X-ray reflectivity study of the heat shock protein Hsp70 interaction with an artificial cell membrane model.

Membrane-bound heat shock protein 70 (Hsp70) apart from its intracellular localization was shown to be specifically expressed on the plasma membrane surface of tumor but not normal cells. Although the association of Hsp70 with lipid membranes is well documented the exact mechanisms for chaperone membrane anchoring have not been fully elucidated. Herein, we addressed the question of how Hsp70 interacts with negatively charged phospholipids in artificial lipid compositions employing the X-ray reflectivity (XRR) studies. In a first step, the interactions between dioleoylphosphatidylcholine (DOPC) in the presence or absence of dioleoylphosphatidylserine (DOPS) and Hsp70 had been assessed using Quartz crystal microbalance measurements, suggesting that Hsp70 adsorbs to the surface of DOPC/DOPS bilayer. Atomic force microscopy (AFM) imaging demonstrated that the presence of DOPS is required for stabilization of the lipid bilayer. The interaction of Hsp70 with DOPC/DOPS lipid compositions was further quantitatively determined by high energy X-ray reflectivity. A systematic characterization of the chaperone-lipid membrane interactions by various techniques revealed that artificial membranes can be stabilized by the electrostatic interaction of anionic DOPS lipids with Hsp70.

A new look at Hsp70 activity in phosphatidylserine-enriched membranes: chaperone-induced quasi-interdigitated lipid phase.

70 kDa heat shock protein Hsp70 (also termed HSP70A1A) is the major stress-inducible member of the HSP70 chaperone family, which is present on the plasma membranes of various tumor cells, but not on the membranes of the corresponding normal cells. The exact mechanisms of Hsp70 anchoring in the membrane and its membrane-related functions are still under debate, since the protein does not contain consensus signal sequence responsible for translocation from the cytosol to the lipid bilayer. The present study was focused on the analysis of the interaction of recombinant human Hsp70 with the model phospholipid membranes. We have confirmed that Hsp70 has strong specificity toward membranes composed of negatively charged phosphatidylserine (PS), compared to neutral phosphatidylcholine membranes. Using differential scanning calorimetry, we have shown for the first time that Hsp70 affects the thermotropic behavior of saturated PS and leads to the interdigitation that controls membrane thickness and rigidity. Hsp70-PS interaction depended on the lipid phase state; the protein stabilized ordered domains enriched with high-melting PS, increasing their area, probably due to formation of quasi-interdigitated phase. Moreover, the ability of Hsp70 to form ion-permeable pores in PS membranes may also be determined by the bilayer thickness. These observations contribute to a better understanding of Hsp70-PS interaction and biological functions of membrane-bound Hsp70 in cancer cells.

Magnetic Relaxation Switching Assay Using IFNα-2b-Conjugated Superparamagnetic Nanoparticles for Anti-Interferon Antibody Detection.

Type I interferons, particularly IFNα-2b, play essential roles in eliciting adaptive and innate immune responses, being implicated in the pathogenesis of various diseases, including cancer, and autoimmune and infectious diseases. Therefore, the development of a highly sensitive platform for analysis of either IFNα-2b or anti-IFNα-2b antibodies is of high importance to improve the diagnosis of various pathologies associated with the IFNα-2b disbalance. For evaluation of the anti-IFNα-2b antibody level, we have synthesized superparamagnetic iron oxide nanoparticles (SPIONs) coupled with the recombinant human IFNα-2b protein (SPIONs@IFNα-2b). Employing a magnetic relaxation switching assay (MRSw)-based nanosensor, we detected picomolar concentrations (0.36 pg/mL) of anti-INFα-2b antibodies. The high sensitivity of the real-time antibodies' detection was ensured by the specificity of immune responses and the maintenance of resonance conditions for water spins by choosing a high-frequency filling of short radio-frequency pulses of the generator. The formation of a complex of the SPIONs@IFNα-2b nanoparticles with the anti-INFα-2b antibodies led to a cascade process of the formation of nanoparticle clusters, which was further enhanced by exposure to a strong (7.1 T) homogenous magnetic field. Obtained magnetic conjugates exhibited high negative MR contrast-enhancing properties (as shown by NMR studies) that were also preserved when particles were administered in vivo. Thus, we observed a 1.2-fold decrease of the T2 relaxation time in the liver following administration of magnetic conjugates as compared to the control. In conclusion, the developed MRSw assay based on SPIONs@IFNα-2b nanoparticles represents an alternative immunological probe for the estimation of anti-IFNα-2b antibodies that could be further employed in clinical studies.

A novel method for purification of biologically active C-terminal fragment of human recombinant anti-mullerian hormone.

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.

Hsp70-containing extracellular vesicles are capable of activating of adaptive immunity in models of mouse melanoma and colon carcinoma.

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.

Anakinra Reduces Epileptogenesis, Provides Neuroprotection, and Attenuates Behavioral Impairments in Rats in the Lithium-Pilocarpine Model of Epilepsy.

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.

Epitope specificity of two anti-morphine monoclonal antibodies: In vitro and in silico studies.

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.

Purification of human recombinant anti-mullerian hormone and its derivatives.

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.

Extracellular Hsp70 Reduces the Pro-Tumor Capacity of Monocytes/Macrophages Co-Cultivated with Cancer Cells.

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.

Ex vivo Hsp70-Activated NK Cells in Combination With PD-1 Inhibition Significantly Increase Overall Survival in Preclinical Models of Glioblastoma and Lung Cancer.

Heat shock protein 70 (Hsp70) which is expressed on the plasma membrane of highly aggressive tumors including non-small cell lung carcinoma and glioblastoma multiforme serves as a target for Hsp70-targeting NK cells. Herein, we aimed to investigate the antitumor effects of a combined therapy consisting of ex vivo Hsp70-peptide TKD/IL-2-activated NK cells in combination with mouse/human anti-PD-1 antibody in a syngeneic glioblastoma and a xenograft lung cancer mouse model. Mice with membrane Hsp70 positive syngeneic GL261 glioblastoma or human xenograft A549 lung tumors were sham-treated with PBS or injected with ex vivo TKD/IL-2-activated mouse/human NK cells and mouse/human PD-1 antibody either as a single regimen or in combination. Tumor volume was assessed by MR scanning and tumor-infiltrating CD8+ T, NK, and PD-1+ cells were quantified by immunohistochemistry (IHC). We could show that the adoptive transfer of ex vivo TKD/IL-2-activated mouse NK cells or the inhibition of PD-1 resulted in tumor growth delay and an improved overall survival (OS) in a syngeneic glioblastoma mouse model. A combination of both therapies was well-tolerated and significantly more effective with respect to both outcome parameters than either of the single regimens. A combined treatment in a xenograft lung cancer model showed identical effects in immunodeficient mice bearing human lung cancer after adoptive transfer of TKD/IL-2-activated human effector cells and a human PD-1 antibody. Tumor control was associated with a massive infiltration with CD8+ T and NK cells in both tumor models and a decreased in PD-1 expression on immune effector cells. In summary, a combined approach consisting of activated NK cells and anti-PD-1 therapy is safe and results in a long-term tumor control which is accompanied by a massive tumor immune cell infiltration in 2 preclinical tumor models.

Developmental prefrontal mRNA expression of D2 dopamine receptor splice variants and working memory impairments in rats after early life Interleukin-1ß elevation.

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 µg/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.

Prefrontal mRNA expression of long and short isoforms of D2 dopamine receptor: Possible role in delayed learning deficit caused by early life interleukin-1ß treatment.

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.

70-kDa heat shock protein coated magnetic nanocarriers as a nanovaccine for induction of anti-tumor immune response in experimental glioma.

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 dendritiс 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.

Recombinant interleukin-1 receptor antagonist conjugated to superparamagnetic iron oxide nanoparticles for theranostic targeting of experimental glioblastoma.

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.

Pilot study of intratumoral injection of recombinant heat shock protein 70 in the treatment of malignant brain tumors in children.

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.

Neurotherapeutic activity of the recombinant heat shock protein Hsp70 in a model of focal cerebral ischemia in rats.

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.

Effective immunotherapy of rat glioblastoma with prolonged intratumoral delivery of exogenous heat shock protein Hsp70.

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.

Superparamagnetic iron oxide nanoparticles conjugated with epidermal growth factor (SPION-EGF) for targeting brain tumors.

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.

Tumor targeting using magnetic nanoparticle Hsp70 conjugate in a model of C6 glioma.

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.

Modified low density lipoprotein stimulates complement C3 expression and secretion via liver X receptor and Toll-like receptor 4 activation in human macrophages.

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.