RESUMO
Lung carcinoma is often incurable and remains the leading cancer killer in both men and women. Recent evidence indicates that tumors contain a small population of cancer stem cells that are responsible for tumor maintenance and spreading. The identification of the tumorigenic population that sustains lung cancer may contribute significantly to the development of effective therapies. Here, we found that the tumorigenic cells in small cell and non-small cell lung cancer are a rare population of undifferentiated cells expressing CD133, an antigen present in the cell membrane of normal and cancer-primitive cells of the hematopoietic, neural, endothelial and epithelial lineages. Lung cancer CD133(+) cells were able to grow indefinitely as tumor spheres in serum-free medium containing epidermal growth factor and basic fibroblast growth factor. The injection of 10(4) lung cancer CD133(+) cells in immunocompromised mice readily generated tumor xenografts phenotypically identical to the original tumor. Upon differentiation, lung cancer CD133(+) cells acquired the specific lineage markers, while loosing the tumorigenic potential together with CD133 expression. Thus, lung cancer contains a rare population of CD133(+) cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. Molecular and functional characterization of such a tumorigenic population may provide valuable information to be exploited in the clinical setting.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/patologia , Neoplasias Pulmonares/patologia , Células-Tronco Neoplásicas/patologia , Antígeno AC133 , Animais , Antígenos CD/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma de Células Pequenas/metabolismo , Diferenciação Celular , Resistencia a Medicamentos Antineoplásicos , Feminino , Glicoproteínas/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/metabolismo , Peptídeos/metabolismo , Fenótipo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The two major cadherins of endothelial cells are neural (N)-cadherin and vascular endothelial (VE)- cadherin. Despite similar level of protein expression only VE-cadherin is located at cell-cell contacts, whereas N-cadherin is distributed over the whole cell membrane. Cotransfection of VE-cadherin and N-cadherin in CHO cells resulted in the same distribution as that observed in endothelial cells indicating that the behavior of the two cadherins was not cell specific but related to their structural characteristics. Similar amounts of alpha- and beta-catenins and plakoglobin were associated to VE- and N-cadherins, whereas p120 was higher in the VE-cadherin complex. The presence of VE-cadherin did not affect N-cadherin homotypic adhesive properties or its capacity to localize at junctions when cotransfectants were cocultured with cells transfected with N-cadherin only. To define the molecular domain responsible for the VE-cadherin-dominant activity we prepared a chimeric construct formed by VE-cadherin extracellular region linked to N-cadherin intracellular domain. The chimera lost the capacity to exclude N-cadherin from junctions indicating that the extracellular domain of VE-cadherin alone is not sufficient for the preferential localization of the molecule at the junctions. A truncated mutant of VE-cadherin retaining the full extracellular domain and a short cytoplasmic tail (Arg621-Pro702) lacking the catenin-binding region was able to exclude N-cadherin from junctions. This indicates that the Arg621-Pro702 sequence in the VE-cadherin cytoplasmic tail is required for N-cadherin exclusion from junctions. Competition between cadherins for their clustering at intercellular junctions in the same cell has never been described before. We speculate that, in the endothelium, VE- and N-cadherin play different roles; whereas VE-cadherin mostly promotes the homotypic interaction between endothelial cells, N-cadherin may be responsible for the anchorage of the endothelium to other surrounding cell types expressing N-cadherin such as vascular smooth muscle cells or pericytes.
Assuntos
Caderinas/análise , Endotélio Vascular/química , Junções Intercelulares/química , Transativadores , Animais , Células CHO/química , Caderinas/biossíntese , Caderinas/química , Adesão Celular/fisiologia , Cricetinae , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Endotélio Vascular/citologia , Humanos , Fosforilação , Estrutura Terciária de Proteína , Transfecção , Tirosina/metabolismo , Veias Umbilicais/citologia , alfa Catenina , beta CateninaRESUMO
mAbs were raised in mice against cultured human endothelial cells (EC) and screened by indirect immunofluorescence for their ability to stain intercellular contacts. One mAb denoted 7B4 was identified which, out of many cultured cell types, specifically decorated cultured human EC. The antigen recognized by mAb 7B4 is bound at the appositional surfaces of cultured EC only as they become confluent and is stably expressed at intercellular boundaries of confluent monolayers. EC recognition specificity was maintained when the antibody was assayed by immuno-histochemistry in tissue sections of many normal and malignant tissues and in blood vessels of different size and type. The antigen recognized by 7B4 was enriched at EC intercellular boundaries similarly in vitro and in situ. In vitro, addition of mAb 7B4 to confluent EC increased permeation of macromolecules across monolayers even without any obvious changes of cell morphology. In addition, when EC permeability was increased by agents such as thrombin, elastase, and TNF/gamma IFN, its distribution pattern at intercellular contact rims was severely altered. mAb 7B4 immunoprecipitated a major protein of 140 kD from metabolically and surface-labeled cultured EC extracts which appeared to be an integral membrane glycoprotein. On the basis of its distribution in cultured cells and in tissues in situ, 7B4 antigen is distinct from other described EC proteins enriched at intercellular contacts. NH2-terminal sequencing of the antigen, immunopurified from human placenta, and sequencing of peptides from tryptic peptide maps revealed identity to the cDNA deduced sequence of a recently identified new member of the cadherin family (Suzuki, S., K. Sano, and H. Tanihara. 1991. Cell Regul. 2:261-270.) These data indicate that 7B4 antigen is an endothelial-specific cadherin that plays a role in the organization of lateral endothelial junctions and in the control of permeability properties of vascular endothelium.
Assuntos
Caderinas/análise , Endotélio Vascular/química , Junções Intercelulares/química , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos de Diferenciação Mielomonocítica/análise , Biomarcadores , Caderinas/química , Adesão Celular/fisiologia , Moléculas de Adesão Celular/análise , Células Cultivadas , Cromatografia de Afinidade , Endotélio Vascular/metabolismo , Imunofluorescência , Humanos , Dados de Sequência Molecular , Mapeamento de Peptídeos , Molécula-1 de Adesão Celular Endotelial a PlaquetasRESUMO
Polymorphonuclear leukocytes (PMN) infiltration into tissues is frequently accompanied by increase in vascular permeability. This suggests that PMN adhesion and transmigration could trigger modifications in the architecture of endothelial cell-to-cell junctions. In the present paper, using indirect immunofluorescence, we found that PMN adhesion to tumor necrosis factor-activated endothelial cells (EC) induced the disappearance from endothelial cell-to-cell contacts of adherens junction (AJ) components: vascular endothelial (VE)-cadherin, alpha-catenin, beta-catenin, and plakoglobin. Immunoprecipitation and Western blot analysis of the VE-cadherin/catenin complex showed that the amount of beta-catenin and plakoglobin was markedly reduced from the complex and from total cell extracts. In contrast, VE-cadherin and alpha-catenin were only partially affected. Disorganization of endothelial AJ by PMN was not accompanied by EC retraction or injury and was specific for VE-cadherin/catenin complex, since platelet/endothelial cell adhesion molecule 1 (PECAM-1) distribution at cellular contacts was unchanged. PMN adhesion to EC seems to be a prerequisite for VE-cadherin/catenin complex disorganization. This phenomenon could be fully inhibited by blocking PMN adhesion with an anti-integrin beta 2 mAb, while it could be reproduced by any condition that induced increase of PMN adhesion, such as addition of PMA or an anti-beta 2-activating mAb. The effect on endothelial AJ was specific for PMN since adherent activated lymphocytes did not induce similar changes. High concentrations of protease inhibitors and oxygen metabolite scavengers were unable to prevent AJ disorganization mediated by PMN. PMN adhesion to EC was accompanied by increase in EC permeability in vitro. This effect was dependent on PMN adhesion, was not mediated by proteases and oxygen-reactive metabolites, and could be reproduced by EC treatment with EGTA. Finally, immunohistochemical analysis showed that VE-cadherin distribution was affected by PMN adhesion to the vessel wall in vivo too. This work suggests that PMN adhesion could trigger intracellular signals in EC that possibly regulate VE-cadherin /catenin complex disorganization. This effect could increase EC permeability and facilitate PMN transmigration during the acute inflammatory reaction.
Assuntos
Adesão Celular , Endotélio Vascular/fisiologia , Junções Intercelulares/fisiologia , Neutrófilos/fisiologia , Transativadores , Western Blotting , Caderinas/análise , Caderinas/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/fisiologia , Desmoplaquinas , Endopeptidases/metabolismo , Endotélio Vascular/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Junções Intercelulares/ultraestrutura , Neutrófilos/ultraestrutura , Veias Umbilicais , alfa Catenina , beta Catenina , gama CateninaRESUMO
Tight junctions are the most apical components of endothelial and epithelial intercellular cleft. In the endothelium these structures play an important role in the control of paracellular permeability to circulating cells and solutes. The only known integral membrane protein localized at sites of membrane-membrane interaction of tight junctions is occludin, which is linked inside the cells to a complex network of cytoskeletal and signaling proteins. We report here the identification of a novel protein (junctional adhesion molecule [JAM]) that is selectively concentrated at intercellular junctions of endothelial and epithelial cells of different origins. Confocal and immunoelectron microscopy shows that JAM codistributes with tight junction components at the apical region of the intercellular cleft. A cDNA clone encoding JAM defines a novel immunoglobulin gene superfamily member that consists of two V-type Ig domains. An mAb directed to JAM (BV11) was found to inhibit spontaneous and chemokine-induced monocyte transmigration through an endothelial cell monolayer in vitro. Systemic treatment of mice with BV11 mAb blocked monocyte infiltration upon chemokine administration in subcutaneous air pouches. Thus, JAM is a new component of endothelial and epithelial junctions that play a role in regulating monocyte transmigration.
Assuntos
Antígenos/fisiologia , Moléculas de Adesão Celular/fisiologia , Movimento Celular , Imunoglobulinas/fisiologia , Monócitos/fisiologia , Junções Íntimas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos/genética , Antígenos/metabolismo , Sequência de Bases , Células COS , Adesão Celular , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Endotélio/citologia , Células Epiteliais/metabolismo , Imunoglobulinas/genética , Imunoglobulinas/metabolismo , Moléculas de Adesão Juncional , Masculino , Camundongos , Dados de Sequência Molecular , Ratos , Ratos Endogâmicos Lew , Pele/imunologia , TransfecçãoRESUMO
An accurate diagnosis of clinically distinct subgroups of aggressive mature B cell lymphomas is crucial for the choice of proper treatment. Presently, precise recognition of these disorders relies on the combination of morphological, immunophenotypical, and cytogenetic/molecular features. The diagnostic workup in such situations implies the application of costly and time-consuming analyses, which are not always required, since an intensified treatment option is reasonably reserved to fit patients. The Italian Group of Haematopathology proposes herein a practical algorithm for the diagnosis of aggressive mature B cell lymphomas based on a stepwise approach, aimed to select cases deserving molecular analysis, in order to optimize time and resources still assuring the optimal management for any patient.
Assuntos
Algoritmos , Linfoma de Células B/diagnóstico , Humanos , Imunofenotipagem/métodos , Hibridização in Situ Fluorescente/métodosRESUMO
The first and family names of the authors were interchanged and are now presented correctly. The original article has been corrected.
RESUMO
Fractalkine (FKN, CX3CL1) is a membrane-bound CX3C chemokine induced by primary proinflammatory signals in vascular endothelial cells (ECs). Here we examined the role of FKN in polarized Th1 or Th2 responses. Proinflammatory signals, including LPS, IL-1, TNF, and CD40 ligand, induced FKN, as did IFN-gamma, which had synergistic activity with TNF. IL-4 and IL-13 did not stimulate the expression of FKN and markedly reduced induction by TNF and IFN-gamma. TNF alone or combined with IFN-gamma also induced release of soluble FKN, which was inhibited by IL-4 and IL-13. In light of this differential regulation of FKN by the master cytokines that control polarized responses, we analyzed the interaction of FKN with natural killer (NK) cells and polarized T-cell populations. NK cells expressed high levels of the FKN receptor CX3CR1 and responded to FKN. CX3CR1 was preferentially expressed in Th1 compared with Th2 cells. Th1 but not Th2 cells responded to FKN. By immunohistochemistry, FKN was expressed on ECs in psoriasis, a Th1-dominated skin disorder, but not in Th2-driven atopic dermatitis. Similarly, ECs in Mycobacterium tuberculosis granulomatous lymphadenitis, but not those in reactive lymph node hyperplasia or in Castelman's disease, showed immunoreactive FKN. These results indicate that regulated expression of FKN in ECs participates in an amplification circuit of polarized type I responses.
Assuntos
Quimiocinas CX3C/biossíntese , Endotélio Vascular/imunologia , Células Matadoras Naturais/imunologia , Proteínas de Membrana/biossíntese , Células Th1/imunologia , Adulto , Ligante de CD40/metabolismo , Receptor 1 de Quimiocina CX3C , Hiperplasia do Linfonodo Gigante/imunologia , Quimiocina CX3CL1 , Quimiotaxia de Leucócito , Dermatite Atópica/imunologia , Endotélio Vascular/efeitos dos fármacos , Humanos , Recém-Nascido , Interferon gama/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Linfadenite/imunologia , Psoríase/imunologia , Receptores de Citocinas/metabolismo , Receptores de HIV/metabolismo , Células Th2/imunologiaRESUMO
This corrects the article DOI: 10.1038/onc.2017.75.
RESUMO
Recent evidences suggest that stearoyl-CoA-desaturase 1 (SCD1), the enzyme involved in monounsaturated fatty acids synthesis, has a role in several cancers. We previously demonstrated that SCD1 is important in lung cancer stem cells survival and propagation. In this article, we first show, using primary cell cultures from human lung adenocarcinoma, that the effectors of the Hippo pathway, Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), are required for the generation of lung cancer three-dimensional cultures and that SCD1 knock down and pharmacological inhibition both decrease expression, nuclear localization and transcriptional activity of YAP and TAZ. Regulation of YAP/TAZ by SCD1 is at least in part dependent upon ß-catenin pathway activity, as YAP/TAZ downregulation induced by SCD1 blockade can be rescued by the addition of exogenous wnt3a ligand. In addition, SCD1 activation of nuclear YAP/TAZ requires inactivation of the ß-catenin destruction complex. In line with the in vitro findings, immunohistochemistry analysis of lung adenocarcinoma samples showed that expression levels of SCD1 co-vary with those of ß-catenin and YAP/TAZ. Mining available gene expression data sets allowed to observe that high co-expression levels of SCD1, ß-catenin, YAP/TAZ and downstream targets have a strong negative prognostic value in lung adenocarcinoma. Finally, bioinformatics analyses directed to identify which gene combinations had synergistic effects on clinical outcome in lung cancer showed that poor survival is associated with high co-expression of SCD1, ß-catenin and the YAP/TAZ downstream target birc5. In summary, our data demonstrate for the first time the involvement of SCD1 in the regulation of the Hippo pathway in lung cancer, and point to fatty acids metabolism as a key regulator of lung cancer stem cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenocarcinoma/metabolismo , Núcleo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias Pulmonares/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Adenocarcinoma/mortalidade , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Complexo de Sinalização da Axina/metabolismo , Regulação para Baixo , Ácidos Graxos/metabolismo , Feminino , Células HEK293 , Via de Sinalização Hippo , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Neoplasias/metabolismo , Cultura Primária de Células , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Estabilidade Proteica , RNA Mensageiro/metabolismo , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/genética , Survivina , Transativadores , Fatores de Transcrição , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteína Wnt3A/metabolismo , Proteínas de Sinalização YAPRESUMO
Charles River mice either untreated or treated at birth with 7,12-dimethylbenz]alpha[anthracene (DMBA) were given allogeneic spleen cells from adult C57BL/Cas donors. These spleen cells were given as a single injection to mice at birth or at 7 or 14 days of age. Allogeneic treatment of mice at birth significantly increased the incidence of DMBA-induced lymphomas and shortened the latency period of the tumors. The incidence of subcutaneous tumors was moderately increased in DMBA-treated mice given grafts of allogeneic cells at 7 days of age. Lung tumors appeared to be decreased in the group given DMBA at birth and allogeneic cells 14 days later. Treatment with only allogeneic cells gave results essentially similar to those observed in untreated controls.
Assuntos
Reação Enxerto-Hospedeiro , Neoplasias Experimentais/imunologia , 9,10-Dimetil-1,2-benzantraceno , Fatores Etários , Animais , Animais Recém-Nascidos , Linfoma/induzido quimicamente , Linfoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Transplante de Neoplasias , Neoplasias Experimentais/induzido quimicamente , Baço/imunologia , Transplante HomólogoRESUMO
Natural killer (NK) activity against K-562 myeloid cell line was evaluated in 21 spleens and 14 lymph nodes from patients with Hodgkin's disease (HD). NK activity of eight HD-involved (HD+) spleens [556 lytic units (LU)] was found 5-fold higher than that of 13 HD-uninvolved (HD-) spleens (112 LU) (p less than 0.01). Moreover, NK activity of HD+ spleens was significantly different (p less than 0.05) from that of three spleens involved by non-HD lymphoma (100 LU). NK activity of four spleens from nonneoplastic patients (250 LU) was intermediate between those of HD+ and HD- spleens. Lymph node cells were about 10-fold less cytotoxic. NK activity of seven HD+ lymph nodes (43 LU) was 3-fold higher than that of three lymph nodes involved by non-HD lymphoma (8 LU). However, these differences were not statistically significant. Our data are compatible with increased NK activity in HD+ tissues as well as with depressed NK activity in HD- tissues. The observation that NK activity of peripheral blood leukocytes from nine HD patients (116 LU) (p less than 0.05 only at 100:1 effector:target cell ratio) may support the latter interpretation. Partial characterization of effector cells in HD+ and in HD- spleens indicated that, in both instances NK cells were nonadherent and almost equally distributed between the erythrocyte-negative and -positive cell fractions. Finally, NK activity of both HD+ and HD- spleen cells could be further potentiated in vitro by interferon.
Assuntos
Doença de Hodgkin/imunologia , Células Matadoras Naturais/imunologia , Linfonodos/imunologia , Baço/imunologia , Adolescente , Adulto , Idoso , Criança , Citotoxicidade Imunológica , Feminino , Humanos , Interferons/farmacologia , Ativação Linfocitária , Masculino , Pessoa de Meia-IdadeRESUMO
Tumor regression induced in cancer patients by i.v. infusion of interleukin-2 (IL-2) is often accompanied by severe side effects. To investigate whether local administration would affect immune response without the side effects, two 5-day cycles of continuous intraarterial [internal iliac artery] infusion of recombinant interleukin-2 (rIL-2) were performed in 12 patients with transitional cell carcinoma (tumor stage 1, node stage 0, metastasis stage 0, and grade 1-2) of the bladder. Four groups of 3 patients were treated at each of 4 escalating doses of rIL-2 (18 x 10(3), 18 x 10(4), 18 x 10(5), and 18 x 10(6) IU/m2/day) throughout the course of the two IL-2 cycles. This treatment was effective in inducing a marked intratumor inflammatory response, consisting mainly of T-lymphocytes and macrophages. A remarkable dose-dependent increase in the levels of soluble CD25 was observed in the urine of all patients, which was associated constantly with an enhanced number of intratumor CD25+ cells. Intratumor macrophages were often immunoreactive for interleukin-1 and/or tumor necrosis factor, suggesting an activated status. Increased levels of soluble CD25 and CD25+ lymphocytes were observed in peripheral blood only at the two highest doses of rIL-2, while increased percentages of circulating HLA-DR+ and CD71+ lymphoid cells and enhancement of CD3+/CD16+ T-lymphocytes were found at lower doses. Peripheral blood eosinophils were augmented in almost all patients but were rarely increased in situ. We provide evidence that continuous intraarterial infusion of rIL-2 activates host immune response, acting preferentially at the tissue level.
Assuntos
Interleucina-2/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Neoplasias da Bexiga Urinária/imunologia , Antígenos CD/imunologia , Relação Dose-Resposta Imunológica , Avaliação de Medicamentos , Humanos , Artéria Ilíaca , Imunidade Celular , Infusões Intra-Arteriais , Interleucina-2/administração & dosagem , Leucócitos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Proteínas Recombinantes/administração & dosagem , Neoplasias da Bexiga Urinária/terapiaRESUMO
OBJECTIVES: To evaluate the impact of response to highly active antiretroviral therapy (HAART) on the natural history of AIDS non-Hodgkin's lymphoma (NHL) and to analyse the feasibility, efficacy and toxicity of HAART in combination with chemotherapy. DESIGN: Prospective observational study in two AIDS clinical centres in Italy. METHODS: All consecutive HIV-infected patients with NHL were included (n = 44; 48% high-risk group) and prospectively followed for 27 months. HAART was administered concomitantly with chemotherapy. The association between response to HAART and clinical presentation, response to chemotherapy and toxicity was analysed by univariate and multivariate models. Survival analysis was performed by Kaplan-Meier estimates and the Cox proportional hazards regression model. RESULTS: A complete response (CR) to chemotherapy was achieved in 71% of HAART responders and 30% of non-responders. Virological response to HAART was the only variable associated with tumour response on multivariate analysis. A higher relative dose intensity (RDI) of chemotherapy was administered in patients with virological response compared with those without. The probability of 1 year survival was higher in patients with virological or immunological response. At Cox regression analysis, immunological response, a higher RDI and a CR to chemotherapy were all associated with a reduced risk of death. CONCLUSION: In HIV-infected patients with NHL, response to HAART was strongly associated with a better response to chemotherapy and prolonged survival. Concurrent treatments were well tolerated, and HAART-responder patients could receive a higher RDI of chemotherapy. In patients with AIDS lymphomas, combining HAART with chemotherapy could be a feasible and effective approach.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Linfoma Relacionado a AIDS/tratamento farmacológico , Linfoma Relacionado a AIDS/mortalidade , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/mortalidade , Adulto , Quimioterapia Combinada , Feminino , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , HIV-1/fisiologia , Humanos , Masculino , Estudos Prospectivos , RNA Viral/sangue , Análise de Sobrevida , Resultado do TratamentoRESUMO
Two monoclonal antibodies (mAb), MEC 7.46 (IgG1) and MEC 13.3 (IgG2a) that specifically recognize mouse endothelial cells (EC) of blood vessels, were produced immunizing a Lewis rat with a polyoma middle T transformed EC line. Antibodies were screened by enzyme-linked immunosorbent assay (ELISA) and by immunofluorescence on different cultured cell lines and by immunoperoxidase staining on frozen sections of various mouse normal and inflammatory tissues. Both mAbs reacted with eight transformed endothelial lines tested in vitro, but were consistently negative on various cell lines of different histological origin. Reactivity was not altered by preexposure of the cell lines to IL-1. Microscopic immunofluorescence analysis showed that the MEC mAbs localized at the cell-cell contacts in EC. Immunohistochemical staining of various mouse tissue was always restricted to the EC of all blood vessels of the organ considered. Staining of the endothelial lining of blood vessels was greater at cell-to-cell contacts. Weak reactivity was detected in bone marrow and spleen megakaryocytes. This picture was not altered in inflamed and tumor tissues. In the developing mouse embryo, MEC 13.3 specifically stained proliferating and sprouting endothelium in all organs and tissues examined. Both MEC 7.46 and MEC 13.3 mAbs were able to precipitate a molecule with an apparent molecular mass of 130 kDa from endothelioma lysates. The protein was synthesized by the cells and exposed on the cell surface. Immunodepletion analysis indicated that MEC 13.3 recognized a molecule related to the murine from of PECAM or CD31. We believe that these mAbs are promising tools for the identification of murine EC and for studying their ontogenesis and functions.
Assuntos
Anticorpos Monoclonais/imunologia , Endotélio Vascular/imunologia , Animais , Antígenos de Diferenciação Mielomonocítica/metabolismo , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Endotélio Vascular/embriologia , Endotélio Vascular/crescimento & desenvolvimento , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Molécula-1 de Adesão Celular Endotelial a Plaquetas , Ratos , Ratos Endogâmicos LewRESUMO
Endothelial cell-specific molecules are potential targets for new therapeutic strategies in the control of inflammatory reactions, immune responses and neoangiogenesis. We describe the production and characterization of MEC 14.7, a monoclonal antibody directed to murine endothelial cells recognizing a glycosylated protein with an apparent molecular mass of about 100 kDa in cultured endothelioma cell lysate and about 80 kDa in lung lysate. MEC 14.7 antigen was selectively expressed by the endothelium in vivo, particularly in small vessels and neoformed capillaries and by developing vascular structures in embryonal bodies. Deglycosylation of the molecule with neuraminidase, O- and N-glycanase showed that the MEC 14.7 epitope is neuraminidase-sensitive. MEC 14.7 antigen was purified from lung lysates by chromatographic techniques, and sequenced internal peptides indicated it was identical with murine CD34. Thus the apparent molecular mass of CD34 is heterogeneous, depending on the glycosylation state in the different cell types. Immunomagnetic isolation and culture of MEC 14.7-positive bone marrow cells showed that this antibody recognizes hematopoietic progenitors (particularly myelomonocytic) and can be used in murine models of bone marrow reconstitution.
Assuntos
Anticorpos Monoclonais , Antígenos CD34/análise , Vasos Sanguíneos/química , Células-Tronco Hematopoéticas/química , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Antígenos CD34/química , Antígenos CD34/isolamento & purificação , Células da Medula Óssea , Endotélio Vascular/química , Epitopos/análise , Feminino , Glicosilação , Separação Imunomagnética , Masculino , Camundongos , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Ratos , Análise de SequênciaRESUMO
The aim of this study was to examine the correlation between intratumoral microvessel density (iMVD) and the presence of cellular fibronectin isoforms, ED-A and ED-B, in order to identify those tumours with a prominent angiogenic phenotype. 91 cases of invasive ductal breast carcinoma were evaluated for TNM, histological grading, percentage of Ki-67+ cells and receptor hormonal status. iMVD was determined as a single microvessel count in a 200 x microscope field from the region of the tumour that appeared to be most densely vascular. When the mean values of iMVD of the various groups were compared, no significant difference was noted (Mann-Whitney test). When tumours were classified as high or low iMVD, based on a cut-off value (99 vessels/0.74 mm2), cases with high iMVD were significantly more numerous in poorly differentiated G3 tumours (P = 0.01, Chi-square test), and in tumours with lymph node metastasis (N0 versus N1 + N2; P = 0.002). The possibility that high iMVD was the expression of prominent vascular neoformation was explored using ED-A and ED-B isoforms of fibronectin as markers of neoformed vessels. ED-A + and/or ED-B + blood vessels were < 10% of total vessels, were detected in approximately 50% of cases independently of iMVD values, and were not more numerous in tumour areas with hot spot vascularisation. Our findings indicate that iMVD and expression of ED-A/ED-B reflect different aspects of tumour-associated angiogenesis.
Assuntos
Neoplasias da Mama/irrigação sanguínea , Carcinoma Ductal de Mama/irrigação sanguínea , Fibronectinas/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/química , Carcinoma Ductal de Mama/química , Feminino , Humanos , Microcirculação , Pessoa de Meia-Idade , Neovascularização Patológica/metabolismoRESUMO
Chemokines are a superfamily of structurally related cytokines involved in leukocyte recruitment in normal and neoplastic tissues. The availability of non-cross-reacting reagents specific for each member of the C-C and C-X-C family is important for careful characterization of their in vitro and in vivo production and relevance. Here we describe a novel, highly specific, mAb against monocyte chemotactic protein-1 (MCP-1). The 5D3-F7 mAb (IgG1,kappa) recognizes human recombinant and natural MCP-1 in ELISA, immunoprecipitation and immunoblot analysis. As a source of natural MCP-1 we used the 8387 human sarcoma line which produces spontaneously MCP-1 and responds to TNF with increased expression and release. The 5D3-F7 mAb inhibited the chemotactic activity of MCP-1 for monocytes. Using the 5D3-F7 mAb and a polyclonal rabbit anti-MCP-1 serum, a sandwich ELISA was developed. In both the direct and the sandwich ELISA, the 5D3-F7 mAb recognized human MCP-1, but not the closely related C-C chemokines MCP-1, MCP-2, MCP-3, MIP-1 alpha, and RANTES and the C-X-C chemokines IL-8, gro alpha and NAP-2. In culture supernatants the sensitivity of the sandwich ELISA was approximately equal to 30 pg/ml. The sandwich ELISA permitted detection of MCP-1 in resting or cytokine-stimulated endothelial, mesothelial and Kaposi's sarcoma cells. Preliminary immunohistochemical analysis revealed production of MCP-1 by macrophage-like cells at sites of inflammation. The 5D3-F7 mAb provides a novel, highly specific reagent with which to investigate the in vitro and in vivo production and role of MCP-1.
Assuntos
Anticorpos Monoclonais/imunologia , Fatores Quimiotáticos/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Quimiocina CCL2 , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Técnicas Imunoenzimáticas , Proteínas Recombinantes/imunologiaRESUMO
The presence of HIV-1 in cystic fluid aspirates from six cases of benign cystic lymphoepithelial lesion (BLL) of the parotid gland, a rare disorder affecting HIV-1-infected patients, has been investigated. HIV-1 p24 protein was present at a concentration ranging from 3 to 15 ng/ml, while it was undetectable in the peripheral blood of the same patients. The number of RNA copies of HIV-1 in the cystic fluids was high, ranging from 0.5 x 10(7) to 7.2 x 10(7) RNA copies/ml. BLL cystic fluid aspirates, despite the high level of HIV-1 RNA, were found to contain only a few infectious virions. The low infectivity correlated with the infrequent detection by electron microscopy of complete HIV-1 particles. The pathogenic mechanism leading to virus accumulation in the cystic fluid was studied by immunohistochemistry of tissue sections. p24 protein was associated with DRC-1+/S-100+ follicular dendritic reticulum cells, which were also present within the cystic cavities. Our findings are consistent with the possibility that the large amounts of virus present in the fluid derive from continuous shedding of HIV-1-infected cells from the surrounding lymphoid tissue.
Assuntos
Cistos/virologia , Reservatórios de Doenças , Células Epiteliais/virologia , HIV-1/isolamento & purificação , Tecido Linfoide/virologia , Doenças Parotídeas/virologia , Adulto , Cistos/patologia , Células Epiteliais/patologia , Feminino , Proteína do Núcleo p24 do HIV/análise , Infecções por HIV/complicações , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Imuno-Histoquímica , Tecido Linfoide/patologia , Masculino , Doenças Parotídeas/patologia , RNA Viral/isolamento & purificaçãoRESUMO
Immunoreactivity for intercellular adhesion molecule-1 (ICAM-1) and for vascular cell adhesion molecule-1 (VCAM-1), two adhesion molecules of the immunoglobulin (Ig) superfamily, was tested and measured on tissue sections from 16 undifferentiated nasopharyngeal carcinomas (U-NPC), 12 keratinizing squamous cell carcinomas (SCCs) of the head and neck region, and 54 malignant epithelial tumors of various origin. Neoplastic cells of all cases of U-NPC were diffusely and intensely stained for ICAM-1 and VCAM-1. Moreover, ICAM-1 messenger RNA (mRNA) and VCAM-1 mRNA were detected by Northern blot analysis of RNA extracts from two tumors. In the other epithelial tumors focal or diffuse staining for ICAM-1 was observed in 40 cases (66%), whereas reactivity for VCAM-1 was detected in a single case of metastatic undifferentiated carcinoma of unknown origin. The biopsy specimens of U-NPC showed variable infiltration by leukocytes, which were positive for the integrins lymphocyte function antigen-1 (LFA-1) and alpha-4/beta-1, the corresponding ligands for ICAM-1 and VCAM-1. The possibility that ICAM-1 and VCAM-1 on neoplastic cells may favor the intratumoral recruitment of leukocytes in a way similar to that occurring in crypt epithelium of the palatine tonsil is discussed.