5% of NPF, 87% of FA, 85% of UEH, and finally 80% of ADH. Although principal component analysis using reactivity indexes explained only 39% of data variability, normal samples appeared grouped and separated from benign breast diseases, which remained spread. Thomsen-Friedenreich was the only antigen that showed an increased tendency for positive expression and intensity from NPF through FA, UEH to ADH, whereas it was not detected in normals. With respect to the pattern of expression, an apical pattern was predominantly found in all the groups.
Assuntos
Antígenos Glicosídicos Associados a Tumores/análise , Neoplasias da Mama/diagnóstico , Mucina-1/análise , Anticorpos , Biomarcadores Tumorais/análise , Feminino , Humanos , Imuno-Histoquímica/métodos , Antígenos CD15/análise , Glândulas Mamárias Humanas/química , Análise de Componente PrincipalRESUMO
Paracoccidioidomycosis (Pmycosis) is one of the most common deep mycoses in many regions of Latin America, particularly in Brazil. Microscopically, it shows granulomatous inflammatory reaction with giant cells, macrophages, lymphocytes, plasma cells, polymorphonuclear neutrophilic leukocytes, and eosinophils. The purpose of this study was to assess the distribution of inflammatory cells in oral Pmycosis. Fifteen cases of oral Pmycosis were studied by immunohistochemistry for the presence of macrophages, CD4(+) and CD8(+) lymphocytes, CD20(+), CD15(+), and S100(+) cells. Macrophages were the main cells in well-organized granulomas and non-granulomatous areas. The CD4 phenotype was predominant in well-organized granulomas and a balance between CD4(+) and CD8(+) cells was observed in non-granulomatous areas. Dendritic, S100(+) cells were found mainly in the epithelium, in subepithelial connective tissue, and at the periphery of organized granulomas. CD15(+) cells were concentrated mainly in areas of intraepithelial microabscess and ulceration. Macrophages and T cells are the predominant cells in oral Pmycosis. Well-organized granulomas contain fewer yeast particles, indicating a more effective host immune response. Better understanding of the histopathological changes in oral Pmycosis might help determine treatment, severity and systemic involvement of the disease.
Assuntos
Células Gigantes/patologia , Leucócitos/patologia , Doenças da Boca/microbiologia , Paracoccidioidomicose/patologia , Fagócitos/patologia , Abscesso/microbiologia , Adulto , Antígenos CD20/análise , Linfócitos B/patologia , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/patologia , Células Dendríticas/patologia , Epitélio/microbiologia , Granulócitos/patologia , Granuloma/microbiologia , Humanos , Inflamação/patologia , Antígenos CD15/análise , Macrófagos/patologia , Masculino , Pessoa de Meia-Idade , Doenças da Boca/patologia , Úlceras Orais/microbiologia , Proteínas S100/análiseRESUMO
Neoplastic cells in classical Hodgkin's lymphomas (cHL) seem to correspond to defective germinal center B-cells, which escape from apoptosis. Epstein-Barr virus (EBV) may be implicated in this protective mechanism. The aim of the present study was to determine the expression of apoptosis-related proteins in cHL among adult patients and correlate them with EBV expression, clinical findings and survival. EBV was detected by in situ hybridization (Epstein-Barr Encoded RNA, EBER, probe). Immunohistochemistry was used on paraffin sections to detect LMP-1/EBV, CD15 and the apoptosis-related proteins (bcl-2, bax, bcl-X, mcl-1 and CD95). Seventy-eight patients seen at our Institution were studied: 36 male and 42 female. Median age was 31 years (15-75 years). Histological types of cHL were: 61 nodular sclerosis (47 NS1 and 14 NS2), 15 mixed cellularity (MC), 1 lymphocyte depletion and 1 unclassified. In 50 cases there was EBV expression (64%). At least one apoptosis-associated protein was expressed in 92% and CD15 in 57.7% of the cases. In the univariate analysis, the following variables were related to a better overall survival: expression of CD15 (p = 0.023), expression of mcl-1 protein (p = 0.029), expression of bcl-2 protein (p = 0.028, only in a Cox model after stratification for histology) and expression of LMP-1 (p = 0.042). EBV expression presented a borderline inverse correlation with bcl-2. A prognostic index (PI) developed in the present study revealed that simultaneous expression of bcl-2, mcl-1 and LMP-1 was significant and independently correlated with an excellent survival.
Assuntos
Apoptose , Doença de Hodgkin/metabolismo , Antígenos CD15/análise , Proteínas de Neoplasias/análise , Proteínas Proto-Oncogênicas c-bcl-2/análise , Adolescente , Adulto , Idoso , Biomarcadores , Infecções por Vírus Epstein-Barr/metabolismo , Feminino , Doença de Hodgkin/mortalidade , Doença de Hodgkin/patologia , Doença de Hodgkin/virologia , Humanos , Tábuas de Vida , Masculino , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Prognóstico , Modelos de Riscos Proporcionais , Proteínas Proto-Oncogênicas/análise , RNA Viral/análise , Estudos Retrospectivos , Análise de Sobrevida , Infecções Tumorais por Vírus/metabolismo , Proteínas da Matriz Viral/análise , Proteína X Associada a bcl-2 , Proteína bcl-X , Receptor fas/análiseRESUMO
The presence of increased number of leukocytes in semen is indicative of inflammation in the male genital tract. Inflammatory processes at this level may lead to marked impairment of sperm function, and finally to a reduction in their fertilizing capability. An immunocytological technique for the detection of seminal leukocytes was evaluated in this study. As part of the standardization technique, different fixation methods were tested to ascertain whether samples could be stored and examined later. It was found that fixation with cold acetone at freezing temperatures retained immunoreactivity until day 11 of storage. All other methods showed a significant loss of immunoreactivity, from as little as day 2 of storage. In 46 specimens with elevated numbers of round cells, number of peroxidase-positive cells and number and type of leukocytes were evaluated by means of indirect immunofluorescence. Determination of peroxidase-positive cells to detect leukocytospermia, the standard procedure recommended by the WHO, was compared with the indirect immunofluorescence technique using monoclonal antibodies. While 19 of 46 patients showed high numbers of leukocytes in the ejaculate, as determined by the immunocytological method, only 9 of these were identified to be leukocytospermic, according to the WHO (standard) procedure. This difference was statistically significant (P < 0.01) and indicates that the standard method of detection of seminal leukocytes may be inaccurate.
Assuntos
Granulócitos/citologia , Leucócitos/citologia , Sêmen/citologia , Acetona , Anticorpos Monoclonais/imunologia , Biomarcadores , Etanol , Fixadores , Técnica Indireta de Fluorescência para Anticorpo , Granulócitos/imunologia , Humanos , Antígenos Comuns de Leucócito/análise , Leucócitos/imunologia , Antígenos CD15/análise , Masculino , Peroxidase , Sêmen/imunologia , Coloração e Rotulagem/métodosRESUMO
OBJECTIVE: To analyze the role of immunochemistry in serous effusions. STUDY DESIGN: We analyzed cell blocks of 18 pleural and 18 peritoneal effusions diagnosed as malignant (18), benign (14) and suspicious (4). They were immunostained by the avidin-biotin complex method with a panel of four monoclonal antibodies--CEA, Ber-EP4, LeuM1 (CD15) and p53--and, for lectins (Ulex europaeus) UEA-l, ConA and ConBr. RESULTS: Seventeen of the 18 cases of adenocarcinoma were positive for CEA (95%), 12 (66.6%) for Ber-EP4, 11 (61%) for CD15 and 11 (61%) for p53. Twelve of the 18 (66.6%) were positive for UEA-1, CEA, Ber-EP4 and CD15. UEA-1 did not react with mesothelial cells. p53 Gave a positive reaction in only one case, reactive mesothelial cells. ConA and ConBr reacted indiscriminately with benign and malignant cells; thus, it was not useful in distinguishing between these cells. CONCLUSION: In this context no antibody used alone is reliable for corroborating a diagnosis, but the selective use of a small panel of three markers (CEA, Ber-EP4 and LeuM1) can be very useful in solving diagnostic difficulties in the cytodiagnosis of serous effusions.
Assuntos
Adenocarcinoma/diagnóstico , Líquido Ascítico/diagnóstico , Biomarcadores Tumorais , Imuno-Histoquímica , Neoplasias Peritoneais/diagnóstico , Lectinas de Plantas , Anticorpos Monoclonais/imunologia , Antígenos de Superfície/análise , Antígenos de Superfície/imunologia , Antígeno Carcinoembrionário/análise , Antígeno Carcinoembrionário/imunologia , Concanavalina A/análise , Concanavalina A/imunologia , Diagnóstico Diferencial , Feminino , Humanos , Lectinas/análise , Lectinas/imunologia , Antígenos CD15/análise , Antígenos CD15/imunologia , Masculino , Neoplasias Mesoteliais/diagnóstico , Derrame Pleural Maligno/diagnóstico , Proteína Supressora de Tumor p53/análise , Proteína Supressora de Tumor p53/imunologiaRESUMO
UNLABELLED: The progression from uncontrolled cell proliferation to invasion and metastasis of epithelial tumors is partially understood. Alteration of epithelial mucin expression have been described in different malignant localizations but only few attempts have been made to identify mucin expression in malignant laryngeal tumors. In the present report, results are shown of studies on the expression of mucins and carbohydrate related antigens in laryngeal cancer and on the isolation of MUC1 mucin from this tumor tissue. Malignant laryngeal specimens were processed for immunohistochemical analysis and for extranuclear membrane fractions (ENM) which were obtained by ultracentrifugation. Subsequently, ENM samples were centrifuged in density-gradient; the analysis of fractions was performed by means of SDS-PAGE and Western-blotting. The panel of monoclonal antibodies (MAbs) included anti MUC1 mucin, anti Lewis x, anti sialyl Lewis x, anti Lewis y, anti MUC-5B, anti oral mucin (gp230), anti Tn hapten, anti p53 and anti cytokeratins. By immunohistochemistry, it was possible to detect MUC1 mucin, Lewis x and Lewis y showing strong reactions while sialyl-Lewis x and Tn antigen only reacted weakly in a few cells; cytokeratins were detected in all samples. In ENM derived fractions obtained by CsCl centrifugation, MUC1 was demonstrated by Western blotting. CONCLUSIONS: (1) laryngeal cancer antigenic expression comprises mostly MUC1 mucin, Lewis x, Lewis y as well as Tn antigen and (2) the methodology here employed is useful to isolate MUC1 from tumor samples.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Laríngeas/metabolismo , Mucina-1/isolamento & purificação , Mucinas/análise , Idoso , Antígenos Glicosídicos Associados a Tumores/análise , Western Blotting , Humanos , Imuno-Histoquímica , Antígenos do Grupo Sanguíneo de Lewis/análise , Antígenos CD15/análise , MasculinoRESUMO
Leukocyte adhesion deficiency II has been described in only 2 patients; herein we report extensive investigation of another patient. The physical stigmata were detected during prenatal ultrasonographic investigation. Sialyl-Lewis X (sLex) was absent from the surface of polymorphonuclear neutrophils, and cell binding to E- and P-selectin was severely impaired, causing an immunodeficiency. The elevation of peripheral neutrophil counts occurred within several days after birth. A severe hypofucosylation of glycoconjugates bearing fucose in different glycosidic links was present in all cell types investigated, demonstrating that leukocyte adhesion deficiency II is not only a disorder of leukocytes but a generalized inherited metabolic disease affecting the metabolism of fucose.
Assuntos
Erros Inatos do Metabolismo dos Carboidratos/metabolismo , Fucose/metabolismo , Síndrome da Aderência Leucocítica Deficitária/metabolismo , Proteína C-Reativa/análise , Cromatografia de Afinidade , Selectina E/metabolismo , Retardo do Crescimento Fetal/diagnóstico por imagem , Humanos , Lactente , Contagem de Leucócitos , Síndrome da Aderência Leucocítica Deficitária/sangue , Síndrome da Aderência Leucocítica Deficitária/diagnóstico por imagem , Antígenos CD15/análise , Masculino , Neutrófilos/imunologia , Selectina-P/metabolismo , Linhagem , Ultrassonografia Pré-NatalRESUMO
Monoclonal antibody (MAb) FC-2.15 recognizes Lewis x antigen (Le(x)-Ag) expressed on the cell surface of most human breast cancer cells. FC-2.15 displays important human complement (C')-mediated cytotoxicity (CMC) against its target cells. In this study the reactivity of FC-2.15 against drug resistant-breast cancer cells was investigated, as well as the possibility to combine the antitumor activities of this MAb with adriamycin (Adr) or taxol. Since resistant clones with altered expression of tumor-associated antigens usually emerge after chemotherapy, the expression of Le(x)-Ag was analyzed in Adr(R) MCF-7 breast cancer cells (Adr resistant subline) and in tumor samples from nine patients with locally advanced breast carcinoma who were treated with FEC chemotherapy. A flow cytometry assay showed that most of Adr(R) MCF-7 cells, as well as wild type (WT) cells, expressed Le(x)-Ag; however, the Le(x) epitope is probably bound to different backbones in these cells. When the cytotoxic ability of FC-2.15 against WT and Adr(R) MCF-7 cells was compared, it was found that a 90 min treatment with FC-2.15 plus C' induced similar CMC against both cell lines. An important cytolysis was obtained at 5 microg/ml FC-2.15, reaching a plateau at 25 microg/ml, at which cell population was diminished to 21.1% for WT and 27.9 for Adr(R) MCF-7 cells. Regarding human tumors, Le(x)-Ag expression was evaluated in samples obtained before and in most cases after chemotherapy, and it could be observed that: 1) before treatment, tumor samples from all patients analyzed (responders and non-responders to chemotherapy) were FC-2.15-positive; 2) the presence of Le(x)-Ag was not modified after treatment. The combined action of Adr or taxol with FC-2.15 was then evaluated. WT and Adr(R) MCF-7 cells were cultured with Adr or taxol followed by an incubation with different FC-2.15 concentrations plus C'. When the effect of Adr alone was determined, ID50 were 1 x 10(-7) M for WT and 4.2 x 10(-5) M for Adr(R) MCF-7 cells. The cytotoxic ability of taxol alone was also tested, and ID50 were 6.4 x 10(-9) M for WT and 3.1 x 10(-6) M for Adr(R) MCF-7 cells. When FC-2.15 was added to Adr or taxol, the cytotoxicity of the drug-FC-2.15 combined treatment was always higher than the isolated effects, showing additive cytotoxicity at the different concentrations tested and with both cell lines. Our results suggest that FC-2.15 may be a useful agent against breast tumor cells which survive chemotherapy with Adr or taxol.