RESUMO
BACKGROUND: Understanding the regional vascular delivery of particles to tumour sites is a prerequisite for developing new diagnostic and therapeutic composites for treatment of oncology patients. We describe a novel imageable 67Ga-radiolabelled polymer composite that is biocompatible in an animal tumour model and can be used for preclinical imaging investigations of the transit of different sized particles through arterial networks of normal and tumour-bearing organs. RESULTS: Radiolabelling of polymer microspheres with 67Ga was achieved using a simple mix and wash method, with tannic acid as an immobilising agent. Final in vitro binding yields after autoclaving averaged 94.7%. In vivo stability of the composite was demonstrated in New Zealand white rabbits by intravenous administration, and intrahepatic artery instillations were made in normal and VX2 tumour implanted rabbit livers. Stability of radiolabel was sufficient for rabbit lung and liver imaging over at least 3 hours and 1 hour respectively, with lung retention of radiolabel over 91%, and retention in both normal and VX2 implanted livers of over 95%. SPECT-CT imaging of anaesthetised animals and planar imaging of excised livers showed visible accumulation of radiolabel in tumours. Importantly, microsphere administration and complete liver dispersal was more easily achieved with 8 µm diameter MS than with 30 µm MS, and the smaller microspheres provided more distinct and localised tumour imaging. CONCLUSION: This method of producing 67Ga-radiolabelled polymer microspheres is suitable for SPECT-CT imaging of the regional vascular delivery of microspheres to tumour sites in animal models. Sharper distinction of model tumours from normal liver was obtained with smaller MS, and tumour resolution may be further improved by the use of 68Ga instead of 67Ga, to enable PET imaging.
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Extracellular histones in neutrophil extracellular traps (NETs) or in chromatin from injured tissues are highly pathological, particularly when liberated by DNases. We report the development of small polyanions (SPAs) (~0.9-1.4 kDa) that interact electrostatically with histones, neutralizing their pathological effects. In vitro, SPAs inhibited the cytotoxic, platelet-activating and erythrocyte-damaging effects of histones, mechanistic studies revealing that SPAs block disruption of lipid-bilayers by histones. In vivo, SPAs significantly inhibited sepsis, deep-vein thrombosis, and cardiac and tissue-flap models of ischemia-reperfusion injury (IRI), but appeared to differ in their capacity to neutralize NET-bound versus free histones. Analysis of sera from sepsis and cardiac IRI patients supported these differential findings. Further investigations revealed this effect was likely due to the ability of certain SPAs to displace histones from NETs, thus destabilising the structure. Finally, based on our work, a non-toxic SPA that inhibits both NET-bound and free histone mediated pathologies was identified for clinical development.
Assuntos
Armadilhas Extracelulares/efeitos dos fármacos , Histonas/metabolismo , Polímeros/farmacologia , Sepse/sangue , Sepse/tratamento farmacológico , Animais , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Feminino , Histonas/toxicidade , Humanos , Bicamadas Lipídicas , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/sangue , Ativação Plaquetária/efeitos dos fármacos , Polieletrólitos , Polímeros/química , Ratos Wistar , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/patologia , Sepse/patologiaRESUMO
PURPOSE: Selective internal radiation therapy (SIRT) is an effective treatment option for liver tumors, using Y-90-loaded polymer microspheres that are delivered via catheterization of the hepatic artery. Since Y-90 is a beta emitter and not conveniently imaged by standard clinical instrumentation, dosimetry is currently evaluated in each patient using a surrogate particle, 99mTechnetium-labeled macroaggregated albumin (99mTc-MAA). We report a new composite consisting of 99mTc-labeled nanoparticles attached to the same polymer microspheres as used for SIRT, which can be imaged with standard SPECT. METHODS: Carbon nanoparticles with an encapsulated core of 99mTc were coated with the polycation protamine sulfate to provide electrostatic attachment to anionic polystyrene sulfonate microspheres of different sizes (30, 12, and 8 µm). The in vivo stability of these composites was determined via intravenous injection and entrapment in the capillary network of normal rabbit lungs for up to 3 hours. Furthermore, we evaluated their biodistribution in normal rabbit livers, and livers implanted with VX2 tumors, following intrahepatic artery instillation. RESULTS: We report distribution tests for three different sizes of radiolabeled microspheres and compare the results with those obtained using 99mTc-MAA. Lung retention of the radiolabeled microspheres ranged from 72.8% to 92.9%, with the smaller diameter microspheres showing the lowest retention. Liver retention of the microspheres was higher, with retention in normal livers ranging from 99.2% to 99.8%, and in livers with VX2 tumors from 98.2% to 99.2%. The radiolabeled microspheres clearly demonstrated preferential uptake at tumor sites due to the increased arterial perfusion produced by angiogenesis. CONCLUSION: We describe a novel use of radiolabeled carbon nanoparticles to generate an imageable microsphere that is stable in vivo under the shear stress conditions of arterial networks. Following intra-arterial instillation in the normal rabbit liver, they distribute in a distinct segmented pattern, with the smaller microspheres extending throughout the organ in finer detail, while still being well retained within the liver. Furthermore, in livers hosting an implanted VX2 tumor, they reveal the increased arterial perfusion of tumor tissue resulting from angiogenesis. These novel composites may have potential as a more representative mimic of the vascular distribution of therapeutic microspheres in patients undergoing SIRT.
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Artérias/diagnóstico por imagem , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/diagnóstico por imagem , Fígado/irrigação sanguínea , Pulmão/irrigação sanguínea , Microesferas , Tecnécio/química , Animais , Linhagem Celular Tumoral , Feminino , Injeções Intra-Arteriais , Injeções Intravenosas , Fígado/diagnóstico por imagem , Neoplasias Hepáticas/tratamento farmacológico , Pulmão/diagnóstico por imagem , Masculino , Poliestirenos/química , Coelhos , Compostos Radiofarmacêuticos/química , Agregado de Albumina Marcado com Tecnécio Tc 99m/administração & dosagem , Agregado de Albumina Marcado com Tecnécio Tc 99m/química , Distribuição Tecidual , Resultado do TratamentoRESUMO
PURPOSE: The aim of the study was to evaluate the prognostic value of soluble urokinase plasminogen activator receptor (suPAR) in preoperatively obtained sera samples (s-suPAR) from breast cancer patients. EXPERIMENTAL DESIGN: suPAR levels were determined by the use of a kinetic ELISA in sera from 274 breast cancer patients and in tumor cytosols (c-suPAR) from 188 of these patients. In addition, s-suPAR levels were analyzed in 174 female blood donors. RESULTS: The mean s-suPAR level was 3.8 ng/ml (range, 1.6-9.2 ng/ml) in the patients and 3 ng/ml (range, 1.3-6.4 ng/ml) in the donors. The mean c-suPAR level was 0.55 ng/mg protein (range, 0.07-2.83 ng/mg protein). A weak but significant linear association was found between s-suPAR and age in the donors; thus, all of the s-suPAR levels were adjusted for this age dependency (aa-s-suPAR). The aa-s-suPAR levels were significantly increased in the patients as compared with the donors (P < 0.0001). No difference was found in aa-s-suPAR levels between the lymph node-positive and -negative patients (P = 0.27), and no correlation was seen between aa-s-suPAR and c-suPAR (sigma = 0.08; P = 0.71). During the follow-up period (5.9 years) 77 patients experienced a relapse and 69 died. aa-s-suPAR as a continuous variable was significantly associated with relapse-free survival [hazard ratio (HR), 1.4; 95% confidence interval (CI), 1.1-1.8; P = 0.003] and overall survival (HR, 1.6; 95% CI, 1.2-2.0; P < 0.0001). In multivariate Cox analysis including the classical prognostic parameters in breast cancer, continuous aa-s-suPAR was significantly associated with both relapse-free survival (HR, 1.4; 95% CI, 1.1-1.7; P = 0.001) and overall survival (HR, 1.4; 95% CI, 1.1-1.8; P = 0.002). In these analyses positive lymph nodes, tumor size >2 cm, and negative estrogen receptor content were also significantly associated with patient outcome. CONCLUSION: This study shows that high preoperative aa-s-suPAR levels are significantly associated with poor outcome for breast cancer patients independent of lymph node status, tumor size, and estrogen receptor status.
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Neoplasias da Mama/patologia , Receptores de Superfície Celular/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Mama/sangue , Neoplasias da Mama/metabolismo , Citosol/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Solubilidade , Análise de SobrevidaRESUMO
PURPOSE: The purpose of this study was to measure total levels of tissue inhibitor of metalloproteinases (TIMP-1) by ELISA in plasma from blood donors, patients with inflammatory bowel disease (IBD), and patients with cancer and to correlate the results to patient diagnosis. EXPERIMENTAL DESIGN: Total TIMP-1 plasma levels were measured by ELISA in blood samples from two different blood donor populations from IBD patients, and preoperative samples from patients with primary colon cancer (CC), rectal cancer (RC), or breast cancer. RESULTS: There were no significant differences in plasma TIMP-1 levels between healthy donors and IBD or breast cancer patients, whereas patients with CC or RC had significantly elevated TIMP-1 levels. Total TIMP-1 levels identified patients with CC with a sensitivity of 63% at 98% specificity, patients with early CC (Dukes' A+B) with a sensitivity of 56% at 98% specificity, and patients with right-sided CC with a sensitivity of 72% at 98% specificity. Combining carcinoembryonic antigen and TIMP-1 measurements increased the sensitivities obtained from TIMP-1 measurements alone. CONCLUSIONS: TIMP-1 was significantly elevated in plasma from CC and RC patients, including those with early-stage disease. Sensitivity and specificity were both sufficiently high to consider TIMP-1 as a marker for the early identification of CC patients, in particular, those with right-sided CC.
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Biomarcadores Tumorais/sangue , Neoplasias do Colo/enzimologia , Neoplasias Retais/enzimologia , Inibidor Tecidual de Metaloproteinase-1/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doadores de Sangue , Neoplasias da Mama/enzimologia , Neoplasias da Mama/patologia , Neoplasias do Colo/patologia , Feminino , Humanos , Doenças Inflamatórias Intestinais/enzimologia , Doenças Inflamatórias Intestinais/patologia , Masculino , Pessoa de Meia-Idade , Neoplasias Retais/patologia , Valores de Referência , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Delivery of chemotherapeutic drugs to tumours by reformulation as nanoparticles has often been proposed as a means of facilitating increased selective uptake, exploiting the increased permeability of the tumour vasculature. However realisation of this improvement in drug delivery in cancer patients has met with limited success. We have compared tumour uptake of soluble Tc99m-pertechnetate and a colloid of nanoparticles with a Tc99m core, using both intra-venous and intra-arterial routes of administration in a rabbit liver VX2 tumour model. The radiolabelled nanoparticles were tested both in untreated and cationised form. The results from this tumour model in an internal organ show a marked advantage in intra-arterial administration over the intra-venous route, even for the soluble isotope. Tumour accumulation of nanoparticles from arterial administration was augmented by cationisation of the nanoparticle surface with histone proteins, which consistently facilitated selective accumulation within microvessels at the periphery of tumours.
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Diagnóstico por Imagem/métodos , Infusões Intra-Arteriais/métodos , Injeções Intravenosas/métodos , Isótopos/administração & dosagem , Neoplasias Hepáticas/diagnóstico , Animais , CoelhosRESUMO
We report here that, clofazimine (CFZ) treatment (0.1-10 microM) led to inhibition of in vitro proliferation of hepatocellular carcinoma (HCC) cell lines Hep3-beta, HuH-7, HepG2, SKHEP-1, PLC/PRF-5 and Novikoff. A 24 h exposure of human HuH-7 cells to various concentrations of CFZ dissolved in lipiodol (CFZ-L 10-160 microM), followed by 4 days treatment with medium alone, also led to dose-dependant inhibition of post-treatment cell growth. In vivo, direct intratumoural and intrahepatic arterial injection (IHA) of CFZ-L led to profound inhibition of orthotopic growth of rat Novikoff liver tumours (P < 0.0001 and P < 0.005, respectively). On the contrary, daily oral administration of 150 mg/kg CFZ for 7 days, did not influence the rate of Novikoff tumour growth. Histological examination of rat tumours, revealed the presence of lipiodol in tumour cells, 7 days after treatment with a single IHA dose. Histopathology did not show any abnormality in liver, lung or bowel sections taken from animals 1 week after IHA administration of CFZ-L. Similarly, liver function tests were all normal compared to saline treated animals. Deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labelling revealed the presence of large numbers of apoptotic cells in the CFZ-L treated tumours. Thus, intraarterial administration of the highly lipophilic antiproliferative agent CFZ in lipiodol solution may represent an effective and yet safe strategy for the regional treatment of HCCs.
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Carcinoma Hepatocelular/tratamento farmacológico , Clofazimina/uso terapêutico , Óleo Iodado/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Administração Oral , Animais , Apoptose , Compostos Azo/farmacologia , Divisão Celular , Linhagem Celular Tumoral , Sobrevivência Celular , Corantes/farmacologia , Meios de Contraste/farmacologia , Relação Dose-Resposta a Droga , Humanos , Marcação In Situ das Extremidades Cortadas , Concentração Inibidora 50 , Óleo Iodado/farmacologia , Metabolismo dos Lipídeos , Neoplasias Hepáticas/patologia , Ratos , Fatores de TempoRESUMO
Soluble tetranectin (TN) was measured preoperatively in serum from 567 patients with primary colorectal cancer and levels were tested for association with prognosis. The prognostic significance of TN was also compared to that of plasminogen-activator inhibitor-1 (PAI-1), urokinase plasminogen activator (uPAR) and carcinoembryonic antigen (CEA). Significantly shorter survival was found for patients with TN levels below a cut-off point of 7.5 mg/l compared to patients with levels above, as illustrated by Kaplan-Meier curves. By Cox analyses, log TN, log soluble uPAR as well as log CEA were found to have an independent prognostic value for survival (log TN: HR = 0.47, 95% CI: 0.29-0.76); log soluble uPAR: HR = 1.65, 95% CI: 1.18-2.31; log CEA: HR = 1.I1, 95% CI: 1.03-1.20). Based on the multivariate model, a patient with a combination of low levels of TN and PAI-1 and elevated levels of soluble uPAR and CEA had a 2.43 increased risk as compared to a patient with median levels of these biochemical markers. Significant correlations were found with Dukes' stages for all the biochemical markers and between the respective biochemical markers. The findings confirm that TN is a strong prognostic factor in patients with colorectal cancer. TN may be valuable as a prognostic variable in future studies evaluating new treatment strategies for colorectal cancer.
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Adenocarcinoma/diagnóstico , Biomarcadores Tumorais/sangue , Antígeno Carcinoembrionário/sangue , Neoplasias Colorretais/diagnóstico , Lectinas Tipo C/sangue , Inibidor 1 de Ativador de Plasminogênio/sangue , Receptores de Superfície Celular/sangue , Ativador de Plasminogênio Tipo Uroquinase/sangue , Adenocarcinoma/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Estadiamento de Neoplasias , Prognóstico , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
Diagnostic imaging of the blood perfusion of the lungs is currently performed using particles of macro-aggregated albumin, which are mechanically arrested at limiting diameters of the capillary bed. While the proportion of blood flow obstructed is typically very low and temporary, it would seem more desirable to image lung perfusion in patients using a non-obstructive method, and using materials that avoid biological hazards. We have characterised the in vitro and in vivo properties of a colloid of a cationised radiolabelled nanocomposite. The nanoparticles comprise a Technetium-99m core encapsulated in graphitic carbon, and coated with low molecular weight poly-lysine to provide a strong charge-based affinity for the endothelial glycocalyx of the lung. Following intravenous injection in rabbits and cats, the nanoparticles rapidly distribute and localise in the lungs, thus enabling gamma camera imaging of lung perfusion. Repeat administration of this colloid in both species over several weeks indicates favourable biocompatibility.
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Pulmão/anatomia & histologia , Nanopartículas , Cátions , Coloides , Humanos , Microscopia Eletrônica de Varredura , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
Recent findings on the role of circulating histone proteins in mediating acute lung injury prompted us to investigate whether there is a specific mechanism for accumulation of histones in the lungs. Binding sites for polycations are already known in the vasculature of the lungs, and we postulated that these could also be involved in histone accumulation, since histones have a high content of positively charged amino acids. Using a histone-coated colloid of a radiolabelled nanocomposite to track histone biodistribution with imaging techniques, it was found that histones bind avidly in the lungs of rabbits after intravenous injection. Blocking experiments with competing polycations in vivo characterised histone lung binding as dependent on a charge interaction with microvessel polyanions. Pretreatment of rabbits with a specific heparinase confirmed that the lung binding sites consist of heparan sulphate in the endothelial glycocalyx. A range of heparan sulphate analogues was accordingly shown to prevent histone accumulation in the lungs by neutralising histones in blood. These findings provide a rational basis for the design of polyanions that can prevent accumulation of cytotoxic histones in the lungs and thereby intervene at an early key step in the development of acute lung injury.
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Capilares/metabolismo , Glicocálix/metabolismo , Heparitina Sulfato/metabolismo , Histonas/metabolismo , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Animais , Anticorpos Neutralizantes/metabolismo , Sítios de Ligação , Bovinos , Eletroforese em Gel de Poliacrilamida , Heparina Liase/metabolismo , Nanopartículas , Poliaminas/metabolismo , Polieletrólitos , Polissacarídeos/metabolismo , Ligação Proteica , Coelhos , Distribuição TecidualRESUMO
Previous studies including various tumor types have shown different associations between tumor tissue levels of plasminogen activator inhibitor 2 (PAI-2) and patient survival. High tumor tissue concentrations of PAI-2 have been associated with good prognosis in patients with breast cancer, small cell lung cancer and ovarian cancer, but with poor histologic differentiation and poor prognosis in patients with colorectal cancer. On the other hand, high tumor tissue concentrations of urokinase plasminogen activator (uPA), uPA receptor (R) and PAI-1 have more consistently been associated with poor histologic differentiation and poor prognosis. Our study quantified PAI-2 and uPAR using specific enzyme-linked immunosorbent assays in homogenates of 274 samples of endometrial cancer tissue. The prognostic power of each factor was analyzed in the subgroup of patients with early stage disease, i.e., International Federation of Gynecology and Oncology (FIGO) surgical stage I-II (n = 188). This group had a median follow-up time of 6.8 years (range 0.7-9.9), and 23 progressions were observed. The 80(th) percentile for PAI-2 and uPAR was used to dichotomize the material, and the results were analyzed for associations with clinical data including progression-free survival. The results were also compared with DNA ploidy status, S-phase fraction, uPA and PAI-1, which we reported in a previous study (Fredstorp Lidebring et al., Eur J Cancer 2001; in press). A high PAI-2 level was associated with shorter progression-free survival in univariate analysis and was an independent prognostic factor in bivariate analyses, which included PAI-1, uPA and DNA ploidy status. In contrast, a high level of uPAR had no association with prognosis in early stage endometrial cancer. The combination of high PAI-2 and PAI-1 levels in tumors revealed a small group of stage I-II patients with an accumulative progression rate of 50%.
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Neoplasias do Endométrio/diagnóstico , Neoplasias do Endométrio/metabolismo , Inibidor 2 de Ativador de Plasminogênio/biossíntese , Prognóstico , Adulto , Idoso , Citosol/metabolismo , Progressão da Doença , Intervalo Livre de Doença , Neoplasias do Endométrio/patologia , Feminino , Humanos , Pessoa de Meia-Idade , Fatores de TempoRESUMO
The urokinase receptor (urokinase plasminogen activator receptor; uPAR) regulates monocyte adhesion by direct binding to vitronectin and by forming complexes with integrins. Therefore, possible up-regulation of uPAR in acute myocardial infarction (AMI) may affect monocyte adhesion. In 20 patients with AMI, uPAR surface expression (measured by flow cytometry) was increased compared with that in patients with chronic stable angina (mean +/- SD fluorescence, 179 +/- 96 vs 80 +/- 53; P =.002). Expression of uPAR correlated with activation of beta(2)-integrins lymphocyte function-associated antigen 1 (LFA-1) and macrophage antigen 1 (Mac-1), measured by using monoclonal antibodies (mAbs) 24 and CBRM1/5. Isolated mononuclear cells (MNCs) from patients with AMI showed enhanced adhesiveness to human umbilical vein endothelial cells (HUVECs), to fibrinogen (Mac-1 ligand), and to vitronectin (uPAR ligand). Excessive adhesion of MNCs to HUVECs was inhibited by mAbs anti-CD18 (84%), anti-CD11a (51%), and anti-CD11b (57%), indicating a major contribution of LFA-1 and Mac-1. The mAb anti-uPAR R3 blocked adhesion of cells from patients with AMI to vitronectin (95%) but also beta(2)-integrin-mediated adhesion to fibrinogen (79%) and HUVECs (66%). Incubation of monocytic MonoMac6 cells with plasma from patients with AMI enhanced uPAR messenger RNA expression and cell adhesion to HUVECs. Thus, released soluble factors may contribute to enhanced monocyte adhesion in AMI. Mouse pre-B lymphocytes (BAF3 cells) transfected with various amounts of uPAR complementary DNA showed a strong correlation of uPAR expression with beta(2)-integrin-dependent adhesion to intercellular adhesion molecule 1, thus providing evidence for the functional relevance of uPAR up-regulation in an isolated in vitro system. In conclusion, we found that uPAR expression is elevated on monocytes in AMI and contributes to enhanced cell adhesion. Thus, uPAR may be a novel target for prevention of unwanted monocyte recruitment as part of inflammatory cardiovascular processes.