RESUMO
AIM: To investigate the proliferation and migration of epithelial cell rests of Malassez (ERM) after stimulation with IL-6. METHODOLOGY: Porcine-derived ERM were seeded on Dulbecco's modified Eagle's Medium, and IL-6 (100 pg mL-1 ) was incorporated into the culture medium. The WST-1 assay was performed to evaluate cell proliferation, and absorption was measured at 450 nm. A wound-healing assay and immunofluorescence assay for integrin α3 were conducted to investigate migration. The Kruskal-Wallis test and the Mann-Whitney U-test with Bonferroni correction were used to analyse data of WST-1 and wound-healing assays. RESULTS: Cell proliferation following the stimulation by IL-6 increased over time, with a significant increase being observed at 6 h (P < 0.05), but not in a concentration-dependent manner. Cell proliferation was significantly greater in IL-6-treated ERM than in nontreated ERM (P < 0.05). The results of the wound-healing assay revealed earlier closure in IL-6-treated ERM (P < 0.05). In the immunofluorescence assay, integrin α3 was detected at the edge of cell processes adjacent to the wound area. A neutralized antibody abrogated the effects of the IL-6 stimulation in cell proliferation and migration. CONCLUSION: IL-6 promoted the proliferation and migration of porcine ERM in vitro.
Assuntos
Células Epiteliais/efeitos dos fármacos , Interleucina-6/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/citologia , Integrina alfa3/análise , Descanso , Suínos , Cicatrização/efeitos dos fármacosRESUMO
BACKGROUND AND OBJECTIVE: Epithelial cells are recognized as the first line of defense against bacterial infection and environmental harmful stimuli such as cigarette smoke (CS). Although previous studies explored the effects of nicotine on host cells, mechanisms by which CS affects cellular functions remain uncertain. The present study investigated the effects of CS condensate (CSC) on in vitro wound closure of gingival epithelial cells and their potential interactions with a major periodontal pathogen, Porphyromonas gingivalis. MATERIAL AND METHODS: Human gingival epithelial cells (Ca9-22) were treated with CSC for 24 h. Cell proliferation was determined using a WST-1 assay. Cell migration was assessed using a wound closure model. The expression of integrins was analyzed by confocal scanning laser microscopy and real-time PCR. Intracellular invasion of P. gingivalis was evaluated by confocal scanning laser microscopy and an antibiotic protection assay. RESULTS: Low concentrations (1-10 µg/mL) of CSC showed no significant effect on cell proliferation. CSC demonstrated dual effects on epithelial wound closure of Ca9-22 cells: high concentrations (i.e. 250 µg/mL) significantly inhibited the wound closure whereas low concentrations (i.e. 10 µg/mL) promoted it (p < 0.01). CSC induced distinct changes in cytoskeleton. When CSC-exposed cells were infected with P. gingivalis for 2 h, a significant inhibition of wound closure was observed concurrent with a decrease in integrin α3 expression near the wound area. A significantly increased P. gingivalis invasion into Ca9-22 was observed when exposed to low concentrations of CSC. CONCLUSION: Low concentrations of CSC increased invasion of human gingival epithelial cells by P. gingivalis and induced changes in cytoskeleton and integrin expression, thereby modulating the cell migration.
Assuntos
Gengiva/citologia , Nicotiana , Porphyromonas gingivalis/fisiologia , Fumaça , Fenômenos Fisiológicos Bacterianos/efeitos dos fármacos , Técnicas de Cultura de Células , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/fisiologia , Gengiva/efeitos dos fármacos , Gengiva/microbiologia , Humanos , Integrina alfa3/análise , Integrina alfa3/efeitos dos fármacos , Nicotina/efeitos adversosRESUMO
BACKGROUND: Gastrin-releasing peptide (GRP) and its receptor, GRP-R, are critically involved in neuroblastoma tumorigenesis; however, the molecular mechanisms and signaling pathways that are responsible for GRP/GRP-R-induced cell migration and invasion remain unclear. In this study, we sought to determine the cell signals involved in GRP/GRP-R-mediated neuroblastoma cell migration and invasion. METHODS: Human neuroblastoma cell lines SK-N-SH, LAN-1, and IMR-32 were used for our study. Transwell migration and invasion assays were performed after GRP (10(-7) M) stimulation. The cDNA GEArray Microarray kit was used to determine GRP-R-induced gene expression changes. Protein and membrane expression of integrin subunits were confirmed by Western blotting and flow cytometry analysis. siRNA transfection was performed using Lipofectamine 2000. For scratch assay, a confluent monolayer of cells in 6-well plates were wounded with micropipette tip and observed microscopically at 24 to 72 h. RESULTS: GRP increased neuroblastoma cell migration and expressions of MMP-2 whereas the TIMP-1 level decreased. GRP-R overexpression stimulated SK-N-SH cell migration and upregulated integrin α2, α3, and ß1 protein as well as mRNA expression. Targeted silencing of integrin ß1 inhibited cell migration. CONCLUSION: GRP/GRP-R signaling contributes to neuroblastoma cell migration and invasion. Moreover, the integrin ß1 subunit critically regulates GRP-R-mediated neuroblastoma cell migration and invasion.
Assuntos
Movimento Celular , Integrina beta1/fisiologia , Neuroblastoma/patologia , Receptores da Bombesina/fisiologia , Linhagem Celular Tumoral , Peptídeo Liberador de Gastrina/farmacologia , Humanos , Integrina alfa2/análise , Integrina alfa3/análise , Invasividade NeoplásicaRESUMO
OBJECTIVES: The study aimed to assess the role of CD151-integrin α3ß1 (INGA3) complex as a potential prognostic indicator in OSCC and to examine whether mapping of its expression in the invasive front separately from that in the rest of the tumour would have an impact on the predictive value of the results. CD151/INGA3 profiles were compared with that of EGFR. MATERIALS AND METHODS: Protein distributions were analysed either in the whole tumour (W) or separately, (i) the main tumour mass (TU) and (ii) the invasive front (IF) in 83 OSCC samples using immunohistochemistry. RESULTS AND CONCLUSION: There was no statistical association between any of the proteins scored in W and clinicopathologic features or patient survival. When examined separately, significant associations were shown for (i) CD151 and EGFR in TU (p=0.036) and (ii) tumour grade and EGFR in both TU (p=0.045) and IF (p=0.030). INGA3 was present predominantly in the tumour-host interface, significantly stronger in IF than TU (p=0.021). An association with 5-year disease-free survival was close to significant for INGA3 (TU and IF) (p=0.050) but not the CD151/INGA3 complex. Expression of CD151/INGA3 at the IF might reflect tumour behaviour pertinent to patient outcome.
Assuntos
Carcinoma de Células Escamosas/patologia , Integrina alfa3beta1/análise , Neoplasias Bucais/patologia , Tetraspanina 24/análise , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Estudos de Coortes , Intervalo Livre de Doença , Epitélio/patologia , Receptores ErbB/análise , Feminino , Seguimentos , Humanos , Imuno-Histoquímica , Integrina alfa3/análise , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Mucosa Bucal/patologia , Gradação de Tumores , Invasividade Neoplásica , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Prognóstico , Adulto JovemRESUMO
The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5γ2 chain as well as α3, ß1 subunits of α3ß1 heterodimer and ß4 subunit of integrin α6ß4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of ß1, ß4 and α3 integrins, and SISCCs lacked ß1, ß4 and α3 integrins in the invasive front. AC cases were negative for the Ln-5γ2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5γ2 chain expression in the invasive front of the tumor. Integrin ß1, ß4 and α3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5γ2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/química , Queilite/metabolismo , Integrinas/análise , Laminina/análise , Neoplasias Labiais/química , Lábio/química , Biópsia , Carcinoma de Células Escamosas/patologia , Adesão Celular , Movimento Celular , Queilite/patologia , Humanos , Imuno-Histoquímica , Integrina alfa3/análise , Integrina beta1/análise , Integrina beta4/análise , Lábio/patologia , Neoplasias Labiais/patologia , Invasividade Neoplásica , FenótipoRESUMO
Prognosis of breast cancer is primarily predicted by the histological grading of the tumor, where pathologists manually evaluate microscopic characteristics of the tissue. This labor intensive process suffers from intra- and inter-observer variations; thus, computer-aided systems that accomplish this assessment automatically are in high demand. We address this by developing an image analysis framework for the automated grading of breast cancer in in vitro three-dimensional breast epithelial acini through the characterization of acinar structure morphology. A set of statistically significant features for the characterization of acini morphology are exploited for the automated grading of six (MCF10 series) cell line cultures mimicking three grades of breast cancer along the metastatic cascade. In addition to capturing both expected and visually differentiable changes, we quantify subtle differences that pose a challenge to assess through microscopic inspection. Our method achieves 89.0% accuracy in grading the acinar structures as nonmalignant, noninvasive carcinoma, and invasive carcinoma grades. We further demonstrate that the proposed methodology can be successfully applied for the grading of in vivo tissue samples albeit with additional constraints. These results indicate that the proposed features can be used to describe the relationship between the acini morphology and cellular function along the metastatic cascade.
Assuntos
Células Acinares/citologia , Neoplasias da Mama/patologia , Mama/citologia , Interpretação de Imagem Assistida por Computador/métodos , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Mama/embriologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Integrina alfa3/análise , Integrina alfa3/metabolismo , Integrina alfa6/análise , Integrina alfa6/metabolismo , Camundongos , Metástase Neoplásica , Máquina de Vetores de Suporte , Transplante HeterólogoRESUMO
Changes in the expression of adhesion proteins involved in myoblast differentiation were investigated in monolayer (two-dimensional) and 3D (three-dimensional) cell cultures. The expression of integrin alpha3 subunit, integrin beta1 subunit, ADAM12 (a disintegrin and metalloproteinase 12), tetraspanins CD9 and CD81 and M-cadherin were examined in the murine myoblast cell line C2C12 and in a primary culture of rat satellite cells. Myoblasts in monolayer and 3D cultures showed significant differences in their morphology and cytoskeletal organization. All of the studied proteins participated in myoblast fusion in each culture examined, but differences in their levels of expression were observed. Satellite cell-derived myoblasts exhibited higher expression of adhesion protein mRNAs than C2C12 cells. Also, C2C12 cells from a 3D culture showed slightly higher expression of adhesion protein transcripts than the same cells cultured as a monolayer. Significantly, the levels of adhesion protein mRNAs were found to change in parallel in all cell culture types. Despite this finding, it is important that differences between satellite cell-derived myoblasts and cell line C2C12 grown in monolayer and 3D cultures are taken into account when studying processes of myoblast differentiation in vitro.
Assuntos
Proteínas de Membrana/metabolismo , Mioblastos/metabolismo , Células Satélites de Músculo Esquelético/metabolismo , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Animais , Antígenos CD/análise , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/análise , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Técnicas de Cultura de Células , Diferenciação Celular , Células Cultivadas , Integrina alfa3/análise , Integrina alfa3/genética , Integrina alfa3/metabolismo , Integrina beta1/análise , Integrina beta1/genética , Integrina beta1/metabolismo , Glicoproteínas de Membrana/análise , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Mioblastos/citologia , RNA Mensageiro/metabolismo , Ratos , Células Satélites de Músculo Esquelético/citologia , Tetraspanina 28 , Tetraspanina 29RESUMO
BACKGROUND AND OBJECTIVE: It remains controversial whether or not the junctional epithelium cells that are directly attached to teeth migrate on the enamel surface, as those cells are able to adhere firmly to the enamel. The aim of this study was to investigate the expression patterns of laminin gamma(2), integrin beta(4) and integrin alpha(3), and to examine their potential function in cell migration. MATERIAL AND METHODS: Oral epithelium cells obtained from Sprague-Dawley rats were established in primary culture. We employed a wound-healing assay to characterize the direction of cell extension at the start of cell migration, and observed different localizations of laminin and integrins using immunofluorescence. For functional analyses of integrins, we employed a phosphatidylinositol-3-kinase (PI3K) activator to promote integrin beta(4) function and used P1B5 to inhibit integrin alpha(3) function, and we analyzed the percentage of re-epithelialization as the migration function. RESULTS: Marked accumulation of laminin gamma(2) was detected in the peripheral cytoplasm of cells adjacent to the wound area, as shown by the results of the migration assay. Integrin beta(4) was detected in the distal cell processes of actively migrating cells, while integrin alpha(3) was found in cell membranes of cells adjacent to the wound area. In the functional analyses, the percentage of re-epithelialization was significantly lower in the PI3K-activator group and in the P1B5-treated group (2.5% and 7.2%, respectively) than in the control group (39.0%) (p < 0.01). CONCLUSION: The results suggest that laminin gamma(2) is secreted as a foothold for cell migration, that integrin beta(4) participates in cell adhesion and that integrin alpha(3) is involved in cell migration in the primary culture cells.
Assuntos
Moléculas de Adesão Celular/fisiologia , Inserção Epitelial/citologia , Integrina alfa3/fisiologia , Integrina beta4/fisiologia , Animais , Adesão Celular/fisiologia , Moléculas de Adesão Celular/análise , Membrana Celular/ultraestrutura , Movimento Celular/fisiologia , Núcleo Celular/ultraestrutura , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Corantes , Citoplasma/ultraestrutura , Ativação Enzimática , Inserção Epitelial/fisiologia , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Imunofluorescência , Integrina alfa3/análise , Integrina alfa3/efeitos dos fármacos , Integrina beta4/análise , Integrina beta4/efeitos dos fármacos , Microscopia Confocal , Fosfatidilinositol 3-Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Cicatrização/fisiologia , CalininaRESUMO
BACKGROUND AND OBJECTIVE: The expression patterns of adhesive proteins and extracellular matrix proteins in regenerating gingival epithelium after gingivectomy are unknown. The aim of this study was to examine the expression of laminin 1, laminin gamma(2) (a specific component of laminin 5), integrin beta(4) and integrin alpha(3) in the regenerating gingival epithelium in order to understand the mechanism of wound healing during reconstitution of the sulcular environment. MATERIAL AND METHODS: The palatal gingivae of the maxillary molars of Institute of Cancer Research mice were excised, and the regenerating tissues were examined 1, 3, 5, 7 and 14 days later. Fresh, non-fixed and non-decalcified frozen sections were prepared and stained using immunofluorescence. RESULTS: At 1 day post-surgery, intense expression of laminin gamma(2), integrin beta(4) and integrin alpha(3) was distinct in the frontal margin of the regenerating oral epithelium. Laminin gamma(2) was diffusely detected on the root surface and in connective tissues beneath the regenerating oral epithelium at 3 and 5 days. At 7 days, laminin gamma(2) was intermittently recognizable in the internal basal lamina (IBL) close to tooth-facing cells, while laminin gamma(2), integrin beta(4) and integrin alpha(3) were observed in the IBL and in the external basal lamina (EBL) of the regenerating junctional epithelium at 14 days. CONCLUSION: These results suggest that secretion of laminin 5 in the connective tissue may induce epithelial cell migration, and that binding of laminin 5 to integrin alpha(6)beta(4) and integrin alpha(3)beta(1) in the IBL may provoke cell adhesion and migration of cells facing the tooth on the enamel surface of the regenerating junctional epithelium.
Assuntos
Inserção Epitelial/patologia , Gengivectomia , Integrinas/análise , Laminina/análise , Regeneração/fisiologia , Animais , Membrana Basal/patologia , Adesão Celular/fisiologia , Moléculas de Adesão Celular/análise , Movimento Celular/fisiologia , Tecido Conjuntivo/patologia , Epitélio/patologia , Gengiva/patologia , Integrina alfa3/análise , Integrina beta4/análise , Masculino , Camundongos , Camundongos Endogâmicos ICR , Fatores de Tempo , Colo do Dente/patologia , Raiz Dentária/patologia , CalininaRESUMO
BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the enamel surface with hemidesmosomes (of which laminin-5 and integrin-alpha(6)beta(4) are the main components) in the internal basal lamina. Laminin-5 is also involved in cell motility with integrin-alpha(3)beta(1), although their functions have not yet been clarified.The purpose of this study was to determine the functions of those adhesive components between the tooth and the junctional epithelium during cell migration.Because an idea has been proposed that directly attached to tooth cells (DAT cells) may not contribute to cell migration, 5-bromo-2-deoxyuridine staining was performed to confirm cell migration. MATERIAL AND METHODS: We investigated laminin-gamma(2) (contained only in laminin-5), integrin-beta(4) (involved in cell-extracellular matrix contact) and integrin-alpha(3) (inducing cell migration) in the junctional epithelium, oral gingival epithelium and gingival sulcus epithelium of 6-wk-old ICR mice using laser microdissection, quantitative real-time reverse transcription-polymerase chain reaction, immunofluorescence and 5-bromo-2-deoxyuridine staining. RESULTS: Laminin and integrins were clearly immuno-localized in the basal lamina of all epithelium. Quantitative analysis of laminin and integrin mRNAs by laser microdissection showed that they were more highly expressed in DAT cells than in basal cells in the oral gingival epithelium. In particular, a 12-fold higher expression of laminin-5 was observed in the junctional epithelium compared with the oral gingival epithelium. 5-Bromo-2-deoxyuridine staining showed rapid coronal migration of DAT cells. CONCLUSION: These results suggest that the abundant expression of laminin-5 and integrin-alpha(6)beta(4) is involved in the attachment of DAT cells to teeth by hemidesmosomes. Abundant expression of laminin-5 and integrin-alpha(3)beta(1) might assist in DAT cell migration, confirmed by 5-bromo-2-deoxyuridine staining during the turnover of junctional epithelium.
Assuntos
Inserção Epitelial/citologia , Integrina alfa3/análise , Integrina beta4/análise , Laminina/análise , Animais , Antimetabólitos , Bromodesoxiuridina , Adesão Celular/fisiologia , Moléculas de Adesão Celular/análise , Movimento Celular/fisiologia , Células Cultivadas , Células Epiteliais/citologia , Técnica Direta de Fluorescência para Anticorpo , Gengiva/citologia , Hemidesmossomos/ultraestrutura , Integrina alfa3beta1/análise , Integrina alfa6beta4/análise , Masculino , Camundongos , Camundongos Endogâmicos ICR , Microdissecção , Reação em Cadeia da Polimerase Via Transcriptase Reversa , CalininaRESUMO
PURPOSE: Patients with glioblastoma usually have a very poor prognosis. Even with a combination of radiotherapy plus temozolomide, the median survival of these patients is only 14.6 months. New treatment approaches to this cancer are needed. Our purpose is to develop new cell surface-binding ligands for glioblastoma cells and use them as targeted imaging and therapeutic agents for this deadly disease. METHODS: One-bead one-compound combinatorial cyclic peptide libraries were screened with live human glioblastoma U-87MG cells. The binding affinity and targeting specificity of peptides identified were tested with in vitro experiments on cells and in vivo and ex vivo experiments on U-87MG xenograft mouse model. RESULTS: A cyclic peptide, LXY1, was identified and shown to be binding to the alpha 3 integrin of U-87MG cells with moderately high affinity (K (d) = 0.5 +/- 0.1 microM) and high specificity. Biotinylated LXY1, when complexed with streptavidin-Cy5.5 (SA-Cy5.5) conjugate, targeted both subcutaneous and orthotopic U-87MG xenograft implants in nude mice. The in vivo targeting specificity was further verified by strong inhibition of tumor uptake of LXY1-biotin-SA-Cy5.5 complex when intravenously injecting the animals with anti-alpha 3 integrin antibody or excess unlabeled LXY1 prior to administrating the imaging probe. The smaller univalent LXY1-Cy5.5 conjugate (2,279 Da) was found to have a faster accumulation in the U-87MG tumor and shorter retention time compared with the larger tetravalent LXY1-biotin-SA-Cy5.5 complex (approximately 64 kDa). CONCLUSIONS: Collectively, the data reveals that LXY1 has the potential to be developed into an effective imaging and therapeutic targeting agent for human glioblastoma.
Assuntos
Glioblastoma/metabolismo , Raios Infravermelhos , Integrina alfa3/metabolismo , Animais , Anticorpos/imunologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Feminino , Corantes Fluorescentes/metabolismo , Glioblastoma/patologia , Humanos , Integrina alfa3/análise , Integrina alfa3/imunologia , Ligantes , Camundongos , Microscopia de Fluorescência , Peptídeos/síntese química , Peptídeos/metabolismo , Peptídeos/farmacocinética , Poliestirenos , Especificidade por Substrato , Distribuição Tecidual , Transplante HeterólogoRESUMO
PURPOSE: To study the processes involved in mediating conjunctival remodelling in vernal keratoconjunctivitis (VKC) by investigating the expression of integrin receptors, epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), transforming growth factor-beta(TGF-beta), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), and Ki67 antigen, which is a marker for cell proliferation. METHODS: Conjunctival biopsy specimens from 16 patients with active VKC and nine control subjects were studied by immunohistochemical techniques using monoclonal and polyclonal antibodies directed against the integrin alpha3 and alpha6 subunits, EGFR, VEGF, TGF-beta, bFGF, PDGF, and Ki67 antigen. The phenotype of inflammatory cells expressing growth factors was examined by double immunohistochemistry. RESULTS: In the normal conjunctiva, very weak immunoreactivity was observed for EGFR and VEGF in epithelial cells, and for alpha3 and alpha6 integrin subunits on basal epithelial cells, and on vascular endothelial cells in the upper substantia propria. There was no immunoreactivity for the other antibodies. In VKC specimens, strong staining for alpha3 and alpha6 integrin subunits was observed on the membranes of basal and suprabasal epithelial cells, and all vascular endothelial cells. Immunoreactivity for Ki67 antigen was observed in the nuclei of the basal and suprabasal epithelial cells. Strong immunoreactivity was observed for EGFR in the deeper layers of the epithelium, and for VEGF in all epithelial cells. Inflammatory cells expressing EGFR, VEGF, TGF-beta, bFGF, and PDGF were noted in 8, 9, 11, 10, and 10 specimens, respectively. The majority of inflammatory cells expressing growth factors were eosinophils (45+/-4%) and monocytes/macrophages (35+/-4%). CONCLUSIONS: Chronic conjunctival inflammation in VKC is associated with increased staining of alpha3, and alpha6 integrin subunits, EGFR, VEGF, TGF-beta, bFGF, and PDGF that might mediate conjunctival remodelling.
Assuntos
Túnica Conjuntiva/química , Conjuntivite Alérgica/imunologia , Integrinas/análise , Receptores de Fatores de Crescimento/análise , Adolescente , Adulto , Análise de Variância , Biomarcadores/análise , Estudos de Casos e Controles , Criança , Túnica Conjuntiva/patologia , Conjuntivite Alérgica/patologia , Eosinófilos/química , Receptores ErbB/análise , Feminino , Fator 2 de Crescimento de Fibroblastos/análise , Humanos , Imuno-Histoquímica/métodos , Integrina alfa3/análise , Integrina alfa6/análise , Antígeno Ki-67/análise , Macrófagos/química , Masculino , Monócitos/química , Fator de Crescimento Derivado de Plaquetas/análise , Fator de Crescimento Transformador beta/análise , Fator A de Crescimento do Endotélio Vascular/análiseRESUMO
We have previously identified 12 surface antigens whose differential expression represented the signature of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B-CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B-CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log-rank statistics were chosen to define the optimal cut-off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut-off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B-CLL patients, into three distinctive prognostic groups (P = 4.78 x 10(-11)) with high- (score 0-3), intermediate- (score 4-6), and low- (score 7-9) risk of death. The three risk groups showed different distribution of cases as for Rai's stages, IgVH mutations, and ZAP-70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B-CLL.
Assuntos
Antígenos CD/análise , Leucemia Linfocítica Crônica de Células B/diagnóstico , ADP-Ribosil Ciclase 1/análise , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD19/análise , Antígenos CD79/análise , Feminino , Citometria de Fluxo , Humanos , Imunofenotipagem/estatística & dados numéricos , Integrina alfa3/análise , Integrina alfa4/análise , Molécula 1 de Adesão Intercelular/análise , Selectina L/análise , Leucemia Linfocítica Crônica de Células B/metabolismo , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Risco , Análise de SobrevidaRESUMO
BACKGROUND/PURPOSE: The maturity of neomucosa growing on a serosal surface for the treatment of short bowel syndrome still is questionable. The aim of this study was to evaluate the intestinal neomucosa to assess its histologic maturity. METHODS: A 6-cm-long isolated ileal segment (IS) was prepared in 8 Wistar albino-type rats. The IS was divided from the antimesenteric side, and 2 intestinal tubes were established, which shared a common wall and a common pedicle. After ileal biopsy sampling for the control group (CG), the IS was fashioned into a mucous fistula. Eight weeks later, all the rats were killed, and the ISs were investigated for neomucosal growth. Sections were prepared with periodic acid shift (PAS) and H & E staining for light microscopy. They also were evaluated by transmission electron microscopy. The microscopic morphology of the 2 groups was evaluated. Immunohistochemical staining was performed to show the expression of the tissue beta1, alpha3 and alpha2beta1 integrin subunits of both the neomucosa (NS) and control group (CG) segments. RESULTS: Sections of the NS showed a well-arranged columnar epithelial cell layer with goblet cells that were generally located superficially and with a complete basement membrane. Under the electron microscope, the sections from the NS group showed an epithelial cell layer with proper microvilli of the same height, although they were shorter than those of the CG, and tight intercellular junctions between the epithelial cells. Significant differences between the NS and CG groups were found in the measurements of villus width at base, microvillus surface, and microvillus height. The lamina propria consisted of rich collagen fibers and active fibroblasts in the NS group. In the immunohistochemical staining, although beta1 integrine showed a dense distribution (+++) in the lamina propria, particularly localizing at the depth of the tunica mucosa layer, alpha3 integrin was observed to have a less dense immunoreactivity (++) in both groups. The expression of alpha2beta1 integrin showed slight and dispersed (+) staining. CONCLUSIONS: The NS showed histologic maturity and ultimate structural similarity with the native small bowel mucosa, which provides strong indirect evidence for the proper functioning of the neomucosa.
Assuntos
Íleo/ultraestrutura , Integrina alfa2beta1/análise , Integrina alfa3/análise , Integrina beta1/análise , Mucosa Intestinal/ultraestrutura , Anastomose Cirúrgica , Animais , Biópsia , Células Epiteliais/química , Células Epiteliais/ultraestrutura , Íleo/cirurgia , Mucosa Intestinal/química , Microscopia Eletrônica , Microvilosidades/química , Microvilosidades/ultraestrutura , Ratos , Ratos Wistar , Síndrome do Intestino Curto/prevenção & controle , Coloração e Rotulagem , Estruturas Criadas Cirurgicamente , CicatrizaçãoRESUMO
BACKGROUND: Pleomorphic adenoma (PA) and adenoid cystic carcinoma (ACC) are neoplasms of distinct behaviour, showing similar origin, cell components and marked presence of extracellular matrix (ECM). Interactions between cells and ECM are important in the biology of tumours, being partially mediated by integrins. This study investigated these interactions on PA and ACC using paraffin-embedded tissue and an in vitro model of these conditions. METHODS: Expression of integrins in paraffin-embedded samples was assessed by immunohistochemistry. Cells from PA and ACC were characterized using immunofluorescence, and integrin patterns of expression were investigated on cells cultivated on different ECM proteins. RESULTS: Luminal cells of both PA and ACC were more intensely positive for integrins than myoepithelial cells. In vitro studies revealed that PA cells expressed more integrins than ACC cells regardless the ECM protein present. CONCLUSIONS: This study revealed particular patterns of integrin expression in both specimens and in vitro models of PA and ACC. This might prove useful for a better understanding of the biology of these lesions.
Assuntos
Adenoma Pleomorfo/patologia , Carcinoma Adenoide Cístico/patologia , Matriz Extracelular/patologia , Integrinas/análise , Actinas/análise , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação a Calmodulina/análise , Colágeno Tipo I/análise , Colágeno Tipo IV/análise , Fibroblastos/patologia , Fibronectinas/análise , Humanos , Integrina alfa2/análise , Integrina alfa3/análise , Integrina alfa5/análise , Integrina beta1/análise , Laminina/análise , Proteínas dos Microfilamentos , Células Estromais/patologia , Vimentina/análise , CalponinasRESUMO
OBJECTIVE: To analyze integrin expression and distribution in different histological types of ameloblastoma, compared with dental germ, dental lamina and adult lining epithelium. MATERIALS AND METHODS: Three-micrometer sections from paraffin-embedded specimens were evaluated employing a streptavidin-biotin immunohistochemical method and anti-integrin alpha2, alpha3, alpha5, alphav, beta1, beta3 and beta4 antibodies. RESULTS: All integrins were present in all specimens, exhibiting different patterns. In follicular ameloblastoma, the integrin staining was stronger in the periphery while integrin alpha2 was not present in the central cells. Acanthomatous ameloblastoma showed a similar pattern, with positive staining for integrins alpha3, alpha5, alphav, beta1 and beta4 in the metaplastic cells. In the unicystic, integrin staining was uniform except for integrins alpha5 and beta3 which showed weaker staining in the upper layers. In the plexiform ameloblastoma, dental germ and lamina integrin staining was uniform. In the adult lining epithelium, staining for integrins alpha2, alpha5 and beta4 was confined to the basal layer, while integrins alphav and beta3 were present in the basal and parabasal, with integrins alpha3 and beta1 in the upper layers. CONCLUSION: Acanthomatous, follicular and unicystic ameloblastomas showed integrin staining patterns similar to the adult lining epithelium while the plexiform ameloblastoma was similar to the dental germ and lamina.
Assuntos
Ameloblastoma/patologia , Cadeias alfa de Integrinas/análise , Cadeias beta de Integrinas/análise , Mucosa Bucal/patologia , Germe de Dente/patologia , Adulto , Ameloblastoma/classificação , Anticorpos , Membrana Basal/patologia , Corantes , Epitélio/patologia , Humanos , Imuno-Histoquímica , Integrina alfa2/análise , Integrina alfa3/análise , Integrina alfa5/análise , Integrina alfaV/análise , Integrina beta1/análise , Integrina beta3/análise , Integrina beta4/análise , MetaplasiaRESUMO
In spite of the attention given to the study of mesenchymal stem cells (MSCs) derived from the bone marrow (BM) of humans and other species, there is a lack of information about murine MSCs. We describe the establishment of conditions for the in vitro expansion of plastic-adherent cells from murine BM for over 50 passages, and provide their characterization regarding morphology, surface marker profile and growth kinetics. These cells were shown to differentiate along osteogenic and adipogenic pathways, and to support the growth and differentiation of haematopoietic stem cells, and were thus operationally defined as murine mesenchymal stem cells (mMSCs). mMSCs were positive for the surface markers CD44, CD49e, CD29 and Sca-1, and exhibited a homogeneous, distinctive morphology. Their frequency in the BM of adult BALB/c and C57Bl/6 mice, normal or knockout for the alpha-L-iduronidase (IDUA) gene, was preliminarily estimated to be 1 per 11,300-27,000 nucleated cells. The emergence of a defined methodology for the culture of mMSCs, as well as a comprehensive understanding of their biology, will make the development of cellular and genetic therapy protocols in murine models possible, and provide new perspectives in the field of adult stem cells research.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Mesenquimais/citologia , Animais , Antígenos Ly/análise , Células da Medula Óssea/imunologia , Diferenciação Celular , Divisão Celular , Separação Celular/métodos , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citometria de Fluxo , Receptores de Hialuronatos , Integrina alfa3/análise , Integrina beta1/análise , Proteínas de Membrana/análise , Células-Tronco Mesenquimais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
GM3 ganglioside inhibits tetraspanin CD9-facilitated cell motility in various cell lines (Ono, M., Handa, K., Sonnino, S., Withers, D. A., Nagai, H., and Hakomori, S. (2001) Biochemistry 40, 6414-6421). We now report the following: (i) CD9 has the novel feature of being soluble in chloroform/methanol, and classifiable as "proteolipid"; (ii) CD9 and alpha(3) integrin were concentrated together in the low-density glycolipid-enriched microdomain (GEM) of ldlD/CD9 cells, and the alpha(3) expression ratio (value for cells grown under +Gal condition divided by the value for cells grown under -Gal condition) in GEM of ldlD/CD9 cells was higher than that in control ldlD/moc cells, suggesting that CD9 recruits alpha(3) in GEM under +Gal condition, whereby GM3 is present. (iii) Chemical levels of alpha(3) and CD9 in the total extract or membrane fractions from cells grown under +Gal versus -Gal condition were nearly identical, whereas alpha(3) expressed at the cell surface, probed by antibody binding in flow cytometry, was higher under -Gal than +Gal condition. These results suggest that GM3 synthesized under +Gal condition promotes interaction of alpha(3) with CD9, which restricts alpha(3) binding to its antibody. A concept of the alpha(3)/CD9 interaction promoted by GM3 was further supported by (i) co-immunoprecipitation of CD9 and alpha(3) under +Gal but not -Gal condition, (ii) enhanced co-immunoprecipitation of CD9 and alpha(3) when GM3 was added exogenously to cells under -Gal condition, and (iii) the co-localization images of CD9 with alpha(3) and of GM3 with CD9 in fluorescence laser scanning confocal microscopy. Based on the promotion of alpha(3)/CD9 interaction by GM3 and the status of laminin-5 as a true ligand for alpha(3), the laminin-5/alpha(3)-dependent motility of ldlD/CD9 cells was found to be greatly enhanced under -Gal condition, but strongly inhibited under +Gal condition. Such a motility difference under +Gal versus -Gal condition was not observed for ldlD/moc cells. The inhibitory effect observed in ldlD/CD9 cells under +Gal condition was reversed upon addition of anti-alpha(3) antibody and is therefore based on interaction between alpha(3), CD9, and GM3 in GEM.