RESUMO
Breast cancer is a leading cancer in women and despite the benefits of the current therapies a significant number of patients with this tumor is at risk of relapse. Some of the alterations taking place in breast cancer cells are currently exploited by molecularly targeted drugs. Different drugs have been developed which target a single molecule but, given that the tumor originates from the dysregulation of many genes, there is the need to find new drugs that have more than one molecular target. Curcumin [1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] (CUR), a polyphenolic compound found in the spice turmeric, is a pleiotropic molecule able to interact with a variety of molecular targets and has antitumor, anti-inflammatory, antioxidant, immunomodulatory and antimicrobial activities. Here we demonstrate that CUR inhibits the growth of breast cancer cell lines in a dose dependent manner, with IC50 values in the micromolar range, and induces an increase in the percentage of cells in sub-G0 phase, representing the apoptotic cell population. The activation of apoptosis was confirmed by PARP-1 cleavage and by the increased ratio between the pro-apoptotic Bax and the anti-apoptotic Bcl-2 protein. In addition, in CUR-treated cells the activity of ERK1/ERK2 MAP kinases was down-regulated. The cytotoxic effects of CUR were observed in breast cancer cells expressing either high or low levels of ErbB2/neu. The in vivo antitumor activity of CUR was tested in BALB-neuT mice transgenic for the neu oncogene, which develop atypical hyperplasia of the mammary gland at 6 weeks of age and invasive carcinoma at 16 weeks of age. CUR, administered to mice both early and in an advanced stage of mammary carcinogenesis, induced a significant prolongation of tumor-free survival and a reduction of tumor multiplicity. In addition, CUR administration was safe, since no modification of hematological and clinical chemistry parameters could be observed in BALB-neuT and BALB/c mice treated with this compound for several weeks. These findings support further studies on the therapeutic potential of CUR in combination with standard therapies in breast cancer patients.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Curcumina/farmacologia , Neoplasias Mamárias Animais/patologia , Receptor ErbB-2/metabolismo , Animais , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Curcumina/efeitos adversos , Curcumina/uso terapêutico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Mamárias Animais/sangue , Neoplasias Mamárias Animais/tratamento farmacológico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Células NIH 3T3 , Fosforilação/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/metabolismo , Ratos , Receptor ErbB-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
Stages of bone turnover during fracture repair can be assessed employing serum markers of osteoblastic and osteoclastic activity, inflammatory cytokines, clinical evaluation and imaging instruments. Our study compare the fracture healing process in fragility fractures and high energy fractures by evaluating serum changes of interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), osteoprotegerin (OPG) and receptor activator of the nuclear factor-kB ligand (RANKL) in combination with radiographic (Radiographic Union Scale for Tibial fractures, RUST) and clinical (Lower extremity measure, LEM) assessments. We enrolled 56 patients divided into four corresponding groups: group A with high energy trauma fracture (tibial/femoral shaft); group B with low energy trauma fracture (femoral fractures); healthy (control A) and osteoporotic subjects (control B). Blood samples were collected before surgery (T0) and after 10 weeks (T10). Serum concentrations of IL-6, TNF-alpha, RANKL and OPG were quantified using commercial enzyme-linked immunosorbent assay (ELISA) kits. Our results show that RANKL values are significantly higher at T10 than at T0 in low energy trauma fractures (group B). OPG is significantly lower in each control group than that of the respective fractured group and its concentration at T0 and at T10 is significantly lower in high than in low energy fractures. RANKL/OPG ratio is significantly higher in both controls than in fractured groups, and significantly increases after 10 weeks. IL-6 and TNF-alpha concentrations significantly decrease during fracture healing and are higher in high (group A) than in low energy fractures (group B). Significant differences were also found in both RUST score and LEM between groups A and B. Changes in TNF-alpha and IL-6 levels correlate with RUST and LEM in fragility and high energy fractures, while RANKL/OPG ratio is associated with these clinical parameters only in fragility fractures. These findings suggest that serum levels of IL-6, TNF-alpha, RANKL and OPG might be used to monitor the stages of fracture repair. Further studies will be needed to confirm the role of these cytokines in fracture repair.
Assuntos
Fraturas do Fêmur/sangue , Interleucina-6/sangue , Osteoprotegerina/sangue , Ligante RANK/sangue , Fraturas da Tíbia/sangue , Fator de Necrose Tumoral alfa/sangue , Adulto , Idoso , Feminino , Fraturas do Fêmur/diagnóstico por imagem , Consolidação da Fratura , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia , Fraturas da Tíbia/diagnóstico por imagemRESUMO
Castleman's disease is a lymphoproliferative disorder thought to be related to deregulated production of IL-6. We have previously shown that mice lacking the trans-acting factor C/EBP beta, a transcriptional regulator of IL-6 and a mediator of IL-6 intracellular signaling, develop a pathology nearly identical to multicentric Castleman's disease, together with increasingly high levels of circulating IL-6. We describe here how the simultaneous inactivation of both IL-6 and C/EBP beta genes prevents the development of pathological traits of Castleman's disease observed in C/EBP beta-deficient mice. Histological and phenotypic analysis of lymph nodes and spleen of double mutant mice did not show either the lymphoadenopathy and splenomegaly or the abnormal expansion of myeloid, B and plasma cell compartments observed in C/EBP beta-/- mice, while B cell development, although delayed, was normal. Our data demonstrate that IL-6 is essential for the development of multicentric Castleman's disease in C/EBP beta-/- mice.
Assuntos
Hiperplasia do Linfonodo Gigante/genética , Proteínas de Ligação a DNA/genética , Interleucina-6/genética , Proteínas Nucleares/genética , Animais , Linfócitos B/patologia , Proteínas Estimuladoras de Ligação a CCAAT , Hiperplasia do Linfonodo Gigante/patologia , Hiperplasia do Linfonodo Gigante/prevenção & controle , Proteínas de Ligação a DNA/metabolismo , Citometria de Fluxo , Linfonodos/patologia , Camundongos , Camundongos Mutantes , Proteínas Nucleares/metabolismo , Fenótipo , Plasmócitos/patologia , Baço/patologia , Timo/patologiaRESUMO
The ability of interleukin (IL)-12 to prevent tumors when administered to individuals with a genetic risk of cancer was studied in two lines of transgenic mice expressing rat HER-2/neu oncogene in the mammary gland. Female BALB/c (H-2(d)) mice carrying the activated HER-2/ neu oncogene show no morphological abnormalities of the mammary gland until 3 wk of age. They then progress through atypical hyperplasia to in situ lobular carcinoma and at 33 wk of age all 10 mammary glands display invasive carcinomas. Adult FVB mice (H-2(q)) carrying the HER-2/neu protooncogene develop mammary carcinomas with a longer latency (38-49 wk) and a lower multiplicity (mean of 2.6 tumors/mice). Treatment with IL-12 (5 daily intraperitoneal injections, 1 wk on, 3 wk off; the first course with 50 ng IL-12/day, the second with 100 ng IL-12/day) begun at 2 wk of age in BALB/c mice and at 21 wk of age in FVB mice markedly delayed tumor onset and reduced tumor multiplicity. Analogous results were obtained in immunocompetent and permanently CD8(+) T lymphocyte-depleted mice. In both transgenic lines, tumor inhibition was associated with mammary infiltration of reactive cells, production of cytokines and inducible nitric oxide synthase, and reduction in microvessel number, in combination with a high degree of hemorrhagic necrosis.
Assuntos
Antineoplásicos/farmacologia , Carcinoma in Situ/prevenção & controle , Carcinoma Lobular/prevenção & controle , Interleucina-12/farmacologia , Neoplasias Mamárias Experimentais/prevenção & controle , Receptor ErbB-2/fisiologia , Animais , Antineoplásicos/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Carcinoma in Situ/genética , Carcinoma in Situ/imunologia , Carcinoma Lobular/genética , Carcinoma Lobular/imunologia , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Feminino , Interferon gama/imunologia , Interleucina-12/imunologia , Depleção Linfocítica , Masculino , Neoplasias Mamárias Experimentais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Óxido Nítrico Sintase/biossíntese , Óxido Nítrico Sintase Tipo II , Ratos , Receptor ErbB-2/genética , Fator de Necrose Tumoral alfa/imunologia , Molécula 1 de Adesão de Célula Vascular/biossínteseRESUMO
In this study, a proteomic approach that combines selective labelling of proteins containing reduced cysteine residues with two-dimensional electrophoresis/mass spectrometry was used to evaluate the redox state of protein cysteines during chronological ageing in Saccharomyces cerevisiae. The procedure was developed on the grounds that biotin-conjugated iodoacetamide (BIAM) specifically reacts with reduced cysteine residues. BIAM-labelled proteins can then be selectively isolated by streptavidin affinity capture. We compared cells grown on 2% glucose in the exponential phase and during chronological ageing and we found that many proteins undergo cysteine oxidation. The target proteins include enzymes involved in glucose metabolism. Both caloric restriction and growth on glycerol resulted in a decrease in the oxidative modification. Furthermore, in these conditions a reduced production of ROS and a more negative glutathione half cell redox potential were observed.
Assuntos
Carbono/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Restrição Calórica , Cisteína/metabolismo , Citocromos/metabolismo , Glucose/metabolismo , Glutationa/metabolismo , Glicerol/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Oxirredução , Consumo de Oxigênio , Proteômica/métodos , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Fatores de TempoRESUMO
AIM: Management of patients with pre-existing coronary heart disease (CHD) relies for the most part on primary care physicians, an endeavour whose success is dependent upon acceptance and day-to-day application of guideline recommendations for secondary CHD prevention. The aim of this study is to analyze the status of secondary CHD prevention in an Italian primary care practice consisting of five partnered general practitioners attending 7006 subjects aged 15 years or more (3137 males, 3869 females) in Pontedera, Tuscany. METHODS: Retrieval of patients with history of CHD (previous myocardial infarction, [MI], and stable angina) from computerized records of the 5987 (2735 men, 3252 women) subjects aged 35-85 years enlisted in the practice. Patients with myocardial infarction <3 months at the time of the query were excluded. RESULTS: Search retrieved 153 (2.6%) subjects with history of CHD, 93 (3.4%) males and 60 (1.8%) females. Females were older and smoked more frequently than men. Antiplatelet drugs, beta-blockers, renin-angiotensin system blockers and statins were prescribed in 84%, 56%, 66% and 68% of the ischemic patients. LDL cholesterol targets of 100 and 70 mg/dL were achieved in only 60 (45%) and 11 (9%) respectively. Systolic blood pressure was above 140 mmHg in 25 out of 146 patients with available data. CONCLUSION: The surveys shows satisfactory uptake of guideline recommendations but also pitfalls in the implementation of secondary CHD prevention requirements. Targeted interventions on primary care physicians are critically needed to enhance further provider adherence to consensus guidelines for CHD risk reduction.
Assuntos
Doença das Coronárias/prevenção & controle , Padrões de Prática Médica , Atenção Primária à Saúde , Prevenção Secundária , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Inquéritos e QuestionáriosRESUMO
We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.
Assuntos
Herpesvirus Humano 8/química , Proteínas Virais , Linfócitos B/virologia , Linhagem Celular , Núcleo Celular , Humanos , Membrana Nuclear , Proteínas Nucleares , Fases de Leitura Aberta , RNA Viral/análise , Homologia de Sequência , Proteínas Virais/análise , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Neural crest-derived cells populate the thymus, and their coexistence with epithelial cells is required for proper organ development and T cell education function. We show here that epidermal growth factor (EGF), a major epithelial cell growth-enhancing agent, has a morphogenetic action to promote the expression of a neuronal phenotype (e.g., neurofilament expression) in cultured thymic epithelial cells that are characterized by a cytokeratin-positive epithelial cell background. The proliferation of such neurodifferentiated cells is also enhanced by EGF. Furthermore, the growth factor enhances cells that express the genes encoding the preprotachykinin A-generated neuropeptides and bipotential neuropoietic and lymphopoietic cytokines ciliary neurotrophic factor and interleukin-6. These cytokines also enhance the neuronal phenotype of thymic epithelial cells. Therefore, EGF appears to be a composite autocrine/paracrine neuromodulator in thymic stroma. This suggests that EGF may regulate thymus-dependent immune functions by promoting neuronal gene expression in neural crest-derived cells.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Citocinas/fisiologia , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Timo/citologia , Animais , Sequência de Bases , Ciclo Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Fator Neurotrófico Ciliar , Primers do DNA/química , Fator de Crescimento Epidérmico/metabolismo , Células Epiteliais , Imunofluorescência , Expressão Gênica , Técnicas In Vitro , Interleucina-6/fisiologia , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/fisiologia , Proteínas de Neurofilamentos/metabolismo , RNA Mensageiro/genéticaRESUMO
A new human retrovirus was isolated from a continuous cell line derived from a patient with CD4+ Tac- cutaneous T cell lymphoma/leukemia. This virus is related to but distinct from human T cell leukemia/lymphoma virus types I and II (HTLV-I and HTLV-II) and human immunodeficiency virus (HIV-1). With the use of a fragment of provirus cloned from one patient with T cell leukemia, closely related sequences were found in DNA of the cell line and of tumor cells from seven other patients with the same disease; these sequences were only distantly related to HTLV-I. The phenotype of the cells and the clinical course of the disease were clearly distinguishable from leukemia associated with HTLV-I. All patients and the wife of one patient showed a weak serological cross-reactivity with both HTLV-I and HIV-1 antigens. None of the patients proved to be at any apparent risk for HIV-1 infection. The name proposed for this virus is HTLV-V, and the date indicate that it may be a primary etiological factor in the major group of cutaneous T cell lymphomas/leukemias, including the sporadic lymphomas known as mycoses fungoides.
Assuntos
Deltaretrovirus/isolamento & purificação , Leucemia/microbiologia , Linfoma/microbiologia , Antígenos Virais/análise , Deltaretrovirus/classificação , Deltaretrovirus/ultraestrutura , Feminino , Humanos , Masculino , Microscopia Eletrônica , Linfócitos T/citologiaRESUMO
Placenta growth factor (PlGF) is a key regulator of pathological angiogenesis and its overexpression has been linked to neoplastic progression. To assess whether PlGF could have a role in malignant mesothelioma (MM), we analyzed the expression of PlGF, VEGF, and their cognate receptors (VEGF-R1 and VEGF-R2) and co-receptors (neuropilin-1 and neuropilin-2) in MM cell lines as well as in resected MM tissues, hyperplastic/reactive mesothelium and normal mesothelium. MM cell cultures expressed both ligands and the associated receptors to a variable extent and released different amounts of PlGF. As assessed by immunohistochemistry, PlGF expression was switched on in hyperplastic/reactive compared to normal mesothelium. Moreover, 74 and 94 percent of MM tissues overexpressed PlGF and VEGF-R1, respectively (p<0.05 MM vs normal mesothelium). Administration of recombinant PlGF-2 did not elicit a significant stimulation of MM cell growth, while it was associated with a transient phosphorylation of Akt, suggesting that PlGF-2 could activate downstream effectors of proliferative and cytoprotective signals via VEGF-R1 in MM cells. Indeed, the administration of an anti-PlGF antibody was found to cause a significant reduction of MM cell survival. In conclusion, our data demonstrate that, by acting as a survival factor, PlGF can play a role which goes beyond the stimulation of angiogenesis in MM. This evidence could help the rational design of new therapeutic interventions for this aggressive tumor.
Assuntos
Epitélio/metabolismo , Mesotelioma/metabolismo , Neoplasias Pleurais/metabolismo , Proteínas da Gravidez/metabolismo , Morte Celular , Linhagem Celular , Proliferação de Células , Sobrevivência Celular , Epitélio/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperplasia , Mesotelioma/irrigação sanguínea , Mesotelioma/genética , Mesotelioma/patologia , Neovascularização Patológica/metabolismo , Neuropilina-1/metabolismo , Neuropilina-2/metabolismo , Fosforilação , Fator de Crescimento Placentário , Neoplasias Pleurais/irrigação sanguínea , Neoplasias Pleurais/genética , Neoplasias Pleurais/patologia , Proteínas da Gravidez/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Endomorphin-1 (EM-1) is an endogenous opioid peptide selectively binding to micro opioid receptors (MORs). Besides its analgesic effects on the central nervous system (CNS), it has been recently reported that EM-1 can cross the blood-brain barrier (BBB) and diffuse into the blood, behaving as an analgesic/anti-inflammatory molecule on peripheral tissues, thus leading to the hypothesis that it could represent a soluble modulator of immune cell functions. Interestingly, nothing is known about its possible effects on monocytes, the main circulating cell-type involved in those systemic responses, such as fever and septic states, involving the release of high amounts of pyrogenic inflammatory factors. The aim of this work is to evaluate possible EM-1effects on lipopolisaccharide (LPS)-stimulated THP-1 monocytes in terms of the production of inflammatory mediators and the instauration of a hyporesponsive-like phenotype which is a main feature of systemic inflammatory responses, and on the development of peripheral monocytes to DC. Our data demonstrate for the first time that EM-1 is able to inhibit both LPS-stimulated monocyte activation, in terms of arachidonic acid, PGE2, ROI and NO2 production and instauration of a hyporesponsive phenotype without any macroscopic effect on DC development. These data support the hypothesis that EM-1 could be involved in modulating monocyte functions during systemic inflammatory reactions, also providing new evidence for its eventual clinical application in endotoxic states.
Assuntos
Lipopolissacarídeos/farmacologia , Monócitos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Ácido Araquidônico/metabolismo , Cálcio/metabolismo , Linhagem Celular , Dinoprostona/metabolismo , Citometria de Fluxo , Humanos , Leucotrieno B4/metabolismo , Monócitos/metabolismo , Dióxido de Nitrogênio/metabolismo , Receptores Opioides mu/metabolismoRESUMO
PURPOSE: The purpose of our study was to investigate the histological, histochemical and ultrastructural aspects of the proximal femoral growth plate in slipped capital femoral epiphysis (SCFE). METHODS: Eight core biopsies of the proximal femoral growth plate were performed during in situ epiphysiodesis in patients with SCFE that was at the pre-slipping stage in two cases and at the mild slipping stage (Southwick angle < 30°) in six cases. After fixation, the specimens were processed for either histological or histochemical or ultrastructural studies. RESULTS: The proximal femoral growth plate was thicker than normal in the SCFE cases, and the 3:1 ratio between the thickness of the resting zone and the other zones of the plate was reversed. Chondrocytes of the proliferating, maturation, hypertrophic and degenerating zones were arranged in large clusters rather than in columns, which were separated by loose fibrillary septae that appeared moderately alcian blue positive and metachromatic. The collagen fibrils of the longitudinal septae were uniformly thin, measuring about 200 Å, whereas in the normal plate collagen fibrils were in the range of 300 to 1200 Å in thickness. Chondrocytes were elongated and smaller than normal, with a dark cytoplasm. In the degenerating zone, mineralisation of the longitudinal and transversal septae was scanty and enchondral ossification was impaired, with a few small osteoblasts forming thin bone trabeculae on the cartilage septae of the degenerating zone. CONCLUSION: In SCFE, the proximal femoral growth plate undergoes several histological, histochemical and ultrastructural changes that precede slipping of the epiphysis since they are already present at a pre-slipping stage of the disease. The loss of solidity of the extracellular matrix and the disarrangement of the normal architecture of the physis very likely cause the consequent slipping of the proximal femoral epiphysis. SCFE aetiology remains unknown.
RESUMO
The aim of the present study was to investigate whether and which cardiac growth factors are involved in human hypertrophy, whether growth factor synthesis is influenced by overload type and/or by the adequacy of the hypertrophy, and the relationships between cardiac growth factor formation and ventricular function. Cardiac growth factor formation was assessed by measuring aorta-coronary sinus concentration gradient in patients with isolated aortic stenosis (n=26) or regurgitation (n=15) and controls (n=12). Gene expression and cellular localization was investigated in ventricular biopsies using reverse transcriptase-polymerase chain reaction and in situ hybridization. Cardiac hypertrophy with end-systolic wall stress <90 kdyne/cm2 was associated with a selective increased formation of insulin-like growth factor (IGF)-I in aortic regurgitation and of IGF-I and endothelin (ET)-1 in aortic stenosis. mRNA levels for IGF-I and preproET-1 were elevated and mainly expressed in cardiomyocytes. At stepwise analysis, IGF-I formation was correlated to the mean velocity of circumferential fiber shortening (r=0.86, P<0.001) and ET-1 formation to relative wall thickness (r=0.82, P<0. 001). When end-systolic wall stress was >90 kdyne/cm2, IGF-I and ET-1 synthesis by cardiomyocytes was no longer detectable, and only angiotensin (Ang) II was generated, regardless of the type of overload. The mRNA level for angiotensinogen was high, and the mRNA was exclusively expressed in the interstitial cells. Ang II formation was positively correlated to end-systolic stress (r=0.89, P<0.001) and end-diastolic stress (r=0.84, P<0.001). Multivariate stepwise analysis selected end-systolic stress as the most predictive variable and left ventricular end-diastolic pressure as the independent variable for Ang II formation (r=0.93, P<0.001). In conclusion, the present results indicate that the course of human left ventricular hypertrophy is characterized by the participation of different cardiac growth factors that are selectively related both to the type of hemodynamic overload and to ventricular function.
Assuntos
Cardiomegalia/metabolismo , Substâncias de Crescimento/metabolismo , Miocárdio/metabolismo , Idoso , Angiotensinas/sangue , Cardiomegalia/sangue , Cardiomegalia/diagnóstico por imagem , Cardiomegalia/fisiopatologia , Ecocardiografia , Endotelinas/sangue , Substâncias de Crescimento/sangue , Coração/fisiopatologia , Hemodinâmica/fisiologia , Humanos , Fator de Crescimento Insulin-Like I/análise , Pessoa de Meia-Idade , Contração Miocárdica/fisiologia , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse MecânicoRESUMO
Employing purified extracellular matrix (ECM) proteins, i.e. type I, III, IV and V collagens (CI, CIII, CIV, CV), laminin (LM) and fibronectin (FN), as antigen sources we detected autoantibodies to conformational and/or denatured ECM antigens among 34 of 50 sera obtained from Hashimotos thyroiditis (HT) patients and 6 of 51 control sera obtained from non-autoimmune thyroid disease patients and healthy donors (HT sera vs. control sera p=4 x 10-9). Reactivity to conformational antigens, mostly due to autoantibodies of the IgG isotype, was observed in 30/50 HT sera and in 6/51 control sera (p=3.5 x 10-7) and was not always concomitant with that to linear antigens, found in 23/50 HT and in 6/51 control sera (p=1.6 x 10-4). Ultrastructural analysis of skin biopsies obtained from 18 HT patients without symptomatic cutaneous diseases revealed defects of the stratified squamous epithelium basement membrane in 11/18, alterations of the stroma in 13/18 and both basement membrane and stromal defects in 9/18. Interestingly, 13/13 (p=0.012) and 9/11 (p=0.012) patients with stromal and basement membrane defects respectively, exhibited serum antibodies to at least one ECM antigen involved in the organization of the altered tissue compartment. Lastly, 10/18 skin biopsies presented immunoglobulin (Ig) and/or complement (C3) deposits along the cutaneous basement membrane zone (BMZ) or in the papillary dermis and 9/10 sera from the same patients simultaneously showed antibodies to at least one ECM antigen involved in the organization of these two skin compartments. Besides, 8/11 HT patients with basement membrane defects exhibited Ig or C3 deposits along the BMZ. Our findings suggest that autoantibodies to ECM molecules might contribute to the development of asymptomatic extra-thyroid skin diseases in HT patients.
Assuntos
Autoanticorpos/sangue , Proteínas da Matriz Extracelular/imunologia , Doença de Hashimoto/imunologia , Pele/ultraestrutura , Membrana Basal/ultraestrutura , Complemento C3/análise , Ensaio de Imunoadsorção Enzimática , Epitélio/ultraestrutura , Doença de Hashimoto/patologia , Humanos , Isotipos de Imunoglobulinas/sangue , Imunoglobulinas/análise , Células Estromais/ultraestruturaRESUMO
BACKGROUND: Numerous animal model studies have examined the ability of genetically engineered tumor cells to release cytokines and to elicit an immune memory against the parental tumor. Often only a single cytokine is studied, and few comparative studies have been conducted. PURPOSE: We evaluated the antitumor efficacy of adenocarcinoma cells engineered to release interleukin (IL)-12 in a mouse model system. The efficacy of this cytokine was compared with that of other cytokines released by engineered adenocarcinoma cells and that of exogenous IL-12 administered both locally and intraperitoneally. METHODS: BALB/cAnCr mice were inoculated with syngeneic parental mammary adenocarcinoma (TSA) cells in quantities sufficient to lead to tumors in all inoculated mice. TSA cells engineered to release IL-12 (TSA-IL12) were also injected into normal and selectively immunosuppressed BALB/cAnCr mice. Tumor incidence, growth, and rejection patterns were evaluated by the measurement of neoplastic masses and by the study of the histologic and ultrastructural features of the tumor site. The effects of local or intraperitoneal administration of recombinant IL-12 (rIL-12) on tumor-bearing animals were also studied. RESULTS: Most mice rejected TSA-IL12 cells through a CD8-positive, T-lymphocyte-dependent reaction associated with macrophage infiltration, vessel damage, and necrosis. The systemic immunity of mice that had rejected TSA-IL12 cells to a subsequent challenge with parental TSA cells was less efficient than that elicited by TSA cells engineered to release IL-4 or IL-10 but equivalent to that elicited by TSA cells engineered to release IL-2, IL-7, and interferon alfa. Compared with TSA cells engineered to produce other cytokines, TSA-IL12 cells were the most efficient in curing mice with established TSA tumors; injection of 0.1 million proliferating cells contralaterally to the tumor growth area cured five of 15 mice bearing 1-day-old tumors; injection of the same dose of proliferating cells into the tumor growth area cured two of 20 tumor-bearing mice. However, two 5-day courses with a nontoxic dose (0.1 microgram) of rIL-12 given intraperitoneally cured a similar proportion of these animals (six of 20). Only two of 20 mice with 7-day-old TSA tumors were cured by vaccination with proliferating TSA-IL12 cells, whereas 24 of 30 mice with such tumors were cured by intraperitoneal administration of rIL-12. CONCLUSIONS: TSA cells engineered to release IL-12 are rejected by most mice; the ensuing immune memory for TSA parental cells, however, was less efficient than that elicited by proliferating TSA cells engineered to release other cytokines (e.g., IL-4, IL-10, and possibly interferon gamma). The immune reaction elicited by TSA-IL12 cells was the most efficient in curing mice with established TSA tumors; notably though, the same or a better cure rate was obtained with rIL-12 given intraperitoneally.
Assuntos
Adenocarcinoma/tratamento farmacológico , Interleucina-12/farmacologia , Interleucina-12/fisiologia , Neoplasias Mamárias Experimentais/tratamento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Modelos Animais de Doenças , Feminino , Infusões Parenterais , Injeções Intralesionais , Interleucina-12/administração & dosagem , Interleucina-12/biossíntese , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Transdução GenéticaRESUMO
A T-lymphoma cell line was established from a lymph node biopsy of a boy currently alive in complete remission. Neoplastic cells from this biopsy did not grow in vitro, whereas they formed a progressively growing s.c. tumor in splenectomized and sublethally irradiated nude mice and became serially transplantable in splenectomized and sublethally irradiated nude mice with a stable latency time. After the fourth transplant, cells were stored in liquid nitrogen and referred to as ST-4 cells. ST-4 cells display a membrane phenotype and a karyotype similar to that of the biopsy cells. After thawing, ST-4 cells grow both in splenectomized and sublethally irradiated nude mice and in vitro. They do not secrete interferon or interleukin 2, do not have natural killer activity, and do not respond to mitogen or alloantigen stimulation. The stable features of these T-lymphoma cells and the availability of normal autologous lymphocytes from the patient make this in vivo system quite unique and of importance for studies in tumor immunotherapy.
Assuntos
Linfoma/patologia , Animais , Criança , Humanos , Ativação Linfocitária , Linfoma/genética , Linfoma/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Linfócitos T , Translocação Genética , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
The potential of interleukin 2-gene-transfected tumor cells to prevent tumor growth and cure established tumors was evaluated using cells from a spontaneous, invasive, and metastasizing mouse mammary adenocarcinoma. Tumor cells engineered to secrete interleukin 2 initially trigger a local inflammatory reaction that leads to inhibition of established parental adenocarcinomas, as well as an antigenically unrelated fibrosarcoma. The ensuing systemic immunity selectively inhibits subsequent parental cell challenges and cures established parental adenocarcinomas and their lung metastases, although less effectively as the neoplastic mass increases. Multiple injections of interleukin 2-gene-transfected tumor cells may thus be considered a new form of vaccination in the management of minimal residual disease and incipient metastases.
Assuntos
Adenocarcinoma/imunologia , Imunização , Interleucina-2/biossíntese , Neoplasias Mamárias Experimentais/imunologia , Transfecção , Adenocarcinoma/patologia , Adenocarcinoma/secundário , Adenocarcinoma/terapia , Animais , Feminino , Fibrossarcoma/imunologia , Fibrossarcoma/patologia , Inflamação , Interleucina-2/genética , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/terapia , Camundongos , Camundongos Endogâmicos BALB C , Linfócitos T Citotóxicos/imunologia , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Until recently, lymphokines were regarded suspiciously as 'ill-defined factors'. Today, however, some of them have been clearly defined in both structural and functional terms. The interleukin-2 (IL-2) molecule and its specific membrane receptors have been the subject of particular attention. Endogenous IL-2 has proved to be an important signal for the activation and expansion of various cell-mediated immunity functions, while exogenous IL-2 has been used to activate numerous cell functions, both in vitro and in vivo, as well as in tumour immunotherapy, both alone or combined with lymphocytes previously activated in vitro (lymphokine-activated killer cells). Adoptive transfer of these cells together with high doses of IL-2 is particularly promising from the clinical standpoint, though by no means free from problems. IL-2 can also be employed in small doses locally in the presence of non-activated lymphocytes from tumour bearing mice to induce a local reaction that subsequently becomes systemic and can lead to the rejection of incipient tumours. Various host immune cells, primarily eosinophils and lymphocytes are involved in this reaction, which can also give rise to tumour-specific immune memory. In this way, the host immune system, despite its inevitable defeat in the first battle against a tumour, may acquire an important role in the long war that lies ahead.
Assuntos
Interleucina-2/uso terapêutico , Neoplasias Experimentais/terapia , Animais , Antígenos de Neoplasias/imunologia , Eosinófilos/imunologia , Imunoterapia , Interleucina-2/fisiologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Microscopia Eletrônica , Neoplasias Experimentais/ultraestrutura , Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologiaRESUMO
A DNA sequence coding for human muscle acylphosphatase has been constructed using 16 chemically synthesized oligonucleotides. The 300-bases long DNA sequence has been cloned in the pT7.7 Escherichia coli expression vector and in the pYEpsec1 Saccharomyces cerevisiae expression vector. In both cases a high level of expression of acylphosphatase has been observed. The recombinant proteins have been purified to homogeneity and assayed in comparison with the natural protein, using benzoylphosphate as a substrate and phosphate as a competitive inhibitor. The recombinant enzymes expressed in the two microorganisms maintain the kinetic properties of the natural protein. In addition, NMR analysis shows that the gross fold of the two recombinant enzymes is correct.
Assuntos
Hidrolases Anidrido Ácido/biossíntese , Hidrolases Anidrido Ácido/genética , DNA/síntese química , Genes Sintéticos , Hominidae/genética , Músculos/enzimologia , Hidrolases Anidrido Ácido/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , DNA/metabolismo , Escherichia coli , Humanos , Cinética , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Peso Molecular , Oligodesoxirribonucleotídeos/síntese química , Conformação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae , AcilfosfataseRESUMO
Three mutants of human muscle acylphosphatase in which arginine-23 was replaced by glutamine, histidine and lysine, respectively, were prepared by oligonucleotide-directed mutagenesis of a synthetic gene coding for the enzyme. All mutants, purified by affinity chromatography, were almost completely unable to catalyze the hydrolysis of the substrate. 1H-NMR spectroscopy experiments showed the absence of any major conformational changes of the three mutants with respect to the wild-type recombinant enzyme. Equilibrium dialysis experiments demonstrated that the mutated proteins lost the ability of binding inorganic phosphate, a competitive inhibitor of the enzyme. These results strongly support an involvement of arginine-23 at the phosphate binding-site of acylphosphatase, confirming the hypothesis of the existence of a phosphate binding structural motif recently proposed by other authors.