RESUMO
BACKGROUND: Due to the close proximity of the prostate and rectum, rectal toxicity remains a major problem in patient treated by radiotherapy for prostate adenocarcinoma. One method of increasing the distance between the prostate and the rectum is to use a spacer implanted into the rectoprostatic space. This report describes the long-term outcomes obtained with a new ballon spacer. METHODS: Patients treated with curative radiotherapy for low- or intermediate-risk prostate adenocarcinoma, who underwent insertion of the ProSpace® (BioProtect Ltd, Tzur Yigal, Israel) rectal-prostate balloon spacer, were included. The main objective was to evaluate the dosimetric benefit of the spacer for OARs. The secondary objectives were to evaluate the feasibility and tolerability of ProSpace® balloon placement and to evaluate its long-term therapeutic efficacy and tolerance. RESULTS: Between October 2013 and March 2015, 16 patients were enrolled in the Pasteur Clinic, Toulouse, France. The median follow-up was 85.5 months. From top to bottom, the space created was a mean of 16.3 mm (range: 11-20.5 mm) at the base of the prostate, 12.1 mm (range: 4-16 mm) at the middle and 8.9 mm at the apex (range: 5-15 mm). On average, rectal volumes receiving a dose of 70 Gy, 60 Gy and 50 Gy were significantly lower after balloon implantation: -4.81 cc (1.5 vs. 6.3; p < 0.0005), -8.08 cc (6.4 vs. 14.5; p = 0.002) and -9.06 cc (16.7 vs. 25.7; p = 0.003), respectively. There were significant differences in coverage after balloon implantation: Median V95% (p < 0.0005), median Dmin (p = 0.01) and median V98% (p < 0.001) were higher after balloon implantation. At 5 years, cumulative gastrointestinal toxicity was grade 1 in 6% (1/16 patients). No toxicity of grade 2 or higher was found. At 5 years, no urinary toxicity grade 3 or 4 toxicity was found. The QoL was not deteriorated. CONCLUSIONS: The use of the ProSpace® balloon seems to be well accepted by patients, allowing a double dosimetric gain: a decrease in doses received by the rectum and an improvement in the coverage of the high-risk PTV. The long-term gastrointestinal toxicity remains low and QoL is preserved in all treated patients.
Assuntos
Neoplasias da Próstata , Reto , Humanos , Masculino , Neoplasias da Próstata/radioterapia , Idoso , Reto/efeitos da radiação , Pessoa de Meia-Idade , Dosagem Radioterapêutica , Adenocarcinoma/radioterapia , Próstata/efeitos da radiação , Próstata/patologia , Idoso de 80 Anos ou mais , Resultado do Tratamento , Lesões por Radiação/etiologia , Lesões por Radiação/prevenção & controle , SeguimentosRESUMO
Deep-learning (DL)-based auto-contouring solutions have recently been proposed as a convincing alternative to decrease workload of target volumes and organs-at-risk (OAR) delineation in radiotherapy planning and improve inter-observer consistency. However, there is minimal literature of clinical implementations of such algorithms in a clinical routine. In this paper we first present an update of the state-of-the-art of DL-based solutions. We then summarize recent recommendations proposed by the European society for radiotherapy and oncology (ESTRO) to be followed before any clinical implementation of artificial intelligence-based solutions in clinic. The last section describes the methodology carried out by three French radiation oncology departments to deploy CE-marked commercial solutions. Based on the information collected, a majority of OAR are retained by the centers among those proposed by the manufacturers, validating the usefulness of DL-based models to decrease clinicians' workload. Target volumes, with the exception of lymph node areas in breast, head and neck and pelvic regions, whole breast, breast wall, prostate and seminal vesicles, are not available in the three commercial solutions at this time. No implemented workflows are currently available to continuously improve the models, but these can be adapted/retrained in some solutions during the commissioning phase to best fit local practices. In reported experiences, automatic workflows were implemented to limit human interactions and make the workflow more fluid. Recommendations published by the ESTRO group will be of importance for guiding physicists in the clinical implementation of patient specific and regular quality assurances.
Assuntos
Aprendizado Profundo , Neoplasias/diagnóstico por imagem , Órgãos em Risco/diagnóstico por imagem , Radioterapia (Especialidade)/métodos , Planejamento da Radioterapia Assistida por Computador/métodos , Europa (Continente) , Humanos , Neoplasias/radioterapia , Guias de Prática Clínica como Assunto , Radioterapia Guiada por Imagem/métodos , Sociedades Médicas , Carga de TrabalhoRESUMO
Modern radiotherapy treatment planning is a complex and time-consuming process that requires the skills of experienced users to obtain quality plans. Since the early 2000s, the automation of this planning process has become an important research topic in radiotherapy. Today, the first commercial automated treatment planning solutions are available and implemented in a growing number of clinical radiotherapy departments. It should be noted that these various commercial solutions are based on very different methods, implying a daily practice that varies from one center to another. It is likely that this change in planning practices is still in its infancy. Indeed, the rise of artificial intelligence methods, based in particular on deep learning, has recently revived research interest in this subject. The numerous articles currently being published announce a lasting and profound transformation of radiotherapy planning practices in the years to come. From this perspective, an evolution of initial training for clinical teams and the drafting of new quality assurance recommendations is desirable.
Assuntos
Aprendizado Profundo , Planejamento da Radioterapia Assistida por Computador/métodos , Fluxo de Trabalho , Automação , Retroalimentação , Previsões , Humanos , Órgãos em Risco , Editoração/estatística & dados numéricos , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador/tendências , SoftwareRESUMO
BACKGROUND: Lectins mediate cell-cell interactions by specifically recognizing oligosaccharide chains. Legume lectins serve as mediators for the symbiotic interactions between plants and nitrogen-fixing microorganisms, an important process in the nitrogen cycle. Lectins from the Viciae tribe have a high affinity for the fucosylated biantennary N-acetyllactosamine-type glycans which are to be found in the majority of N-glycosylproteins. While the structures of several lectins complexed with incomplete oligosaccharides have been solved, no previous structure has included the complete glycoprotein. RESULTS: We have determined the crystal structures of Lathyrus ochrus isolectin II complexed with the N2 monoglycosylated fragment of human lactotransferrin (18 kDa) and an isolated glycopeptide (2.1 kDa) fragment of human lactotransferrin (at 3.3 A and 2.8 A resolution, respectively). Comparison between the two structures showed that the protein part of the glycoprotein has little influence on either the stabilization of the complex or the sugar conformation. In both cases the oligosaccharide adopts the same extended conformation. Besides the essential mannose moiety of the monosaccharide-binding site, the fucose-1' of the core has a large surface of interaction with the lectin. This oligosaccharide conformation differs substantially from that seen in the previously determined isolectin I-octasaccharide complex. Comparison of our structure with that of concanavalin A (ConA) suggests that the ConA binding site cannot accommodate this fucose. CONCLUSIONS: Our results explain the observation that Viciae lectins have a higher affinity for fucosylated oligosaccharides than for unfucosylated ones, whereas the affinity of ConA for these types of oligosaccharides is similar. This explanation is testable by mutagenesis experiments. Our structure shows a large complementary surface area between the oligosaccharide and the lectin, in contrast with the recently determined structure of a complex between the carbohydrate recognition domain of a C-type mammalian lectin and an oligomannoside, where only the non-reducing terminal mannose residue interacts with the lectin.
Assuntos
Fucose , Glicopeptídeos/química , Lactoferrina/química , Lactoferrina/metabolismo , Lectinas/química , Lectinas/metabolismo , Oligossacarídeos/química , Fragmentos de Peptídeos/química , Lectinas de Plantas , Estrutura Secundária de Proteína , Sítios de Ligação , Configuração de Carboidratos , Sequência de Carboidratos , Glicopeptídeos/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismoRESUMO
The uptake of iron (III) mediated by lactotransferrin to human biopsies from upper intestine has suggested the presence of specific receptors for human lactotransferrin at the brush border (Cox, T., Mazurier, J., Spik, G., Montreuil, J. and Peters, T.J. (1979) Biochim. Biophys. Acta 588, 120-128). In the present data, using 125I-radiolabeled transferrins, we have demonstrated that a preparation of microvillous membrane vesicles, from rabbit jejunal brush-border specifically binds human lactotransferrin. This binding is specific, saturable and calcium dependent. Scatchard plots analysis of lactotransferrin binding indicates 1.5 X 10(13) sites per mg of membrane proteins with an equilibrium constant of 1.2 X 10(6) M-1. Sodium dodecyl sulfate solubilization of the brush-border proteins allows the lactotransferrin receptor to retain its binding activity. Moreover, the ligand blotting of the detergent solubilized membrane proteins on nitrocellulose sheet and after incubation with 125I-labeled lactotransferrin, has shown that the receptor is a protein of about 100 kDa. In the same experimental conditions, the rabbit microvillous membrane vesicles do not specifically bind rabbit serotransferrin indicating the absence of serotransferrin receptors at the brush border.
Assuntos
Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Lactoferrina/metabolismo , Lactoglobulinas/metabolismo , Microvilosidades/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Fracionamento Celular , Humanos , Cinética , Microscopia Eletrônica , Microvilosidades/ultraestrutura , Peso Molecular , Plásticos , Coelhos , Soroalbumina Bovina/metabolismoRESUMO
In order to determine the conformational relationship of iron binding of human serotransferrin and lactotransferrin, ultraviolet difference spectral studies were performed in the presence of guanidine chloride and perturbants as deuterium oxide, ethylene glycol, glycerol and polyethylene glycol. In the presence of guanidine chloride solution the molar absorption differences at 292 nm of iron-saturated forms versus iron-free forms of human serotransferrin and lactotransferrin are respectively -16000 +/- 1000 and -14000 +/- 775. These modifications may be attributed to the involvement of tryptophan residues in the iron-binding sites of the two proteins. However, the results do not demonstrate that these tryptophan residues are bound directly to iron. Difference spectral studies in the presence of perturbants show that the apparent exposed tryptophan and tyrosine residues are higher with shorter range perturbants in iron-free forms of both transferrin molecules. The most important modification of exposed tyrosine residues has been noticed upon removing iron from human lactotransferrin than from serotransferrin.
Assuntos
Lactoferrina , Lactoglobulinas , Transferrina , Apoproteínas , Humanos , Peso Molecular , Conformação Proteica , Solventes , Espectrofotometria Ultravioleta , Triptofano/análise , Tirosina/análiseRESUMO
Human lactotransferrin is able to bind two vanadyl(IV) ions in specific metal-binding sites. The EPR signals of the two vanadyl bound ions, however, appear as one. This result suggests that the environments of the binding sites of human lactotransferrin are similar. The binding activity is promoted to pH 4 using carbonate or bicarbonate as synergistic anion. This unusual stability of the anion-binding site, which is destroyed below pH 6 for other transferrins, can explain in part the great stability of the metallic complexes of human lactotransferrin. However, the different sensitivities of the two metal-binding sites towards protonation permit the preparation of mixed vanadyl(IV), iron(III) complexes with VO2+ bound either on the N-terminal (acid-labile or B site) or on the C-terminal (acid-stable or A site) site. Analysis of the spectra of such mixed complexes shows the presence of a third nonspecific VO2+-binding site termed A'. The nonspecific A' site seems to be located on the outer surface of the protein close to the C-terminal site.
Assuntos
Lactoferrina/análise , Lactoglobulinas/análise , Metais/análise , Proteínas de Transporte/análise , Fenômenos Químicos , Química , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Concentração de Íons de Hidrogênio , Proteínas de Ligação ao Ferro , Proteínas de Ligação a Transferrina , Vanádio/análiseRESUMO
The ability of a range of homologous transferrin-like proteins to donate iron to pieces of human duodenal mucosa, was examined with an in vitro incubation technique. In contrast to serum transferrin and ovotransferrin, only lactotransferrin was able to yield its iron to intestinal tissue, but in an autologous system this protein was unable to donate iron to human reticulocyte preparations. Studies with 125I-labelled lactotransferrin and lactotransferrin dual-labelled with 59Fe and 125I, indicated that the intact protein is excluded from entry into the enterocytes. The experiments suggest that iron may be transported across the brush border after delivery to specific protein binding sites at the cell surface.
Assuntos
Membrana Celular/metabolismo , Duodeno/metabolismo , Lactoferrina/metabolismo , Lactoglobulinas/metabolismo , Microvilosidades/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sítios de Ligação , Bovinos , Galinhas , Conalbumina/metabolismo , Citocalasinas/farmacologia , Duodeno/ultraestrutura , Feminino , Humanos , Ferro/metabolismo , Reticulócitos/metabolismo , Fatores de TempoRESUMO
Human lactotransferrin contains six residues of methionine per mol. Seven different fragments were characterized after treatment with cyanogen bromide (CNBr) and large parts of their sequences were determined. The alignment of the CNBr fragments was established by the determination of N- and C-terminal sequences, by the study of the C-terminal domain obtained by peptic digestion of the protein and by taking into account the internal homology as well as homology with human serum transferrin. The two glycopeptides were situated in the N- and C-terminal parts of the protein, respectively, a situation quite different from that encountered in serum transferrin. The sequence studies allowed us to suggest a 4- and perhaps 6-fold internal homology.
Assuntos
Lactoferrina/análise , Lactoglobulinas/análise , Fragmentos de Peptídeos/análise , Sequência de Aminoácidos , Aminoácidos/análise , Cromatografia em Gel , Brometo de Cianogênio , Humanos , Pepsina ARESUMO
Human lactotransferrin contains two prosthetic sugar groups situated in two different cyanogen bromide fragments: the amino acid sequences around the two polysaccharide attachment sites were established. The location of the prosthetic groups was quite different in serum and lactotransferrins and no sequence homology could so far be characterized between the glycopeptides of these two transferrins, whereas a close relationship between a glycopeptide of human lactotransferrin and a glycopeptide of hen ovotransferrin was noted.
Assuntos
Lactoferrina , Lactoglobulinas , Filogenia , Sequência de Aminoácidos , Animais , Galinhas , Conalbumina , Brometo de Cianogênio , Feminino , Glicopeptídeos , Humanos , Especificidade da EspécieRESUMO
Mild treatment of iron-saturated human lactotransferrin by trypsin at pH 8.2 cleaves the molecule into a N-tryptic (Mr approximately equal to 30000) and a C-tryptic (Mr approximately equal to 50000) fragment, which have been isolated. Each of them carries a glycan moiety and keeps the property to bind reversibly one Fe3+. The N-tryptic fragment has been submitted to a second tryptic digestion which led to an iron-binding glycopeptide fragment with a molecular weight of about 18500. This fragment, the smallest iron-binding peptide isolated up to now from a transferrin, includes the ND2 domain of human lactotransferrin.
Assuntos
Glicopeptídeos/isolamento & purificação , Ferro/metabolismo , Lactoferrina/metabolismo , Lactoglobulinas/metabolismo , Carboidratos/análise , Humanos , Ferro/análise , Cinética , Peso Molecular , Fragmentos de Peptídeos/isolamento & purificação , Espectrofotometria , TripsinaRESUMO
Lactoferrin is an iron-binding glycoprotein implicated in particular in the control of immune functions and cell proliferation. We have investigated its involvement, at inflammatory concentrations, in cancer progression. We report that lactoferrin has a significant effect on natural killer (NK) cell cytotoxicity against haematopoietic and breast epithelial cell lines. Lactoferrin increases cytolysis at a low concentration (10 micrograms/ml) while at a high concentration (100 micrograms/ml) it modulates cytolysis depending on the target cell phenotype. By pre-treatment of either NK cells or target cells with lactoferrin, we have demonstrated that the lactoferrin effect is due both to a modulation of NK cell cytotoxicity and the target cell sensitivity to lysis. Lactoferrin binds to 91% of the naturally heterogeneous CD56dim/bright NK cell population and increases the NK cell cytotoxic activity at low concentrations. High concentrations of lactoferrin seem to be toxic for the CD56bright NK cells and decrease NK cell cytotoxicity. Lactoferrin also exerts an effect on target cells depending on the cell phenotype. It does not modify the susceptibility to lysis of haematopoietic cells such as Jurkat and K-562 cells, but does significantly increase that of the breast and colon epithelial cells. We have also demonstrated that lactoferrin inhibits epithelial cell proliferation by blocking the cell cycle progression.
Assuntos
Citotoxicidade Imunológica/efeitos dos fármacos , Neoplasias Hematológicas/imunologia , Células Matadoras Naturais/imunologia , Lactoferrina/farmacologia , Neoplasias Epiteliais e Glandulares/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/imunologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/imunologia , Humanos , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/metabolismo , Lactoferrina/metabolismo , Ligação Proteica/imunologia , Células Tumorais CultivadasRESUMO
Isolectin II (LOL II) isolated from the seeds of Lathyrus ochrus has been crystallized in the presence of the N2 fragment (18,500 Da) isolated from human lactotransferrin, which contains an N-acetyllactosamine type biantennary glycan linked to Asn137. This is the first example of a legume lectin crystallized with an N-glycosylprotein. Crystals of the LOL II-N2 complex belong to the tetragonal space group (P4(1)2(1)2 or the enantiomorph) with cell dimensions: a = b = 63.5 A, c = 251.9 A. They diffract well up to at least 3.5 A resolution and more weakly up to 2.8 A resolution. Assuming one functional half-entity in the asymmetric unit, an alpha, beta monomer complexed to one N2 fragment (24,500 Da + 18,500 Da) would give a Vm of 2.95 A3/Da and a solvent content of approximately 58%. SDS/polyacrylamide gels of the dissolved crystals show the presence of both the LOL II and N2 fragment.
Assuntos
Fabaceae/química , Lactoferrina/química , Lectinas/química , Plantas Medicinais , Cristalização , Humanos , Substâncias Macromoleculares , Lectinas de Plantas , Conformação Proteica , Estrutura Terciária de Proteína , Difração de Raios XRESUMO
In August 2013, the French nuclear safety agency (ASN) requested the permanent group of experts in radiation protection in medicine (GPMED) to propose recommendations on the implementation of new technology and techniques in radiation oncology. These recommendations were finalized in February 2015 by the GPMED. In April 2015, the ASN sent a letter to the French ministry of health (DGS/DGOS), and its national health agencies (ANSM, INCa, HAS). In these letters, ASN proposed that, from the 12 recommendations made by the GPMED, an action plan should be established, whose control could be assigned to the French national cancer institute (INCa), as a pilot of the national committee for radiotherapy and that this proposal has to be considered at the next meeting of the national committee of radiotherapy.
Assuntos
Neoplasias/radioterapia , Segurança do Paciente/normas , Radioterapia (Especialidade)/normas , Humanos , Radioterapia/normasRESUMO
Monoclonal antibodies have been raised against the soluble lactotransferrin binding protein purified from the cell culture supernatant of Jurkat cell line, a human T-lymphoblastic cell. All monoclonal antibodies were able to specifically bind to the membrane of Jurkat cells. One of the monoclonal antibodies, DP5B3G10, recognized both the soluble lactotransferrin-binding protein and the membrane lymphocyte lactotransferrin receptor after SDS-PAGE in presence of 2-mercaptoethanol and electrotransfer on nitrocellulose. The monoclonal antibody DP5B3G10 inhibited the binding of lactotransferrin to Jurkat cells and human peripheral activated lymphocytes. In addition, lactotransferrin inhibited the binding of the monoclonal antibody to the cell surface. These results suggest that the 95 kDa lactotransferrin-binding protein isolated from the cell culture medium corresponds to the soluble form of the 105 kDa lymphocyte lactotransferrin receptor. Corresponding proteins of 105 kDa molecular mass were identified in Jurkat and CEM T-cells and Raji B-cells. Finally, the monoclonal antibody DP5B3G10 was used to immunolocalize the lactotransferrin receptor on the Jurkat cells. Using fluorescence and electron microscopy, the receptor was localized both inside and at the cell surface. The cell membrane receptor was associated into clusters. After permeabilization of the plasma membrane, the staining was positive in the peri-membrane area. The region near the nucleus was devoid of receptor.
Assuntos
Receptores de Superfície Celular/análise , Linfócitos T/química , Anticorpos Monoclonais , Ligação Competitiva , Linhagem Celular , Meios de Cultura , Imunofluorescência , Humanos , Imunoglobulinas/biossíntese , Microscopia Eletrônica , SolubilidadeRESUMO
Binding of either iron-saturated or iron-free lactoferrin to the Jurkat human lymphoblastic T-cell line was saturable with a dissociation constant Kd of 40 nM. The total number of binding sites was estimated to be approximately 300,000. Non-specific binding did not exceed 30% of the total binding. Removal of the 4 clustered arginine residues of lactoferrin at position 2 to 5, which are involved in the interactions with heparan sulfate, did not modify the binding parameters. Therefore, the high number of low affinity binding sites previously described as responsible for the interaction of lactoferrin with either hepatocytes, enterocytes or the U937 monocytic cell line, is not involved in the binding of lactoferrin to Jurkat cells. After binding at 4 degrees C, a shift to 37 degrees C causes cell to internalize lactoferrin, with the maximum intracellular concentration found at 3 to 8 and 5 to 15 min for iron-saturated and iron-free forms, respectively. Addition of colchicine had no effect on binding or internalization. These results suggest that endocytosis of lactoferrin by Jurkat cells occurs through a receptor-mediated process. Jurkat cells internalize lactoferrin monophasically with a first-order endocytic constant K(in) of 0.060 min-1 at 37 degrees C. Confocal microscopic analysis, using fluorescein-carbohydrate-labeled lactoferrin showed that lactoferrin was mainly localized in intracellular vesicles. Following uptake, the endocytic path utilized by fluorescein-carbohydrate-labeled lactoferrin was shown to diverge from that of rhodamine-labeled serum transferrin; after internalization, lactoferrin and serum transferrin did not fully colocalize. Intracellular lactoferrin was found in endosome vesicles as assessed by electron microscopy. Raising the pH in endosomes using chloroquine led to the accumulation of lactoferrin into endosomes (acidic compartment). After internalization, Jurkat cells released both degraded and intact lactoferrin into the culture medium, suggesting that a fraction (30-40%) of the ligand is degraded at each round of endocytosis.
Assuntos
Lactoferrina/metabolismo , Linfócitos T/metabolismo , Linhagem Celular , Endocitose , Humanos , Cinética , Microscopia Imunoeletrônica , Linfócitos T/citologiaRESUMO
We previously demonstrated that lactoferrin increases breast cell sensitivity to natural killer cell cytotoxicity whereas haematopoietic cells are unaffected by lactoferrin. It has been described that lactoferrin binds to various glycosaminoglycans. Compared to haematopoietic cells, breast cancer cells and particularly the breast cell line MDA-MB-231, possess a high level of proteoglycans. Scatchard analysis of 125I-lactoferrin binding to MDA-MB-231 cells revealed the presence of two classes of binding sites: a low affinity site with a Kd of about 700 nM and 3.9 x 10(6) sites and a higher affinity class with a Kd of 45 nM and 2.9 x 10(5) sites per cell. To investigate the potential regulation of lactoferrin activity by proteoglycans expressed on the MDA-MB-231 cells, we treated these cells with glycosaminoglycan-degrading enzymes or sodium chlorate, a metabolic inhibitor of proteoglycan sulphation. We showed that chondroitinase treatment has no effect, while heparinase or chlorate treatment significantly reduces both the binding of lactoferrin to cell surface sulphated molecules such as heparan sulphate proteoglycans (HSPG) and the affinity of lactoferrin for the higher affinity binding sites. The modulation of the lactoferrin binding was correlated with a decrease in lactoferrin activities on both MDA-MB-231 cell sensitisation to lysis and proliferation. Taken together, these results suggest that the presence of adequately sulphated molecules, in particular HSPG, is important for lactoferrin interaction and activity on the breast cancer cells MDA-MB-231.
Assuntos
Proteoglicanas de Heparan Sulfato/fisiologia , Lactoferrina/metabolismo , Neoplasias da Mama , Divisão Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cloratos/farmacologia , Condroitina ABC Liase/metabolismo , Feminino , Glicosaminoglicanos/metabolismo , Heparina Liase/metabolismo , Humanos , Radioisótopos do Iodo , Células Matadoras Naturais/imunologia , Células Tumorais CultivadasRESUMO
The activation of leukocytes by lipopolysaccharides (LPS), resulting in the oxidative burst, contributes to the pathogenesis of septic shock. The binding of LPS to L-selectin, which was reported as a serum-independent LPS receptor on neutrophils, induces the production of oxygen free radicals. Human lactoferrin (hLf), an anti-inflammatory glycoprotein released from neutrophil granules during infection, binds to LPS. In this study, we investigated the capacity of hLf to inhibit the L-selectin-mediated activation of neutrophils. Our experiments revealed that hLf prevents the binding of LPS to L-selectin in a concentration-dependent manner. Inhibition was maximum (87.7+/-0.5%) at a concentration of 50 microg/ml of hLf. Furthermore, hLf inhibited up to 55.4+/-0.5% of the intracellular hydrogen peroxide production induced by LPS in neutrophils. These findings suggest that the anti-inflammatory properties of hLf are due, at least in part, to their ability to prevent the binding of LPS to neutrophil L-selectin.
Assuntos
Selectina L/metabolismo , Lactoferrina/farmacologia , Lipopolissacarídeos/metabolismo , Neutrófilos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Escherichia coli , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Peróxido de Hidrogênio/metabolismo , Inflamação/metabolismo , Neutrófilos/efeitos dos fármacos , Ligação ProteicaRESUMO
It has previously been suggested that volume-regulated anion channels (VRACs) and store-operated channels (SOCs) interact with each other according to their expected colocalization in the plasma membrane of LNCaP cells. In order to study interactions between these two channels, we used 2-aminoethoxydiphenyl borate (2-APB) as a regular SOC inhibitor. Surprisingly 2-APB reduced VRAC activity in a dose-dependent manner (IC(50)=122.8 microM), but not 2,2-diphenyltetrahydrofuran (a structural analog of 2-APB). This effect was also present in keratinocytes. We conclude that 2-APB is an inhibitor of the VRAC family, and is also a potent tool to study the SOC-VRAC interaction in LNCaP cells.
Assuntos
Compostos de Boro/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Canais de Cloreto/antagonistas & inibidores , Ácido Egtázico/análogos & derivados , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Furanos/química , Furanos/farmacologia , Humanos , Soluções Hipotônicas/farmacologia , Concentração Inibidora 50 , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Neoplasias da Próstata/metabolismoRESUMO
Human lactotransferrin receptors have been recently characterized on mitogen-stimulated human lymphocytes [(1989) Eur. J. Biochem. 179, 481-487]. In order to define the lactotransferrin recognition site by these receptors, the binding to lymphocytes of several tryptic fragments, isolated from human lactotransferrin by mild tryptic hydrolysis [(1984) Biochim. Biophys. Acta 787, 90-96], has been investigated. The 30 kDa N-tryptic fragment (residues 4-281) and the re-associated N,C-tryptic complex bind to lactotansferrin lymphocyte receptor with a dissociation constant of 44 nM and 39 nM, respectively, similar to the value obtained for the native lactotransferrin (Kd = 46 nM). However, neither the N-terminal domain II (residues 91-257) nor the 50 kDa C-tryptic fragment (residues 282-703) are recognized. These results suggest that the binding site of human lactotransferrin by the lymphocyte receptor is located in the N-terminal lobe and more precisely in the N-terminal domain I (residues 4-90 and/or 258-281).