RESUMEN
Anti-mullerian hormone (AMH) is one of the least studied members of transforming growth factor beta superfamily showing pro-apoptotic activity against cells positive for hormone type II receptor overexpressed by malignant cells in many cancer cases. Here, we propose an improved method for isolation of recombinant C-terminal AMH fragment (C-rAMH) to obtain homogeneous preparations of this protein with high biological activity. In contrast to our previously developed C-rAMH purification technology based on reversed-phase HPLC, the key stage of the new approach is hydrophobic interaction chromatography using Toyopearl Butyl-650S resin performed under more benign conditions. This modification of the previously developed method allowed highly purified C-rAMH to be obtained that is characterized by twice the specificity estimated as the ability to bind to the recombinant analog of AMH type II receptor and by significantly higher biological activity, that is, the ability to induce the death of target cells. Thus, we made the purification technology even more cost-effective and suitable for the production of drug forms based on C-rAMH.
Asunto(s)
Hormona Antimülleriana , Cromatografía Líquida de Alta Presión/métodos , Proteínas Recombinantes , Animales , Hormona Antimülleriana/química , Hormona Antimülleriana/aislamiento & purificación , Hormona Antimülleriana/farmacología , Células CHO , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cromatografía de Fase Inversa/métodos , Cricetinae , Cricetulus , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
Monoclonal antibodies (mAbs) against morphine are important in the development of immunotherapeutic and diagnostic methods for the treatment and prevention of drug addiction. By the surface plasmon resonance (SPR) and enzyme immunoassay techniques, we characterized two previously obtained mAbs 3K11 and 6G1 and showed their ability to recognize free morphine and morphine-containing antigens in different ways because of the epitope specificity thereof. Using the defined amino acid sequences, we obtained three-dimensional models of the variable regions of Fab fragments of these antibodies and compared them with the known sequence and spatial structure of the anti-morphine antibody 9B1. Docking simulations are performed to obtain models of the antibodies complexes with morphine. Differences in the models of 3K11 and 6G1 complexes with morphine correlate with their experimentally detected epitope specificity. The results, in particular, can be used for the structure-based design of the corresponding humanized antibodies. According to our modeling and docking results, the very different modes of morphine binding to mAbs 3K11 and 6G1 are qualitatively similar to those previously reported for cocaine and two anti-cocaine antibodies. Thus, the obtained structural information brings more insight into the hapten recognition diversity.
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Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Simulación por Computador , Epítopos/inmunología , Morfina/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Sitios de Unión , Inmunoensayo , Cinética , Ratones , Modelos Moleculares , Simulación del Acoplamiento Molecular , Resonancia por Plasmón de SuperficieRESUMEN
Anti-mullerian hormone (AMH) is a cytokine of transforming growth factor ß (TGF-ß) superfamily able to induce apoptosis in cells bearing specific AMH type II receptors (AMHRII). AMHRII is overexpressed in some malignant cells, so at present recombinant AMH (rAMH) is considered as a new candidate antineoplastic drug. The use of rAMH may be especially effective in case of such severe diseases as ovarian, prostate and breast cancer. However, the development of a new drug is hampered by the laboriousness of obtaining highly purified rAMH and by the lack of data about the pharmacological characteristics of rAMH derivatives. In this work, we obtained preparations of prohormone, half-cleaved rAMH and a C-terminal fragment of rAMH, which was confirmed by qualitative and quantitative analyses. To obtain rAMH and its derivatives we used a previously developed highly effective producer strain containing the optimized human AMH gene. The production process has been divided into several stages: (a) rAMH biosynthesis in the bioreactor; (b) culture media preparation; (c) purification of rAMH and its derivatives using immunoaffinity chromatography and reversed-phase HPLC; (d) identification of the purified proteins by immunoblotting and analytical reversed-phase HPLC; and (e) evaluation of the hormone forms activity. The obtained proteins may be used in preclinical trials and in vitro study of rAMH derivatives properties.
Asunto(s)
Hormona Antimülleriana/genética , Hormona Antimülleriana/aislamiento & purificación , Hormona Antimülleriana/metabolismo , Técnicas de Cultivo de Célula , Cromatografía de Afinidad , Cromatografía de Fase Inversa , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Cancer cells are known to contain high levels of the heat shock protein 70 kDa (Hsp70), which mediates increased cell proliferation, escape from programmed cell death, enhanced invasion, and metastasis. A part of Hsp70 molecules may release from cancer cells and affect the behavior of adjacent stromal cells. To explore the effects of Hsp70 on the status of monocytes/macrophages in the tumor locale, we incubated human carcinoma cells of three distinct lines with normal and reduced content of Hsp70 with THP1 monocytes. Using two methods, we showed that the cells with knock-down of Hsp70 released a lower amount of protein in the extracellular medium. Three cycles of the co-cultivation of cancer and monocytic cells led to the secretion of several cytokines typical of the tumor microenvironment (TME) and to pro-cancer activation of the monocytes/macrophages as established by elevation of F4/80 and arginase-1 markers. Unexpectedly, the efficacy of epithelial-mesenchymal transition and resistance of carcinoma cells to anticancer drugs after incubation with monocytic cells were more pronounced in cells with lower Hsp70, e.g., releasing less Hsp70 into the extracellular milieu. These data suggest that Hsp70 released from tumor cells into the TME is able, together with the development of an anti-cancer immune response, to limit the conversion of a considerable part of monocytic cells to the pro-tumor phenotype.
Asunto(s)
Carcinogénesis/inmunología , Proteínas HSP70 de Choque Térmico/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Microambiente Tumoral , Células A549 , Carcinogénesis/patología , Línea Celular Tumoral , Proliferación Celular , Técnicas de Cocultivo , Transición Epitelial-Mesenquimal , Humanos , Inmunidad , Macrófagos/patología , Monocitos/patologíaRESUMEN
Long (D2L) and Short (D2S) isoforms of D2 dopamine receptor differ in their biochemical and physiological properties, which could affect functioning of prefrontal cortex. Contribution of distinct D2 dopamine receptor isoforms to cognitive dysfunctions and its developmental regulation are currently not fully elucidated. In the present study, we evaluated developmental mRNA expression of D2S/D2L dopamine receptor isoforms within the rat medial prefrontal cortex (mPFC) in the model of neurodevelopmental cognitive dysfunction. Working memory performance (Y-maze spontaneous alternations) and D2S/D2L mRNA expression in the mPFC (by qRT-PCR) were evaluated in juvenile (P27), adolescent (P42-47) and adult (P75-90) rats after chronic early life treatment with proinflammatory cytokine interleukin (IL)-1ß (1⯵g/kg i.p. daily P15-21). It was shown that IL-1ß elevation during the 3rd week of life leads to working memory deficit originating in juvenile animals and persisting into adulthood. D2S mRNA expression was strongly downregulated during adolescence, and such downregulation was exaggerated in animals injected with IL-1ß during P15-21. Early life IL-1ß administrations influenced developmental changes in the D2S/D2L mRNA ratio. This measure was found to be decreased in adolescent and adult control (intact and vehicle-treated) rats compared to juvenile control, while in the case of IL-1ß-treated animals, the decrease in D2S/D2L ratio was observed only in adulthood but not in adolescence compared to juvenile rats. During the adolescence, D2S mRNA expression was downregulated and D2S/D2L ratio was upregulated in the mPFC of rats treated with IL-1ß during the 3rd week of life compared to controls. Based on these data we conclude that changes in the developmental expression of D2 dopamine receptor splice variants within mPFC may underlie long-lasting cognitive deficit associated with neonatal pathology.
Asunto(s)
Encefalitis/inducido químicamente , Interleucina-1beta/administración & dosificación , Memoria a Corto Plazo/fisiología , Corteza Prefrontal/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Modelos Animales de Enfermedad , Interleucina-1beta/fisiología , Masculino , Memoria a Corto Plazo/efectos de los fármacos , Trastornos del Neurodesarrollo/inducido químicamente , Corteza Prefrontal/efectos de los fármacos , Isoformas de Proteínas/metabolismo , ARN Mensajero/metabolismo , Ratas WistarRESUMEN
Chaperone Hsp70 can activate adaptive immunity suggesting its possible application as an antitumor vaccine. To assess the therapeutic capacity of Hsp70 we administered purified chaperone into a C6 glioblastoma brain tumor and explored the viability and tumor size as well as interferon gamma (IFNγ) production and cytotoxicity of lymphocytes in the treated animals. Targeted intratumoral injection of Hsp70 resulted in its distribution within the area of glioblastoma, and caused significant inhibition of tumor progression as confirmed by magnetic resonance imaging. The delay in tumor growth corresponded to the prolonged survival of tumor-bearing animals of up to 31 days versus 20 days in control. Continuous administration of Hsp70 with an osmotic pump increased survival even further (39 days). Therapeutic efficacy was associated with infiltration to glioblastoma of NK cells (Ly-6c+) and T lymphocytes (CD3+, CD4+ and CD8+) as well as with an increase in the activity of NK cells (granzyme B production) and CD8+ T lymphocytes as shown by IFNγ ELISPOT assay. Furthermore, we found that Hsp70 treatment caused concomitantly, with a tenfold elevated IFNγ production, an increase in anti-C6 tumor cytotoxicity of lymphocytes. In conclusion, continuous intratumoral delivery of Hsp70 demonstrates high therapeutic potential and therefore could be applied in the treatment of glioblastoma.
Asunto(s)
Neoplasias Encefálicas/terapia , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/administración & dosificación , Glioblastoma/terapia , Proteínas HSP70 de Choque Térmico/metabolismo , Inmunoterapia , Animales , Apoptosis , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Proliferación Celular , Sistemas de Liberación de Medicamentos , Citometría de Flujo , Glioblastoma/inmunología , Glioblastoma/metabolismo , Proteínas HSP70 de Choque Térmico/administración & dosificación , Humanos , Técnicas para Inmunoenzimas , Inyecciones Intralesiones , Interferón gamma/metabolismo , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , Ratas , Linfocitos T Citotóxicos/inmunología , Células Tumorales CultivadasRESUMEN
Complement C3 is a pivotal component of three cascades of complement activation. C3 is expressed in human atherosclerotic lesions and is involved in atherogenesis. However, the mechanism of C3 accumulation in atherosclerotic lesions is not well elucidated. We show that acetylated low density lipoprotein and oxidized low density lipoprotein (oxLDL) increase C3 gene expression and protein secretion by human macrophages. Modified LDL (mLDL)-mediated activation of C3 expression mainly depends on liver X receptor (LXR) and partly on Toll-like receptor 4 (TLR4), whereas C3 secretion is increased due to TLR4 activation by mLDL. LXR agonist TO901317 stimulates C3 gene expression in human monocyte-macrophage cells but not in human hepatoma (HepG2) cells. We find LXR-responsive element inside of the promoter region of the human C3 gene, which binds to LXRß in macrophages but not in HepG2 cells. We show that C3 expression and secretion is decreased in IL-4-treated (M2) and increased in IFNγ/LPS-stimulated (M1) human macrophages as compared with resting macrophages. LXR agonist TO901317 potentiates LPS-induced C3 gene expression and protein secretion in macrophages, whereas oxLDL differently modulates LPS-mediated regulation of C3 in M1 or M2 macrophages. Treatment of human macrophages with anaphylatoxin C3a results in stimulation of C3 transcription and secretion as well as increased oxLDL accumulation and augmented oxLDL-mediated up-regulation of the C3 gene. These data provide a novel mechanism of C3 gene regulation in macrophages and suggest new aspects of cross-talk between mLDL, C3, C3a, and TLR4 during development of atherosclerotic lesions.
Asunto(s)
Complemento C3/genética , Complemento C3/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Lipoproteínas LDL/farmacología , Macrófagos/efectos de los fármacos , Receptores Nucleares Huérfanos/metabolismo , Receptor Toll-Like 4/metabolismo , Secuencia de Bases , Transporte Biológico , Complemento C3/biosíntesis , Células Hep G2 , Humanos , Lipoproteínas LDL/metabolismo , Receptores X del Hígado , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/citología , Macrófagos/metabolismo , FN-kappa B/metabolismo , Receptores Nucleares Huérfanos/agonistas , Regiones Promotoras Genéticas/genética , Transcripción Genética/efectos de los fármacosRESUMEN
The release of Hsp70 chaperone from tumor cells is found to trigger the full-scale anti-cancer immune response. Such release and the proper immune reaction can be induced by the delivery of recombinant Hsp70 to a tumor and we sought to explore how the endogenous Hsp70 can be transported to extracellular space leading to the burst of anti-cancer activity. Hsp70 transport mechanisms were studied by analyzing its intracellular tracks with Rab proteins as well as by using specific inhibitors of membrane domains. To study Hsp70 forms released from cells we employed the assay consisting of two affinity chromatography methods. Hsp70 content in culture medium and extracellular vesicles (EVs) was measured with the aid of ELISA. The properties and composition of EVs were assessed using nanoparticle tracking analysis and immunoblotting. The activity of immune cells was studied using an assay of cytotoxic lymphocytes, and for in vivo studies we employed methods of affinity separation of lymphocyte fractions. Analyzing B16 melanoma cells treated with recombinant Hsp70 we found that the chaperone triggered extracellular transport of its endogenous analog in soluble and enclosed in EVs forms; both species efficiently penetrated adjacent cells and this secondary transport was corroborated with the strong increase of Natural Killer (NK) cell toxicity towards melanoma. When B16 and CT-26 colon cancer cells before their injection in animals were treated with Hsp70-enriched EVs, a powerful anti-cancer effect was observed as shown by a two-fold reduction in tumor growth rate and elevation of life span. We found that the immunomodulatory effect was due to the enhancement of the CD8-positive response and anti-tumor cytokine accumulation; supporting this there was no delay in CT-26 tumor growth when Hsp70-enriched EVs were grafted in nude mice. Importantly, pre-treatment of B16 cells with Hsp70-bearing EVs resulted in a decline of arginase-1-positive macrophages, showing no generation of tumor-associated macrophages. In conclusion, Hsp70-containing EVs generated by specifically treated cancer cells give a full-scale and effective pattern of anti-tumor immune responses.
Asunto(s)
Inmunidad Adaptativa , Vesículas Extracelulares , Proteínas HSP70 de Choque Térmico/farmacología , Animales , Carcinoma/inmunología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Células HEK293 , Humanos , Células Asesinas Naturales/inmunología , Melanoma Experimental/inmunología , RatonesRESUMEN
Temporal lobe epilepsy is a widespread chronic disorder that manifests as spontaneous seizures and is often characterized by refractoriness to drug treatment. Temporal lobe epilepsy can be caused by a primary brain injury; therefore, the prevention of epileptogenesis after a primary event is considered one of the best treatment options. However, a preventive treatment for epilepsy still does not exist. Neuroinflammation is directly involved in epileptogenesis and neurodegeneration, leading to the epileptic condition and cognitive decline. In the present study, we aimed to clarify the effect of treatment with a recombinant form of the Interleukin-1 receptor antagonist (anakinra) on epileptogenesis and behavioral impairments in rats using the lithium-pilocarpine model. We found that anakinra administration during the latent phase of the model significantly suppressed the duration and frequency of spontaneous recurrent seizures in the chronic phase. Moreover, anakinra administration prevented some behavioral impairments, including motor hyperactivity and disturbances in social interactions, during both the latent and chronic periods. Histological analysis revealed that anakinra administration decreased neuronal loss in the CA1 and CA3 areas of the hippocampus but did not prevent astro- and microgliosis. The treatment increased the expression level of the solute carrier family 1 member 2 gene (Slc1a2, encoding excitatory amino acid transporter 2 (EAAT2)) in the hippocampus, potentially leading to a neuroprotective effect. However, the increased gene expression of proinflammatory cytokine genes (Interleukin-1ß (Il1b) and tumor necrosis factor α (Tnfa)) and astroglial marker genes (glial fibrillary acidic protein (Gfap) and inositol 1,4,5-trisphosphate receptor type 2 (Itpr2)) in experimental rats was not affected by anakinra treatment. Thus, our data demonstrate that the administration of anakinra during epileptogenesis has some beneficial disease-modifying effects.
RESUMEN
The feasibility of efficient aerosol delivery of the human IL-1 receptor antagonist (IL-1Ra) for reduction of acute lung inflammation was demonstrated in a mouse model study. The therapeutic efficacy of dry powder formulations PM(2), PM(10) of IL-1Ra was studied at nonforced inhalation in an aerosol chamber using the DPI "Spinhaler". Micronized powder formulations for insufflation were produced by air-jet milling. The anti-inflammatory effect of IL-Ra preparation was assessed by differential cell counts and biochemical composition of bronchoalveolar lavage (BAL). Reactive oxygen species (ROS), metabolic parameters of BAL (pH, redox potential, total protein, and lactate), and morphological lung changes were investigated by methods of luminol-dependent chemoluminescence, electrochemistry, microscopy, optical, and NMR spectroscopy. Inhalation of IL-1Ra aerosol ensured the systemic absorption of IL-1Ra in the circulatory system and reduced the acute inflammatory response to intranasal lipopolysaccharide challenge. The inhaled anti-inflammatory dosage in aerosol administration appeared to be comparable with i.p. injection. The mechanism of positive action of pulmonary aerosol delivery of Il-Ra includes normalization of the oxidative activity of bronchoalveolar cells, prevention of neutrophil recruitment to the bronchoalveolar tract, and improving of cell respiration. The results were used to develop mathematical models of the anti-inflammatory effects of IL-Ra as functions of the doses and dispersion grades of IL-Ra preparations. Aerosol application of IL-Ra may be an apparent way for prophylactic treating of respiratory inflammation caused by bacterial antigens.
Asunto(s)
Antiinflamatorios/administración & dosificación , Inflamación/tratamiento farmacológico , Proteína Antagonista del Receptor de Interleucina 1/administración & dosificación , Administración por Inhalación , Animales , Química Farmacéutica , Modelos Animales de Enfermedad , Estudios de Factibilidad , Proteína Antagonista del Receptor de Interleucina 1/antagonistas & inhibidores , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Long (D2L) and short (D2S) isoform of the D2 dopamine receptor are believed to play different roles in behavioral regulation. However, little is known about differential regulation of these isoforms mRNA expression during the process of learning in physiological and pathological states. In this study, we have investigated the combined effect of training in active avoidance (AA) paradigm and chronic early life treatment with pro-inflammatory cytokine interleukin (IL)-1ß (1µg/kg i.p., P15-21) on D2S and D2L dopamine receptor mRNA expression in the medial prefrontal cortex (mPFC) of adult rats. We have shown differential regulation of D2 short and long mRNA isoform expression in the mPFC. There was no effect of AA-training on D2S mRNA expression, while D2L mRNA was downregulated in AA-trained control (intact and saline-treated) animals, and this effect was not observed in rats treated with IL-1ß. D2S mRNA expression level negatively correlated with learning ability within control (saline-treated and intact) groups but not in IL-1ß-treated animals. Thus, prefrontal expression of distinct D2 dopamine receptor splice variants is supposed to be implicated in cognitive decline caused by early life immune challenge.
Asunto(s)
Interleucina-1beta/farmacología , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismo , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Receptores de Dopamina D2/genética , Análisis de Varianza , Animales , Animales Recién Nacidos , Reacción de Prevención/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Discapacidades para el Aprendizaje/inducido químicamente , Discapacidades para el Aprendizaje/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas WistarRESUMEN
Cerebral edema commonly accompanies brain tumors and contributes to neurologic symptoms. The role of the interleukin-1 receptor antagonist conjugated to superparamagnetic iron oxide nanoparticles (SPION-IL-1Ra) was assessed to analyze its anti-edemal effect and its possible application as a negative contrast enhancing agent for magnetic resonance imaging (MRI). Rats with intracranial C6 glioma were intravenously administered at various concentrations of IL-1Ra or SPION-IL-1Ra. Brain peritumoral edema following treatment with receptor antagonist was assessed with high-field MRI. IL-1Ra administered at later stages of tumor progression significantly reduced peritumoral edema (as measured by MRI) and prolonged two-fold the life span of comorbid animals in a dose-dependent manner in comparison to control and corticosteroid-treated animals (P < .001). Synthesized SPION-IL-1Ra conjugates had the properties of negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION-IL-1Ra nanoparticles demonstrated high intracellular incorporation and absence of toxic influence on C6 cells and lymphocyte viability and proliferation. Retention of the nanoparticles in the tumor resulted in enhanced hypotensive T2-weighted images of glioma, proving the application of the conjugates as negative magnetic resonance contrast agents. Moreover, nanoparticles reduced the peritumoral edema confirming the therapeutic potency of synthesized conjugates. SPION-IL-1Ra nanoparticles have an anti-edemal effect when administered through a clinically relevant route in animals with glioma. The SPION-IL-1Ra could be a candidate for theranostic approach in neuro-oncology both for diagnosis of brain tumors and management of peritumoral edema.
Asunto(s)
Neoplasias Encefálicas/diagnóstico , Compuestos Férricos , Glioblastoma/diagnóstico , Nanopartículas de Magnetita , Receptores de Interleucina-1/antagonistas & inhibidores , Proteínas Recombinantes/administración & dosificación , Animales , Barrera Hematoencefálica/metabolismo , Encéfalo/patología , Edema Encefálico/tratamiento farmacológico , Edema Encefálico/metabolismo , Edema Encefálico/patología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/mortalidad , Línea Celular Tumoral , Medios de Contraste , Compuestos Férricos/química , Glioblastoma/tratamiento farmacológico , Glioblastoma/mortalidad , Imagen por Resonancia Magnética , Nanopartículas de Magnetita/química , Masculino , Neoplasias Experimentales , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinéticaRESUMEN
Nanovaccines based on superparamagnetic iron oxide nanoparticles (SPIONs) provide a novel approach to induce the humoral and cell-based immune system to fight cancer. Herein, we increased the immunostimulatory capacity of SPIONs by coating them with recombinant heat shock protein 70 (Hsp70) which is known to chaperone antigenic peptides. After binding, Hsp70-SPIONs deliver immunogenic peptides from tumor lysates to dendritiÑ cells (DCs) and thus stimulate a tumor-specific, CD8+ cytotoxic T cell response. We could show that binding activity of Hsp70-SPIONs to the substrate-binding domain (SBD) is highly dependent on the ATPase activity of its nucleotide-binding domain NBD), as shown by (31)P NMR spectroscopy. Immunization of C6 glioma-bearing rats with DCs pulsed with Hsp70-SPIONs and tumor lysates resulted in a delayed tumor progression (as measured by MRI) and an increased overall survival. In parallel an increased IFNγ secretion were detected in the serum of these animals and immunohistological analysis of subsequent cryosections of the glioma revealed an enhanced infiltration of memory CD45RO+ and cytotoxic CD8+ T cells. Taken together the study demonstrates that magnetic nanocarriers such as SPIONs coated with Hsp70 can be applied as a platform for boosting anti-cancer immune responses.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Vacunas contra el Cáncer/administración & dosificación , Dextranos/administración & dosificación , Portadores de Fármacos , Glioma/tratamiento farmacológico , Proteínas HSP70 de Choque Térmico/administración & dosificación , Nanopartículas de Magnetita/administración & dosificación , Animales , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Vacunas contra el Cáncer/química , Vacunas contra el Cáncer/inmunología , Vacunas contra el Cáncer/metabolismo , Técnicas de Cocultivo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Dextranos/química , Dextranos/inmunología , Dextranos/metabolismo , Composición de Medicamentos , Glioma/sangre , Glioma/inmunología , Glioma/metabolismo , Glioma/patología , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Inmunización , Interferón gamma/sangre , Células K562 , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Imagen por Resonancia Magnética , Nanopartículas de Magnetita/química , Masculino , Melanoma Experimental , Ratones , Nanomedicina , Dominios y Motivos de Interacción de Proteínas , Espectroscopía de Protones por Resonancia Magnética , Ratas Wistar , Linfocitos T Citotóxicos/efectos de los fármacos , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Factores de Tiempo , Carga Tumoral/efectos de los fármacosRESUMEN
Superparamagnetic iron oxide nanoparticles (SPIONs) conjugated with recombinant human epidermal growth factor (SPION-EGF) were studied as a potential agent for magnetic resonance imaging contrast enhancement of malignant brain tumors. Synthesized conjugates were characterized by transmission electron microscopy, dynamic light scattering, and nuclear magnetic resonance relaxometry. The interaction of SPION-EGF conjugates with cells was analyzed in a C6 glioma cell culture. The distribution of the nanoparticles and their accumulation in tumors were assessed by magnetic resonance imaging in an orthotopic model of C6 gliomas. SPION-EGF nanosuspensions had the properties of a negative contrast agent with high coefficients of relaxation efficiency. In vitro studies of SPION-EGF nanoparticles showed high intracellular incorporation and the absence of a toxic influence on C6 cell viability and proliferation. Intravenous administration of SPION-EGF conjugates in animals provided receptor-mediated targeted delivery across the blood-brain barrier and tumor retention of the nanoparticles; this was more efficient than with unconjugated SPIONs. The accumulation of conjugates in the glioma was revealed as hypotensive zones on T2-weighted images with a twofold reduction in T2 relaxation time in comparison to unconjugated SPIONs (P<0.001). SPION-EGF conjugates provide targeted delivery and efficient magnetic resonance contrast enhancement of EGFR-overexpressing C6 gliomas.
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Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Dextranos/administración & dosificación , Dextranos/química , Factor de Crecimiento Epidérmico/farmacocinética , Glioma/tratamiento farmacológico , Glioma/metabolismo , Nanopartículas de Magnetita/administración & dosificación , Nanopartículas de Magnetita/química , Animales , Apoptosis , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Supervivencia Celular , Dextranos/ultraestructura , Factor de Crecimiento Epidérmico/química , Factor de Crecimiento Epidérmico/genética , Glioma/patología , Nanopartículas de Magnetita/ultraestructura , Nanocápsulas/administración & dosificación , Nanocápsulas/química , Nanocápsulas/ultraestructura , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Resultado del TratamientoRESUMEN
Intratumoral injections of recombinant heat shock protein (Hsp)70 were explored for feasibility in patients with brain tumors. Patients aged 4.5-14 years with untreated newly diagnosed tumors (n=12) were enrolled. After tumor resection, five injections of recombinant Hsp70 (total 2.5 mg) were administered into the resection cavity through a catheter. Before administration of Hsp70 and after the last injection, specific immune responses to the autologous tumor lysate were evaluated using the delayed-type hypersensitivity test. Further, peripheral blood was monitored to identify possible changes in lymphocyte subpopulations, cytokine levels, and the cytolytic activity of natural killer cells. The follow-up period in this trial was 12 months. Intratumoral injections of Hsp70 were well tolerated by patients. One patient had a complete clinical response documented by radiologic findings and one patient had a partial response. A positive delayed-type hypersensitivity test was observed in three patients. In peripheral blood, there was a shift from cytokines provided by Th2 cells toward cytokines of a Th1-cell-mediated response. These data corresponded to changes in lymphocyte subpopulations. Immunosuppressive T-regulatory cell levels were also reduced after injection of Hsp70, as well as production of interleukin-10. The cytolytic activity of natural killer cells was unchanged. The present study demonstrates the feasibility of intratumoral delivery of recombinant Hsp70 in patients with cancer. Further randomized clinical trials are recommended to assess the optimum dose of the chaperone, the treatment schedule, and clinical efficacy.
RESUMEN
BACKGROUND: Superparamagnetic iron oxide nanoparticles (SPIONs), due to their unique magnetic properties, have the ability to function both as magnetic resonance (MR) contrast agents, and can be used for thermotherapy. SPIONs conjugated to the heat shock protein Hsp70 that selectively binds to the CD40 receptor present on glioma cells, could be used for MR contrast enhancement of experimental C6 glioma. METHODS: The magnetic properties of the Hsp70-SPIONs were measured by NMR relaxometry method. The uptake of nanoparticles was assessed on the C6 glioma cells by confocal and electron microscopes. The tumor selectivity of Hsp70-SPIONs being intravenously administered was analyzed in the experimental model of C6 glioma in the MRI scanner. RESULTS: Hsp70-SPIONs relaxivity corresponded to the properties of negative contrast agents with a hypointensive change of resonance signal in MR imaging. A significant accumulation of the Hsp70-SPIONs but not the non-conjugated nanoparticles was observed by confocal microscopy within C6 cells. Negative contrast tumor enhancement in the T2-weighted MR images was higher in the case of Hsp70-SPIONs in comparison to non-modified SPIONs. Histological analysis of the brain sections confirmed the retention of the Hsp70-SPIONs in the glioma tumor but not in the adjacent normal brain tissues. CONCLUSION: The study demonstrated that Hsp70-SPION conjugate intravenously administered in C6 glioma model accumulated in the tumors and enhanced the contrast of their MR images.
Asunto(s)
Neoplasias Encefálicas/metabolismo , Modelos Animales de Enfermedad , Glioma/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Nanopartículas de Magnetita/administración & dosificación , Animales , Neoplasias Encefálicas/patología , Glioma/patología , Humanos , Inyecciones Intravenosas , Imagen por Resonancia Magnética , Nanopartículas de Magnetita/química , Masculino , Microscopía Confocal , Microscopía Electrónica , Ratas , Ratas Wistar , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Tumorales CultivadasRESUMEN
Recombinant 70 kDa heat shock protein (Hsp70) is an antiapoptotic protein that has a cell protective activity in stress stimuli and thus could be a useful therapeutic agent in the management of patients with acute ischemic stroke. The neuroprotective and neurotherapeutic activity of recombinant Hsp70 was explored in a model of experimental stroke in rats. Ischemia was produced by the occlusion of the middle cerebral artery for 45 minutes. To assess its neuroprotective capacity, Hsp70, at various concentrations, was intravenously injected 20 minutes prior to ischemia. Forty-eight hours after ischemia, rats were sacrificed and brain tissue sections were stained with 2% triphenyl tetrazolium chloride. Preliminary treatment with Hsp70 significantly reduced the ischemic zone (optimal response at 2.5 mg/kg). To assess Hsp70's neurotherapeutic activity, we intravenously administered Hsp70 via the tail vein 2 hours after reperfusion (2 hours and 45 minutes after ischemia). Rats were then kept alive for 72 hours. The ischemic region was analyzed using a high-field 11 T MRI scanner. Administration of the Hsp70 decreased the infarction zone in a dose-dependent manner with an optimal (threefold) therapeutic response at 5 mg/kg. Long-term treatment of the ischemic rats with Hsp70 formulated in alginate granules with retarded release of protein further reduced the infarct volume in the brain as well as apoptotic area (annexin V staining). Due to its high neurotherapeutic potential, prolonged delivery of Hsp70 could be useful in the management of acute ischemic stroke.