RESUMO
Engrailed-2 (En2), a homeodomain transcription factor involved in regionalization and patterning of the midbrain and hindbrain regions has been associated to autism spectrum disorders (ASDs). En2 knockout (En2(-/-)) mice show ASD-like features accompanied by a significant loss of GABAergic subpopulations in the hippocampus and neocortex. Brain-derived neurotrophic factor (BDNF) is a crucial factor for the postnatal development of forebrain GABAergic neurons, and altered GABA signaling has been hypothesized to underlie the symptoms of ASD. Here we sought to determine whether interneuron loss in the En2(-/-) forebrain might be related to altered expression of BDNF and its signaling receptors. We first evaluated the expression of different BDNF mRNA isoforms in the neocortex and hippocampus of wild-type (WT) and En2(-/-) mice. Quantitative RT-PCR showed a marked down-regulation of several splicing variants of BDNF mRNA in the neocortex but not hippocampus of adult En2(-/-) mice, as compared to WT controls. Accordingly, levels of mature BDNF protein were lower in the neocortex but not hippocampus of En2(-/-) mice, as compared to WT. Increased levels of phosphorylated TrkB and decreased levels of p75 receptor were also detected in the neocortex of mutant mice. Accordingly, the expression of low density lipoprotein receptor (LDLR) and RhoA, two genes regulated via p75 was significantly altered in forebrain areas of mutant mice. These data indicate that BDNF signaling alterations might be involved in the anatomical changes observed in the En2(-/-) forebrain and suggest a pathogenic role of altered BDNF signaling in this mouse model of ASD.
Assuntos
Hipocampo/metabolismo , Proteínas de Homeodomínio/metabolismo , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Processamento Alternativo , Animais , Transtorno do Espectro Autista/metabolismo , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Feminino , Hipocampo/patologia , Proteínas de Homeodomínio/genética , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neocórtex/patologia , Proteínas do Tecido Nervoso/genética , Fosforilação , RNA Mensageiro/metabolismo , Receptor trkB/metabolismo , Receptores de LDL/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Long-term potentiation (LTP) is accompanied by increased spine density and dimensions triggered by signaling cascades involving activation of the neurotrophin brain-derived neurotrophic factor (BDNF) and cytoskeleton remodeling. Chemically-induced long-term potentiation (c-LTP) is a widely used cellular model of plasticity, whose effects on spines have been poorly investigated. We induced c-LTP by bath-application of the N-methyl-d-aspartate receptor (NMDAR) coagonist glycine or by the K(+) channel blocker tetraethylammonium (TEA) chloride in cultured hippocampal neurons and compared the changes in dendritic spines induced by the two models of c-LTP and determined if they depend on BDNF/TrkB signaling. We found that both TEA and glycine induced a significant increase in stubby spine density in primary and secondary apical dendrites, whereas a specific increase in mushroom spine density was observed upon TEA application only in primary dendrites. Both TEA and glycine increased BDNF levels and the blockade of tropomyosin-receptor-kinase receptors (TrkRs) by the nonselective tyrosine kinase inhibitor K-252a or the selective allosteric TrkB receptor (TrkBR) inhibitor ANA-12, abolished the c-LTP-induced increase in spine density. Surprisingly, a blockade of TrkBRs did not change basal spontaneous glutamatergic transmission but completely changed the synaptic plasticity induced by c-LTP, provoking a shift from a long-term increase to a long-term depression (LTD) in miniature excitatory postsynaptic current (mEPSC) frequency. In conclusion, these results suggest that BDNF/TrkB signaling is necessary for c-LTP-induced plasticity in hippocampal neurons and its blockade leads to a switch of c-LTP into chemical-LTD (c-LTD).
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Espinhas Dendríticas/fisiologia , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptor trkB/metabolismo , Transdução de Sinais/fisiologia , Animais , Fator Neurotrófico Derivado do Encéfalo/antagonistas & inibidores , Células Cultivadas , Espinhas Dendríticas/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Glicina/farmacologia , Glicinérgicos/farmacologia , Hipocampo/citologia , Potenciação de Longa Duração/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Ratos Wistar , Receptor trkB/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/agonistas , Transdução de Sinais/efeitos dos fármacos , Tetraetilamônio/farmacologiaRESUMO
Alternatively spliced brain-derived neurotrophic factor (BDNF) transcripts are targeted to distinct cellular compartments in neurons but the mechanisms underlying this sorting are unknown. Although only some BDNF isoforms are targeted to dendrites, we have found that the coding region common to all BDNF transcripts contains a constitutively active dendritic targeting signal and that this signal is suppressed in transcripts containing exons 1 or 4, which are restricted to the cell soma and proximal dendrites. This dendritic targeting signal is mediated by translin, an RNA-binding protein implicated in RNA trafficking, and is disrupted by the G196A mutation associated with memory deficits and psychiatric disorders. Molecular modeling and mutational studies indicate that the G196A mutation blocks dendritic targeting of BDNF mRNA by disrupting its interaction with translin. These findings implicate abnormal dendritic trafficking of BDNF mRNA in the pathophysiology of neuropsychiatric disorders linked to the G196A mutation.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Proteínas de Ligação a DNA/metabolismo , Dendritos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Western Blotting , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Compartimento Celular , Linhagem Celular Tumoral , Células Cultivadas , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Hibridização In Situ , Camundongos , Modelos Moleculares , Mutação de Sentido Incorreto , Neurônios/citologia , Neurônios/metabolismo , Ligação Proteica , Interferência de RNA , Transporte de RNA , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Brain-derived neurotrophic factor (BDNF) may exert contrasting effects depending on its different subcellular sites of action (soma, dendrites, axons). These contrasting effects may explain contradictory findings, for example that BDNF may favour or oppose epileptogenesis. We determined the distribution of five BDNF splice variants in the soma and dendrites of rat hippocampal principal neurons, after application of stimuli that prompt BDNF mRNA accumulation in dendrites (epileptogenic seizures). Under basal conditions, no BDNF mRNA splice variant was detectable in dendrites, while specific splice variants were found in dendrites in response to epileptogenic seizures. Three hours after pilocarpine administration, exon VI and exon II splice variants were found in dendrites, while exons I and IV transcripts displayed a strictly somatic localization. Three hours after kainate administration, only exon VI was found in dendrites. These data suggest that the regulated expression of different splice variants may provide a spatial code to ensure the delivery of BDNF to precise destinations in the cell soma or along the dendrites.
Assuntos
Processamento Alternativo/fisiologia , Fator Neurotrófico Derivado do Encéfalo/genética , Hipocampo/citologia , Neurônios/citologia , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Análise de Variância , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dendritos , Epilepsia/induzido quimicamente , Epilepsia/patologia , Éxons/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hibridização In Situ , Ácido Caínico , Masculino , Pilocarpina , Ratos , Ratos Sprague-DawleyRESUMO
We present an analysis of the antitumour effects of a library of ingenol derivatives synthesized in our laboratory and published elsewhere. Fluoro-ingenol (1), ingenol-20-deoxy-20-phtalimido (2), ingenol-3-benzoate-20-deoxy-20-benzamide (3), ingenol-3-benzoate (4), ingenol-3,5-dibenzoate (5), ingenol-3,20-dibenzoate (6), 20-deoxy-20-benylureidoingenol-3-benzoate (7), ingenol-20-deoxy-20-fluoro-3-benzoate (8), ingenol-20-deoxy-20-fluoro-3,5-dibenzoate (9), ingenol-20-phenylcarbamate (10), ingenol-20-benzoate (11), ingenol-3-benzoate-20-phenylcarbamate (12) were tested in vitro on two well characterized breast cancer cell (BCC) lines, namely T47D and MDA-MB-231, as representative of two opposite types of hormone-sensitiveness and differentiation stage. These experiments led us to identify ingenol-20-benzoate (11) as a promising antitumour compound characterized by a relevant inhibition of cell growth and apoptotic cell death involving a p53-mediated pathway.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Diterpenos/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Neoplasias da Mama/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Diterpenos/administração & dosagem , Diterpenos/uso terapêutico , Feminino , Humanos , Proteína Supressora de Tumor p53/efeitos dos fármacosRESUMO
Elevated levels of prostaglandin E2 (PGE2) have been reported in many high metastatic human breast cancers, but no relationship between exogenous PGE2 activity, expression of matrix metalloproteinases (MMPs) and metastasis in human tumor cells has been reported. The poorly invasive human breast cancer cell line MCF-7 was cultured for 24h in the presence of both phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 nM) and PGE2 (1 microM) and the activity of MMP-9, one of the MMPs involved in metastasis, was measured, in growth medium by gelatin substrate zymography. TPA induced a strong production of MMP-9 while exogenous PGE2 had no effect on the basal MMP-9 level, but inhibited the TPA induced enzyme expression and matrigel invasiveness. We showed that MCF-7 cells expressed EP2, EP3 and EP4 receptors for PGE2 and that its action was probably mediated by EP4 receptor and adenylyl cyclase activation while cAMP dependent PKA was not involved in the process of inhibition of MMP-9 production. These findings suggest a possible inhibitory role for exogenous PGE2 in the metastatic process development.
Assuntos
Dinoprostona/farmacologia , Metaloproteinase 9 da Matriz/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colágeno , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Dinoprostona/fisiologia , Combinação de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Indução Enzimática/efeitos dos fármacos , Humanos , Laminina , Inibidores de Metaloproteinases de Matriz , Metástase Neoplásica , Proteoglicanas , Receptores de Prostaglandina/biossíntese , Receptores de Prostaglandina E/agonistas , Receptores de Prostaglandina E/biossíntese , Receptores de Prostaglandina E Subtipo EP4RESUMO
We evaluated whether insulin signaling modulates plasma cell glycoprotein (PC-1) plasma membrane recruitment, posttranslational processing, and gene expression in human cultured cell lines. Insulin induced a fourfold increase (P < 0.01) of membrane PC-1 expression by rapid and sensitive mechanism(s). This effect was reduced (P < 0.05-0.01) by inhibition of phosphatidylinositol 3-kinase (200 nmol/l wortmannin) and S6 kinase (50 nmol/l rapamycin) activities and intracellular trafficking (50 micromol/l monensin) and was not accompanied by PC-1 gene expression changes. Moreover, at Western blot, insulin elicited the appearance, in both plasma membrane and cytosol, of a PC-1-related 146-kDa band (in addition to bands of 163, 117, 106, and 97 kDa observed also in absence of insulin) that was sensitive to endoglycosidase H. Finally, inhibition of PC-1 translocation to plasma membrane, by wortmannin pretreatment, increases insulin-stimulated receptor autophosphorylation. Our data indicate that insulin stimulates PC-1 posttranslational processing and translocation to the plasma membrane, which in turn impairs insulin receptor signaling. Bidirectional cross talk between insulin and PC-1, therefore, takes place, which may be part of the hormone self-desensitization mechanism.
Assuntos
Insulina/fisiologia , Diester Fosfórico Hidrolases/metabolismo , Processamento de Proteína Pós-Traducional , Pirofosfatases/metabolismo , Androstadienos/farmacologia , Transporte Biológico/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Citosol/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Insulina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/efeitos dos fármacos , Diester Fosfórico Hidrolases/genética , Fosforilação/efeitos dos fármacos , Pirofosfatases/química , Pirofosfatases/efeitos dos fármacos , Pirofosfatases/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , WortmaninaRESUMO
AIMS/HYPOTHESIS: Serum anti-CD38 autoantibodies (aAbs) have been reported in 17 to 19% of patients with long-standing Type I (insulin-dependent) diabetes mellitus and Type II (non-insulin-dependent) diabetes mellitus. Whether these aAbs are also found in new-onset Type I diabetes and in Latent Autoimmune Diabetes in Adults (LADA) is not known, as is their relationship with conventional islet aAbs. METHODS: These issues were addressed by studying new-onset Type I and LADA diabetic cohorts with a recently developed anti-CD38 enzymatic immuno-assay. RESULTS: Anti-CD38 aAb prevalence among new-onset Type I patients (3.8%) was lower than previously found in long-standing Type I diabetes (11.7%, as defined with the 97.5 percentile cutoff; p=0.01), suggesting a late appearance of these aAbs. Among LADA patients, 14.9% were anti-CD38(+). Anti-CD38 were only associated with anti-GAD aAbs in new-onset Type I diabetes. Although the CD38 target molecule was expressed in human pancreatic islets, anti-CD38 aAbs did not contribute to the islet cell antibody (ICA) immunofluorescence reactivity. All the positive sera analysed for Ca(2+) release were found to mobilise it. In agreement with these agonistic features, anti-CD38(+) new-onset Type I patients showed higher fasting C-peptide values as compared to negative counterparts; the association was stronger when the analysis was limited to the agonistic anti-CD38(+) sera. A similar trend was found among LADA patients. CONCLUSION/INTERPRETATION: Anti-CD38 aAbs are distinct markers of islet autoimmunity which are more prevalent in long-standing disease, as opposed to the other known islet aAbs. Their in vitro agonistic properties could be operating in vivo as well, as they identify sub-groups of patients with higher residual beta-cell function.
Assuntos
ADP-Ribosil Ciclase/imunologia , Antígenos CD/imunologia , Autoanticorpos/análise , Diabetes Mellitus Tipo 1/imunologia , ADP-Ribosil Ciclase 1 , Adolescente , Idoso , Peptídeo C/sangue , Cálcio/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , Estudos de Coortes , Diabetes Mellitus Tipo 1/metabolismo , Feminino , Glutamato Descarboxilase/imunologia , Humanos , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-IdadeRESUMO
CD38 is involved in transmembrane signaling in many cell types; anti-CD38 autoantibodies have been described in diabetic patients. We tested whether human anti-CD38 antibodies possess signaling properties by measuring their ability to raise intracellular calcium ([Ca2+]i) using the fluo-3-acetoxymethyl ester method in a human-derived T-cell line (Jurkat T-cells, expressing high levels of surface CD38) and in dispersed human islet cells from normal donors. In Jurkat T-cells, 11 of 19 anti-CD38-positive sera raised [Ca2+]i (by > or =20% of baseline), whereas no [Ca2+]i-mobilizing activity was found in 27 anti-CD38-negative sera (chi2 = 20.5, P < 0.0001). In dispersed human islet cells, 5 of 11 anti-CD38-positive sera (and none of three anti-CD38-negative sera) raised [Ca2+]i significantly. When preincubated with Staphylococcus aureus protein A to remove IgG, anti-CD38-positive sera showed a 70 +/- 5% reduction in [Ca2+]i-mobilizing activity. Preincubation with CD38-transfected NIH-3T3 fibroblasts, but not with mock-transfected NIH-3T3 cells, abolished [Ca2+]i mobilization. In blocking experiments, preincubation with nonagonistic anti-CD38 monoclonal antibodies also prevented [Ca2+]i mobilization. In cultured human islets, anti-CD38-positive sera exhibiting [Ca2+]i-mobilizing activity in Jurkat T-cells (n = 6) significantly stimulated insulin release at 3.3 mmol/l glucose (median [interquartile range] 738 microU/ml [234], P = 0.0001 vs. 320 [52] microU/ml of control), whereas 6 anti-CD38-positive sera without [Ca2+]i-mobilizing activity and 10 anti-CD38-negative did not. In further incubations, the five anti-CD38-positive sera displaying [Ca2+]i-mobilizing activity in dispersed islet cells significantly stimulated insulin release at both 3.3 mmol/l glucose (2.2 +/- 0.3% of insulin islet content, P < 0.002 vs. 1.2 +/- 0.1% of control) and 16.7 mmol/l glucose (3.7 +/- 0.3 vs. 2.3 +/- 0.3%, P < 0.002). We conclude that human anti-CD38 autoantibodies with agonistic properties on the CD38 effector system occur in nature; in human islets, their [Ca2+]i-mobilizing activity is coupled with the ability to stimulate insulin release.
Assuntos
Antígenos CD/imunologia , Antígenos de Diferenciação/imunologia , Autoanticorpos/farmacologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/fisiologia , NAD+ Nucleosidase/imunologia , Células 3T3 , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Animais , Anticorpos Monoclonais/farmacologia , Autoanticorpos/sangue , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Enterotoxinas/farmacologia , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Células Jurkat , Glicoproteínas de Membrana , Camundongos , Proteínas Recombinantes/imunologia , Superantígenos/farmacologia , Linfócitos T/imunologia , TransfecçãoRESUMO
Insulin secretion is one of the functions mediated by CD38, a nonlineage pleiotropic cell surface receptor. The molecule is the target of an autoimmune response, because serum autoantibodies (aAbs) to CD38 have been detected in diabetic patients. In the healthy Caucasian population, the CD38 gene is bi-allelic (86% CD38*B and 14% CD38*A), whereas an Arg140Trp mutation has been identified in Japanese diabetic patients. We investigated the relationship between CD38 and diabetes in Caucasian patients by characterizing anti-CD38 aAbs in terms of prevalence and function (agonistic/nonagonistic activity) and by exploring the potential influence of the CD38 genetic background. A novel enzymatic immunoassay, using recombinant soluble CD38 as the target antigen, was developed for the analysis of anti-CD38 aAb titers. Sera from 19.15% of type 1 and 16.67% of type 2 diabetic patients were positive. The majority of anti-CD38 aAbs (57.14%) displayed agonistic properties, i.e., they demonstrated the capability to trigger Ca2+ release in lymphocytic cell lines. In agreement with these functional features, the presence of anti-CD38 aAbs in type 2 diabetic patients was associated with significantly higher levels of fasting plasma C-peptide and insulin, as compared with anti-CD38-counterparts. No diabetic subject carrying the Arg140Trp mutation and no preferential association between diabetes or aAb status and the CD38*A allele was found in the study population. These results show the significance of anti-CD38 aAbs as a new diagnostic marker of beta-cell autoimmunity in diabetes. Moreover, the prevalent agonistic activity of these aAbs suggests that they could mediate relevant effects on target cells by means of Ca2+ mobilization.
Assuntos
Antígenos CD , Antígenos de Diferenciação/imunologia , Autoanticorpos/análise , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/imunologia , NAD+ Nucleosidase/imunologia , População Branca/genética , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Idoso , Antígenos de Diferenciação/genética , Autoanticorpos/fisiologia , Cálcio/metabolismo , Diabetes Mellitus Tipo 1/fisiopatologia , Diabetes Mellitus Tipo 2/fisiopatologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Glicoproteínas de Membrana , Pessoa de Meia-Idade , NAD+ Nucleosidase/genética , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Human CD38 is a signal transduction molecule, and, concurrently, an ectoenzyme catalyzing the synthesis and degradation of cyclic ADP-ribose (cADPR), a potent Ca2+ mobilizer. One facet of CD38 that has not yet been addressed is its role in NK cells. To this end, the events triggered by CD38 ligation with agonistic mAb were analyzed on freshly purified human NK cells. Ligation was followed by (i) a significant rise in the intracellular level of Ca2+, (ii) increased expression of HLA class II and CD25, and (iii) tyrosine phosphorylation of discrete cytoplasmic substrates. The phosphorylation cascade involved CD3-zeta and FcepsilonRIgamma chains, zeta-associated protein (ZAP)-70 and the proto-oncogene product c-Cbl. NK effector functions were then analyzed: CD38 signaling was able (iv) to induce release of IFN-gamma and, more prominently, of granulocyte macrophage colony stimulating factor, as assessed by measuring both mRNA and protein products; and, lastly, (v) to induce cytolytic effector functions on target cells after IL-2 activation, as shown both by cytotoxicity assays and ultrastructural changes. The tyrosine-phosphorylated substrates and all the effects mediated by CD38 were similar to those observed following triggering via CD16 (FcgammaRIIIA); moreover, Ca2+ mobilization via CD38 no longer operated in NK-derived cell lines lacking CD16. These results suggest that the activation signals transduced by CD38 in NK cells elicit relevant cellular events. The effects are similar to those elicited via CD16 and possibly rely on common signaling pathways.
Assuntos
Antígenos CD , Antígenos de Diferenciação/imunologia , Células Matadoras Naturais/imunologia , Ativação Linfocitária , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Complexo CD3/análise , Cálcio/metabolismo , Citotoxicidade Imunológica , Fator Estimulador de Colônias de Granulócitos e Macrófagos/análise , Antígenos de Histocompatibilidade Classe II/análise , Humanos , Interferon gama/análise , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Glicoproteínas de Membrana , NAD+ Nucleosidase/imunologia , Fosforilação , Proteínas Tirosina Quinases/análise , Proteínas Tirosina Quinases/metabolismo , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/análise , RNA Mensageiro/análise , Receptores de IgE/análise , Receptores de IgG/imunologia , Receptores de Interleucina-2/análise , Transdução de Sinais , Tirosina/metabolismo , Proteína-Tirosina Quinase ZAP-70RESUMO
CD38, a nonlineage-restricted surface glycoprotein, is an ecto-enzyme (ADP ribosyl cyclase/cADPR hydrolase/EC 3.2.2.6) that regulates cytoplasmic Ca2+ and cell-cell interactions. The molecule also delivers trans-membrane signals, despite a structural ineptitude to the scope. To reconcile these issues in a unitarian model, we compared the effects of CD38 signaling in circulating and residential T lymphocytes, the latter represented by those colonizing the intestinal lamina propria. Results are as follows: 1) LP T cells express an enzymatically active form of CD38, characterized by a modified ratio between cyclase and hydrolase functions; 2) LP T cells do not mobilize Ca2+ upon CD38 ligation, as seen in PB T cells (this condition is due to a lack in activation of PLC- g, constantly observed in PB T lymphocytes); 3) The early steps of CD38 signaling involve activation of lck, syk, and LAT; 4) Late events include synthesis and release of IL-2, IL-4, IL-5, IL-10, IFN-g and GM-CSF; 5) The uniqueness of the CD38 pathway in LP T cells is not caused by impaired interactions with the CD31 ligand. The differences observed concern the signaling machinery that CD38 exploits for its own use and not the interplay with its ligand.
Assuntos
Antígenos CD , Antígenos de Diferenciação/metabolismo , Cálcio/metabolismo , Mucosa/metabolismo , NAD+ Nucleosidase/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Subpopulações de Linfócitos T/metabolismo , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Anticorpos Monoclonais/metabolismo , Antígenos de Diferenciação/genética , Western Blotting , Separação Celular , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Isoenzimas/metabolismo , Células Jurkat , Glicoproteínas de Membrana , Modelos Biológicos , Mucosa/citologia , Complexos Multienzimáticos , NAD+ Nucleosidase/genética , Fosfolipase C gama , Fosforilação , Testes de Precipitina , Fosfolipases Tipo C/metabolismoRESUMO
Exposure of RPMI 8226, Karpas 707 and U266 human myeloma-like lines to low doses of As(2)O(3) was followed by a marked increase in lymphokine activated killers (LAK)-mediated killing and up- modulation of CD38 and CD54, two molecules involved in cell-cell interactions. Moreover, simultaneous exposure of effectors and targets to As(2)O(3) yielded the most effective condition for lysis. The expression of CD31 (CD38 ligand) and CD11a (CD54 ligand) was also up-regulated by LAK, suggesting that increased adhesion was responsible for the improved killing. Similar results were obtained using freshly isolated myeloma cells. These findings indicate that As(2)O(3) may be useful to boost the immune system against myelomas.
Assuntos
Antígenos CD , Arsenicais/imunologia , Células Matadoras Ativadas por Linfocina/efeitos dos fármacos , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/patologia , Óxidos/imunologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos de Diferenciação/efeitos dos fármacos , Antígenos de Diferenciação/metabolismo , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Trióxido de Arsênio , Arsenicais/uso terapêutico , Citotoxicidade Imunológica , Relação Dose-Resposta a Droga , Inibidores do Crescimento/imunologia , Inibidores do Crescimento/uso terapêutico , Humanos , Imunofenotipagem , Molécula 1 de Adesão Intercelular/efeitos dos fármacos , Molécula 1 de Adesão Intercelular/metabolismo , Células Matadoras Ativadas por Linfocina/imunologia , Células Matadoras Ativadas por Linfocina/metabolismo , Glicoproteínas de Membrana , Mieloma Múltiplo/metabolismo , NAD+ Nucleosidase/efeitos dos fármacos , NAD+ Nucleosidase/metabolismo , Óxidos/uso terapêutico , Células Tumorais CultivadasRESUMO
During the last three decades, research focused on cancer treatment has led to the development of many cytotoxic agents. Despite the fact that these efforts have significantly improved the prognosis of certain malignancies such as some lymphomas, leukemias and testicular carcinomas, other tumors such as ovarian, lung and metastatic breast cancer are still associated with a poor prognosis. An innovative approach has recently emerged, thanks to a better understanding of tumor cell biology and many efforts are aimed at finding compounds capable of restoring a more differentiated phenotype to tumor cells, thereby reducing the tumor's aggressiveness and ultimately reverting it to its normal counterpart [1, 2]. Retinoids are the prototype of this new therapeutical approach called "differentiation therapy".
Assuntos
Anticarcinógenos/uso terapêutico , Neoplasias da Mama/prevenção & controle , Retinoides/uso terapêutico , Animais , Feminino , Humanos , RatosRESUMO
For many years, we and others have reported the efficacy of cervical cerclage in the prevention of miscarriage in patients with uterine malformations. In this paper the experience of 275 cases collected between 1978 and 1998 is reported. Our data indicate that cervical cerclage is effective in preventing miscarriages, prevalently in those pregnancies bearing uterine malformations with simultaneous cervical incompetence.
Assuntos
Aborto Espontâneo/prevenção & controle , Colo do Útero/cirurgia , Procedimentos Cirúrgicos Obstétricos , Incompetência do Colo do Útero/cirurgia , Útero/anormalidades , Adulto , Feminino , Humanos , Procedimentos Cirúrgicos Obstétricos/métodos , Gravidez , Resultado do TratamentoRESUMO
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that stimulates the proliferation and differentiation of bone marrow progenitors. Moreover, the presence and activity of GM-CSF and its receptor (GM-CSF-r) has been documented on tissues and cell lines of a non-hemopoietic origin. In this paper we studied the expression and putative role of GM-CSF and GM-CSF-r in endometrial cancer. The modulation of GM-CSF-r alpha-chain upon progesterone treatment suggests a role for GM-CSF and its receptor in the pathogenesis and development of endometrial cancer.
Assuntos
Neoplasias do Endométrio/química , Progesterona/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação para Baixo , Neoplasias do Endométrio/etiologia , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/fisiologia , Humanos , Mifepristona/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/análise , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/fisiologia , Células Tumorais CultivadasAssuntos
Antígenos CD , Antígenos de Diferenciação/fisiologia , Leucócitos/fisiologia , Complexos Multienzimáticos/fisiologia , NAD+ Nucleosidase/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/fisiologia , Transdução de Sinais , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Adesão Celular , Humanos , Glicoproteínas de MembranaRESUMO
In addition to its catalytic activities as ecto-NAD+ glycohydrolase (NADase), CD38 displays the ability to transduce signals of biological relevance. Indeed, ligation of CD38 on peripheral blood mononuclear cells (PBMC) by agonistic monoclonal antibodies (mAbs) is followed by the transcription and secretion of a vast array of regulatory cytokines. The present work addresses the issue of whether the signals leading to calcium (Ca2+) mobilization, lymphocyte proliferation and release of cytokines is dependent on the epitopes recognized by the individual mAbs. Competition binding analysis identifies two families of mAbs, namely IB4, IB6 and AT2 on one side and OKT10, SUN-4B7 and AT1 on the other. Each mAb family binds epitopes that are completely or partially common. However, the functional activities of the CD38 molecule can not be simply attributed to the epitopes engaged: for instance, IB4 and OKT10 mAbs, which bind different epitopes, perform as agonistic mAbs in inducing PBMC proliferation and interferon (IFN)-gamma secretion. SUN-4B7 yields intermediate effects, whereas IB6, AT1 and AT2 mAbs are totally ineffective. The effects mediated by IB4 and OKT10 mAbs are apparent in 80% of the healthy individuals studied, whereas the effects of SUN-4B7 mAb operate only in 25% of the donors. Interleukin (IL)-6 secretion was observed in all individuals analyzed, irrespective of the epitopes triggered and of mAbs used to ligate the CD38 molecule. In addition, IB4 is the only mAb able to induce significant intracellular Ca2+ fluxes.
Assuntos
Antígenos CD , Antígenos de Diferenciação/química , Antígenos de Diferenciação/imunologia , Leucócitos Mononucleares/imunologia , NAD+ Nucleosidase/química , NAD+ Nucleosidase/imunologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antibacterianos/farmacologia , Anticorpos Monoclonais , Antígenos de Diferenciação/metabolismo , Ligação Competitiva/imunologia , Cálcio/metabolismo , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Mapeamento de Epitopos , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Ligantes , Lipopolissacarídeos/antagonistas & inibidores , Glicoproteínas de Membrana , NAD+ Nucleosidase/metabolismo , Polimixina B/farmacologia , Estrutura Terciária de Proteína , Transdução de Sinais/imunologiaRESUMO
All-trans retinoic acid (ATRA) is currently used in clinical trials for breast cancer, in virtue of its ability to inhibit cell growth and to promote cell differentiation. Elucidation of the molecular mechanism(s) underlying the pleiotropic pharmacological activity of ATRA is of fundamental relevance for an effective use of the compound in clinics. This paper reports on the effects of ATRA treatment on the cell surface expression of a panel of adhesion molecules known to regulate the interactions between the effectors of the immune system and tumor targets. Results indicate that breast cancer (BC) cell lines exposed to ATRA selectively up-modulate the surface expression of ICAM-1/CD54, a molecule regulating cell/cell contacts. Such effect could be reproduced in all the BC cell lines analyzed, independently of their hormone receptor status, indicating that estrogens and progesterone are irrelevant in this process. The regulatory effects on ICAM-1 expression are time- and dose-dependent and reversible. Moreover, other differentiating and proliferating agents comparatively tested, e.g. dimethyl sulfoxide, estradiol or dexamethas one, are ineffective, indicating that ICAM-1 up-modulation is uniquely featured by ATRA. A second observation is that ATRA treated cells are, only apparently, less sensitive to lysis by lymphocytes activated by IL-2, as determined by means of a standard 51Cr release assay. In fact, notwithstanding this effect, a marked reduction in the ability to form colonies was highlighted in ATRA treated versus control lines after incubation with LAK. Finally, the clonogenic killing effect could be reversed using anti-CD54 mAbs as blocking tools, indicating that ICAM-1 plays a key role in the phenomena.