Membrane-bound heat shock protein mHsp70 is required for migration and invasion of brain tumors.

Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n=23) and pediatric (n=9) neurooncological patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass-spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in chaperone cluster (including representatives of other families such as Hsp70, Hsc70, Hsp105, Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, myosin-9). The use of small-molecule inhibitors of HSP70 (PES, JG-98) led to a significant decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG-98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies.

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.

Granzyme B Functionalized Nanoparticles Targeting Membrane Hsp70-Positive Tumors for Multimodal Cancer Theranostics.

Functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have emerged as potential clinical tools for cancer theranostics. Membrane-bound 70 kDa heat shock protein (mHsp70) is ubiquitously expressed on the cell membrane of various tumor types but not normal cells and therefore provides a tumor-specific target. The serine protease granzyme B (GrB) that is produced as an effector molecule by activated T and NK cells has been shown to specifically target mHsp70 on tumor cells. Following binding to Hsp70, GrB is rapidly internalized into tumor cells. Herein, it is demonstrated that GrB functionalized SPIONs act as a contrast enhancement agent for magnetic resonance imaging and induce specific tumor cell apoptosis. Combinatorial regimens employing stereotactic radiotherapy and/or magnetic targeting are found to further enhance the therapeutic efficacy of GrB-SPIONs in different tumor mouse models.

Evaluation of the temporary effect of physical vapor deposition silver coating on resistance to infection in transdermal skin and bone integrated pylon with deep porosity.

Periprosthetic infection via skin-implant interface is a leading cause of failures and revisions in direct skeletal attachment of limb prostheses. Implants with deep porosity fabricated with skin and bone integrated pylons (SBIP) technology allow for skin ingrowth through the implant's structure creating natural barrier against infection. However, until the skin cells remodel in all pores of the implant, additional care is required to prevent from entering bacteria to the still nonoccupied pores. Temporary silver coating was evaluated in this work as a means to provide protection from infection immediately after implantation followed by dissolution of silver layer in few weeks. A sputtering coating with 1 µm thickness was selected to be sufficient for fighting infection until the deep ingrowth of skin in the porous structure of the pylon is completed. In vitro study showed less bacterial (Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa) growth on silver coated tablets compared to the control group. Analysis of cellular density of MG-63 cells, fibroblasts, and mesenchymal stem cells (MSCs) showed that silver coating did not inhibit the cell growth on the implants and did not affect cellular functional activity. The in vivo study did not show any postoperative complications during the 6-month observation period in the model of above-knee amputation in rabbits when SBIP implants, either silver-coated or untreated were inserted into the bone residuum. Three-phase scintigraphy demonstrated angiogenesis in the pores of the pylons. The findings suggest that a silver coating with well-chosen specifications can increase the safety of porous implants for direct skeletal attachment. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 169-177, 2019.

Detection of experimental myocardium infarction in rats by MRI using heat shock protein 70 conjugated superparamagnetic iron oxide nanoparticle.

Superparamagnetic iron-oxide based contrast agents can provide important diagnostic information regarding the assessment of cardiac inflammatory diseases. The aim of the study was to analyze whether nanoparticles conjugated to recombinant 70-kDa heat shock protein (Hsp70-SPION) can be applied for the detection of acute myocardium infarct by MRI. Cellular experiments demonstrated increased CD40-mediated uptake of Hsp70-SPIONs in comparison to non-conjugated SPIONs. Following induction of an acute infarct in rats by ligation of the left anterior descending artery SPIONs and Hsp70-SPION conjugates were injected intravenously on day 4. The animals underwent sequential MRI that showed the presence of the particles in the infarcted zone. Subsequent biodistribution analyses with the help of method on non-linear magnetic response indicated the preferential accumulation of the Hsp70-SPIONs in the heart tissue that was further confirmed with histological analyses. The study demonstrated that an acute infarct can be visualized by MRI using Hsp70-functionalized SPION conjugates. FROM THE CLINICAL EDITOR: Superparamagnetic iron oxides nanoparticles (SPIONs) have been studied extensively as a contrast agent for MRI. Their tissue specificity can be further enhanced by conjugation with various ligands. In this study, the authors conjugated superparamagnetic nanoparticles to 70-kDa heat shock protein (Hsp70-SPION) to investigate the feasibility for the detection of acute myocardium infarct. The positive findings would suggest that this approach might be used clinically in the future.

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.

Ionizing radiation improves glioma-specific targeting of superparamagnetic iron oxide nanoparticles conjugated with cmHsp70.1 monoclonal antibodies (SPION-cmHsp70.1).

The stress-inducible 72 kDa heat shock protein Hsp70 is known to be expressed on the membrane of highly aggressive tumor cells including high-grade gliomas, but not on the corresponding normal cells. Membrane Hsp70 (mHsp70) is rapidly internalized into tumor cells and thus targeting of mHsp70 might provide a promising strategy for theranostics. Superparamagnetic iron oxide nanoparticles (SPIONs) are contrast negative agents that are used for the detection of tumors with MRI. Herein, we conjugated the Hsp70-specific antibody (cmHsp70.1) which is known to recognize mHsp70 to superparamagnetic iron nanoparticles to assess tumor-specific targeting before and after ionizing irradiation. In vitro experiments demonstrated the selectivity of SPION-cmHsp70.1 conjugates to free and mHsp70 in different tumor cell types (C6 glioblastoma, K562 leukemia, HeLa cervix carcinoma) in a dose-dependent manner. High-resolution MRI (11 T) on T(2)-weighted images showed the retention of the conjugates in the C6 glioma model. Accumulation of SPION-cmHsp70.1 nanoparticles in the glioma resulted in a nearly 2-fold drop of T*(2) values in comparison to non-conjugated SPIONs. Biodistribution analysis using NLR-M(2) measurements showed a 7-fold increase in the tumor-to-background (normal brain) uptake ratio of SPION-cmHsp70.1 conjugates in glioma-bearing rats in comparison to SPIONs. This accumulation within Hsp70-positive glioma was further enhanced after a single dose (10 Gy) of ionizing radiation. Elevated accumulation of the magnetic conjugates in the tumor due to radiosensitization proves the combination of radiotherapy and application of Hsp70-targeted agents in brain tumors.

Minimally Invasive Port Access Approach for Reoperations on the Mitral Valve.

BACKGROUND: In patients requiring a second-time or more operation on the mitral valve (MV), we assessed whether the outcomes of the minimally invasive port access approach (port access group) were equivalent to those of the traditional redo sternotomy approach (redo sternotomy group). METHODS: In a retrospective review (1998-2011), 409 patients had previous MV operations requiring a second-time or more MV reintervention. Of those, 67 patients had the port access approach, and 342 had the redo sternotomy approach. Of the latter, 220 met the inclusion criteria because emergencies, patients with endocarditis, and those requiring concomitant procedures involving aortic valve and aorta were excluded. RESULTS: New York Heart Association class 2 or above, age, atrial fibrillation, and surgical indications were similar in both groups. The port access group had more patients with previous MV repair (78% [n = 52] vs 41% [n = 90], p < 0.01) than with MV replacement (19% [n = 13) vs 53% [n = 116], p < 0.01). Concomitant procedures were similar (20% [n = 14] vs 27% [n = 59], p = 0.4). The MV re-repair rates were similar (19% [n = 10] vs 22% [n = 20], p = 1). The cardiopulmonary bypass times (153 ± 42 minutes vs 172 ± 83 minutes, p = 0.07) and aortic cross-clamping times (104 ± 38 minutes versus 130 ± 71 minutes, p < 0.01) were lower in the port access group. Mortality was lower in the port access group, although not significantly (3.0% [n = 2] vs 6.0% [n = 13], p = 0.5). The rates of postoperative stroke were similar (3.0% [n = 2] vs 3.2% [n = 7], p = 1). On postoperative echocardiography, freedom from mitral regurgitation >2+ was 100% in the port access group and 99% in the redo sternotomy group. The mean hospital length of stay was 11 ± 15 days versus 14 ± 12 days (p = 0.07). CONCLUSIONS: The port access approach can be safely adopted for reoperations on the MV without compromising postoperative mortality or MV function.

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.

Changes of fractional anisotropy (FA) and apparent diffusion coefficient (ADC) in the model of experimental acute hydrocephalus in rabbits.

BACKGROUND: To study the integrity of white matter, we investigated the correlation between the changes in neuroradiological and morphological parameters in an animal model of acute obstructive hydrocephalus. METHODS: Hydrocephalus was induced in New Zealand rabbits (n = 10) by stereotactic injection of kaolin into the lateral ventricles. Control animals received saline in place of kaolin (n = 10). The progression of hydrocephalus was assessed using magnetic resonance imaging. Regional fractional anisotropy (FA) and the apparent diffusion coefficient (ADC) were measured in several white matter regions before and after the infusion of kaolin. Morphology of myelinated nerve fibers as well as of the blood-brain barrier were studied with the help of transmission electron microscopy (TEM) and light microscopy. RESULTS: Compared with control animals, kaolin injection into the ventricles resulted in a dramatic increase in ventricular volume with compression of basal cisterns, brain shift and periventricular edema (as observed on magnetic resonance imaging [MRI]). The values of ADC in the periventricular and periaqueductal areas significantly increased in the experimental group (P < 0.05). FA decreased by a factor of 2 in the zones of periventricular, periaqueductal white matter and corpus collosum. Histological analysis demonstrated the impairment of the white matter and necrobiotic changes in the cortex. Microsctructural alterations of the myelin fibers were further proved with the help of TEM. Blood-brain barrier ultrastructure assessment showed the loss of its integrity. CONCLUSIONS: The study demonstrated the correlation of the neuroradiological parameters with morphological changes. The abnormality of the FA and ADC parameters in the obstructive hydrocephalus represents a significant implication for the diagnostics and management of hydrocephalus in patients.

Subperiosteal transmission of intra-articular pressure between articulated and stationary joints.

Hydrostatic pressures can be transmitted between synovial capsules. In each of ten rabbits, we simultaneously measured pressure in two joints, one of which was passively ranged, and the other of which was kept stationary. The intra-articular pressure inside the stationary joint changed every time its companion joint was ranged. But the pressure in the stationary joint did not change when the periosteum was transected above the ranged joint. This phenomenon was observed in all four animals that served as their own controls. The study suggests that the intra-articular pressure was transmitted through the space between the periosteum and the bone surface. Alternative explanations, like measurements of venous blood pressure, did not show correlation with hydrostatic pressure changes in the joints. The Floating Skeleton concept suggests a biomechanical rationale for this newly observed phenomenon: that there exists a subperiosteal hydrostatic connection of synovial joints, and that this "net" distributes excess pressures among joints through the periosteal sheath to sustain the integrity of the joint contacting surfaces over a lifetime.

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.

Two-stage implantation of the skin- and bone-integrated pylon seeded with autologous fibroblasts induced into osteoblast differentiation for direct skeletal attachment of limb prostheses.

Angio- and osteogenesis following the two-stage (TS) implantation of the skin- and bone-integrated pylon seeded with autologous fibroblasts was evaluated. Two consecutive animal substudies were undertaken: intramedullary subcutaneous implantation (15 rabbits) and a TS transcutaneous implantation (12 rabbits). We observed enhanced osseointegrative properties of the intramedullary porous component seeded with fibroblasts induced into osteoblast differentiation, as compared to the untreated porous titanium pylon. The three-phase scintigraphy and subsequent histological analysis showed that the level of osteogenesis was 1.5-fold higher than in the control group, and significantly so (p < 0.05). The biocompatibility was further proved by the absence of inflammatory response or encapsulation and sequestration on the histology assay. Treatment of the transcutaneous component with autologous fibroblasts was associated with nearly a 2-fold decrease in the period required for the ingrowth of dermal and subdermal soft tissues into the implant surface, as compared to the untreated porous titanium component. Direct dermal attachment to the transcutaneous implant prevented superficial and deep periprosthetic infections in rabbits in vivo.

Covariance adjustments for the analysis of randomized field experiments.

BACKGROUND: It has become common practice to analyze randomized experiments using linear regression with covariates. Improved precision of treatment effect estimates is the usual motivation. In a series of important articles, David Freedman showed that this approach can be badly flawed. Recent work by Winston Lin offers partial remedies, but important problems remain. RESULTS: In this article, we address those problems through a reformulation of the Neyman causal model. We provide a practical estimator and valid standard errors for the average treatment effect. Proper generalizations to well-defined populations can follow. CONCLUSION: In most applications, the use of covariates to improve precision is not worth the trouble.