The presence of circulating human apolipoprotein J reduces the occurrence of cerebral microbleeds in a transgenic mouse model with cerebral amyloid angiopathy.

BACKGROUND: Cerebral amyloid angiopathy (CAA) is characterized by amyloid-ß (Aß) deposition in cerebral vessels, leading to lobar cerebral microbleeds (CMB) and intracerebral hemorrhages (ICH). Apolipoprotein J (ApoJ) is a multifunctional chaperone related to Aß aggregation and clearance. Our study investigated the vascular impact of chronic recombinant human Apolipoprotein J (rhApoJ) treatment in a transgenic mouse model of ß-amyloidosis with prominent CAA. METHODS: Twenty-month-old APP23 C57BL/6 mice received 25 doses of rhApoJ (1 mg/kg) (n = 9) or saline (n = 8) intraperitoneally for 13 weeks, while Wild-type (WT) mice received saline (n = 13). Postmortem brains underwent T2*-weighted magnetic resonance imaging (MRI) to detect hemorrhagic lesions. Aß levels and distribution, cerebral fibrinogen leakage, brain smooth muscle actin (sma), and plasma matrix metalloproteinases and inflammatory markers were analyzed after treatments. Additionally, plasma samples from 22 patients with lobar ICH were examined to determine the clinical relevance of the preclinical findings. RESULTS: rhApoJ-treated APP23 presented fewer cortical CMBs (50-300 µm diameter) (p = 0.012) and cortical larger hemorrhages (> 300 µm) (p = 0.002) than saline-treated mice, independently of Aß brain levels. MRI-detected hemorrhagic lesions correlated with fibrinogen cerebral extravasation (p = 0.011). Additionally, rhApoJ-treated mice presented higher number of sma-positive vessels than saline-treated mice (p = 0.038). In rhApoJ-treated mice, human ApoJ was detected in plasma and in occasional leptomeningeal vessels, but not in the parenchyma, suggesting that its mechanism of action operates through the periphery. The administration of rhApoJ induced an increase in plasma Groα (p = 0.035) and MIP-1α (p = 0.035) levels, while lower MMP-12 (p = 0.046) levels, compared to the saline-treated group. In acute lobar ICH patients, MMP-12 plasma levels correlated with larger hemorrhage volume (p = 0.040) and irregular ICH shape (p = 0.036). CONCLUSIONS: Chronic rhApoJ treatment in aged APP23 mice ameliorated CAA-related neurovascular damage by reducing the occurrence of CMB. We propose that rhApoJ may prevent blood-brain barrier (BBB) leakage and CMB appearance partly through circulating MMP-12 modulation.

Efficient Tumor Eradication at Ultralow Drug Concentration via Externally Controlled and Boosted Metallic Iron Magnetoplasmonic Nanocapsules.

With the aim to locally enhance the efficacy of cancer nanotherapies, here we present metal iron based magnetoplasmonic drug-loaded nanocapsules (MAPSULES), merging powerful external magnetic concentration in the tumor and efficient photothermal actuation to locally boost the drug therapeutic action at ultralow drug concentrations. The MAPSULES are composed of paclitaxel-loaded polylactic-co-glycolic acid (PLGA) nanoparticles partially coated by a nanodome shape iron/silica semishell. The iron semishell has been designed to present a ferromagnetic vortex for incorporating a large quantity of ferromagnetic material while maintaining high colloidal stability. The large iron semishell provides very strong magnetic manipulation via magnetophoretic forces, enabling over 10-fold higher trapping efficiency in microfluidic channels than typical superparamagnetic iron oxide nanoparticles. Moreover, the iron semishell exhibits highly damped plasmonic behavior, yielding intense broadband absorbance in the near-infrared biological windows and photothermal efficiency similar to the best plasmonic nanoheaters. The in vivo therapeutic assays in a mouse xenograft tumor model show a high amplification of the therapeutic effects by combining magnetic concentration and photothermal actuation in the tumor, leading to a complete eradication of the tumors at ultralow nanoparticle and drug concentration (equivalent to only 1 mg/kg PLGA nanoparticles containing 8 µg/kg of paclitaxel, i.e., 100-500-fold lower than the therapeutic window of the free and PLGA encapsulated drug and 13-3000-fold lower than current nanotherapies combining paclitaxel and light actuation). These results highlight the strength of this externally controlled and amplified therapeutic approach, which could be applied to locally boost a wide variety of drugs for different diseases.

Metal-Free Radical Dendrimers as MRI Contrast Agents for Glioblastoma Diagnosis: Ex Vivo and In Vivo Approaches.

Simultaneously being a nonradiative and noninvasive technique makes magnetic resonance imaging (MRI) one of the highly required imaging approaches for the early diagnosis and follow-up of tumors, specifically for brain cancer. Paramagnetic gadolinium (Gd)-based contrast agents (CAs) are the most widely used ones in brain MRI acquisitions with special interest when assessing blood-brain barrier (BBB) integrity, a characteristic of high-grade tumors. However, alternatives to Gd-based contrast agents (CAs) are highly required to overcome their established toxicity. Organic radicals anchored on a dendrimer macromolecule surface (radical dendrimers) are promising alternatives since they also exhibit paramagnetic properties and can act as T1 CAs like Gd-based CAs while being organic species (mitigating concerns about toxic metal accumulation). Here, we studied the third generation of a water-soluble family of poly(phosphorhydrazone) radical dendrimers, with 48 PROXYL radical units anchored on their branches, exploring their potential of ex vivo and in vivo contrast enhancement in brain tumors (in particular, of immunocompetent, orthotopic GL261 murine glioblastoma (GB)). Remarkably, this radical species provides suitable contrast enhancement on murine GL261 GB tumors, which was comparable to that of commercial Gd-based CAs (at standard dose 0.1 mmol/kg), even at its 4 times lower administered dose (0.025 mmol/kg). Importantly, no signs of toxicity were detected in vivo. In addition, it showed a selective accumulation in brain tumor tissues, exhibiting longer retention within the tumor, which allows performing imaging acquisition over longer time frames (≥2.5 h) as opposed to Gd chelates. Finally, we observed high stability of the radicals in biological media, on the order of hours instead of minutes, characteristic of the isolated radicals. All of these features allow us to suggest that the G3-Tyr-PROXYL-ONa radical dendrimer could be a viable alternative to metal-based MRI contrast agents, particularly on MRI analysis of GB, representing, to the best of our knowledge, the first case of organic radical species used for this purpose and one of the very few examples of these types of radical species working as MRI CAs in vivo.

Efficacy of adeno-associated virus gene therapy in a MNGIE murine model enhanced by chronic exposure to nucleosides.

BACKGROUND: Preclinical studies have shown that gene therapy is a feasible approach to treat mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). However, the genetic murine model of the disease (Tymp/Upp1 double knockout, dKO) has a limited functional phenotype beyond the metabolic imbalances, and so the studies showing efficacy of gene therapy have relied almost exclusively on demonstrating correction of the biochemical phenotype. Chronic oral administration of thymidine (dThd) and deoxyuridine (dUrd) to dKO mice deteriorates the phenotype of the animals, providing a better model to test therapy approaches. METHODS: dKO mice were treated with both dThd and dUrd in drinking water from weaning until the end of the study. At 8 - 11 weeks of age, mice were treated with several doses of adeno-associated virus (AAV) serotype 8 vector carrying the human TYMP coding sequence under the control of different liver-specific promoters (TBG, AAT, or HLP). The biochemical profile and functional phenotype were studied over the life of the animals. FINDINGS: Nucleoside exposure resulted in 30-fold higher plasma nucleoside levels in dKO mice compared with non-exposed wild type mice. AAV-treatment provided elevated TP activity in liver and lowered systemic nucleoside levels in exposed dKO mice. Exposed dKO mice had enlarged brain ventricles (assessed by magnetic resonance imaging) and motor impairment (rotarod test); both were prevented by AAV treatment. Among all promoters tested, AAT showed the best efficacy. INTERPRETATION: Our results show that AAV-mediated gene therapy restores the biochemical homeostasis in the murine model of MNGIE and, for the first time, demonstrate that this treatment improves the functional phenotype. FUNDING: This work was funded in part by the Spanish Instituto de Salud Carlos III, and the Generalitat de Catalunya. The disclosed funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Microwave-Assisted Synthesis of SPION-Reduced Graphene Oxide Hybrids for Magnetic Resonance Imaging (MRI).

Magnetic resonance imaging (MRI) is a useful tool for disease diagnosis and treatment monitoring. Superparamagnetic iron oxide nanoparticles (SPION) show good performance as transverse relaxation (T2) contrast agents, thus facilitating the interpretation of the acquired images. Attachment of SPION onto nanocarriers prevents their agglomeration, improving the circulation time and efficiency. Graphene derivatives, such as graphene oxide (GO) and reduced graphene oxide (RGO), are appealing nanocarriers since they have both high surface area and functional moieties that make them ideal substrates for the attachment of nanoparticles. We have employed a fast, simple and environmentally friendly microwave-assisted approach for the synthesis of SPION-RGO hybrids. Different iron precursor/GO ratios were used leading to SPION, with a median diameter of 7.1 nm, homogeneously distributed along the RGO surface. Good relaxivity (r2*) values were obtained in MRI studies and no significant toxicity was detected within in vitro tests following GL261 glioma and J774 macrophage-like cells for 24 h with SPION-RGO, demonstrating the applicability of the hybrids as T2-weighted MRI contrast agents.

Differential effects of apoE and apoJ mimetic peptides on the action of an anti-Aß scFv in 3xTg-AD mice.

Anti-Aß immunotherapy has emerged as a promising approach to treat Alzheimer's disease (AD). The single-chain variable fragment scFv-h3D6 is an anti-Aß antibody fragment that lacks the Fc region, which is associated with the induction of microglial reactivity by the full-length monoclonal antibody bapineuzumab. ScFv-h3D6 was previously shown to restore the levels of apolipoprotein E (apoE) and apolipoprotein J (apoJ) in a triple-transgenic-AD (3xTg-AD) mouse model. Since apoE and apoJ play an important role in the development of AD, we aimed to study the in vivo effect of the combined therapy of scFv-h3D6 with apoE and apoJ mimetic peptides (MPs). Four-and-a-half-month-old 3xTg-AD mice were treated for six weeks with scFv-h3D6, apoE-MP, apoJ-MP, or a combination of scFv-h3D6 with each of the MPs, or a vehicle, and then the results were compared to non-transgenic mice. Magnetic Resonance Imaging showed a general tendency of the different treatments to protect against the reduction in brain volume. Aß burden decreased after treatment with scFv-h3D6, apoE-MP, or apoJ-MP, but the effect was not as evident with the combined therapies. In terms of glial reactivity, apoE-MP showed a potent anti-inflammatory effect that was eased by the presence of scFv-h3D6, whereas the combination of apoJ-MP and scFv-h3D6 was not detrimental. ScFv-h3D6 alone did not induce microglial reactivity, as full-length antibodies do; rather, it reduced it. Endogenous apoE and apoJ levels were decreased by scFv-h3D6, but the MPs lead to a simultaneous increase of both apolipoproteins. While apoE-MP and apoJ-MP demonstrated different effects in the combined therapies with scFv-h3D6, they did not improve the overall protective effect of scFv-h3D6 in reducing the Aß burden, apolipoproteins levels or microglial reactivity.

Assessment of biodistribution using mesenchymal stromal cells: Algorithm for study design and challenges in detection methodologies.

BACKGROUND AIMS: Biodistribution of candidate cell-based therapeutics is a critical safety concern that must be addressed in the preclinical development program. We aimed to design a decision tree based on a series of studies included in actual dossiers approved by competent regulatory authorities, noting that the design, execution and interpretation of pharmacokinetics studies using this type of therapy is not straightforward and presents a challenge for both developers and regulators. METHODS: Eight studies were evaluated for the definition of a decision tree, in which mesenchymal stromal cells (MSCs) were administered to mouse, rat and sheep models using diverse routes (local or systemic), cell labeling (chemical or genetic) and detection methodologies (polymerase chain reaction [PCR], immunohistochemistry [IHC], fluorescence bioimaging, and magnetic resonance imaging [MRI]). Moreover, labeling and detection methodologies were compared in terms of cost, throughput, speed, sensitivity and specificity. RESULTS: A decision tree was defined based on the model chosen: (i) small immunodeficient animals receiving heterologous MSC products for assessing biodistribution and other safety aspects and (ii) large animals receiving homologous labeled products; this contributed to gathering data not only on biodistribution but also on pharmacodynamics. PCR emerged as the most convenient technique despite the loss of spatial information on cell distribution that can be further assessed by IHC. DISCUSSION: This work contributes to the standardization in the design of biodistribution studies by improving methods for accurate assessment of safety. The evaluation of different animal models and screening of target organs through a combination of techniques is a cost-effective and timely strategy.

Metronomic treatment in immunocompetent preclinical GL261 glioblastoma: effects of cyclophosphamide and temozolomide.

Glioblastoma (GBM) causes poor survival in patients even when applying aggressive treatment. Temozolomide (TMZ) is the standard chemotherapeutic choice for GBM treatment, but resistance always ensues. In previous years, efforts have focused on new therapeutic regimens with conventional drugs to activate immune responses that may enhance tumor regression and prevent regrowth, for example the "metronomic" approaches. In metronomic scheduling studies, cyclophosphamide (CPA) in GL261 GBM growing subcutaneously in C57BL/6 mice was shown not only to activate antitumor CD8+ T-cell response, but also to induce long-term specific T-cell tumor memory. Accordingly, we have evaluated whether metronomic CPA or TMZ administration could increase survival in orthotopic GL261 in C57BL/6 mice, an immunocompetent model. Longitudinal in vivo studies with CPA (140 mg/kg) or TMZ (range 140-240 mg/kg) metronomic administration (every 6 days) were performed in tumor-bearing mice. Tumor evolution was monitored at 7 T with MRI (T2 -weighted, diffusion-weighted imaging) and MRSI-based nosological images of response to therapy. Obtained results demonstrated that both treatments resulted in increased survival (38.6 ± 21.0 days, n = 30) compared with control (19.4 ± 2.4 days, n = 18). Best results were obtained with 140 mg/kg TMZ (treated, 44.9 ± 29.0 days, n = 12, versus control, 19.3 ± 2.3 days, n = 12), achieving a longer survival rate than previous group work using three cycles of TMZ therapy at 60 mg/kg (33.9 ± 11.7 days, n = 38). Additional interesting findings were, first, clear edema appearance during chemotherapeutic treatment, second, the ability to apply the semi-supervised source analysis previously developed in our group for non-invasive TMZ therapy response monitoring to detect CPA-induced response, and third, the necropsy findings in mice cured from GBM after high TMZ cumulative dosage (980-1400 mg/kg), which demonstrated lymphoma incidence. In summary, every 6 day administration schedule of TMZ or CPA improves survival in orthotopic GL261 GBM with respect to controls or non-metronomic therapy, in partial agreement with previous work on subcutaneous GL261.

Metabolomics of Therapy Response in Preclinical Glioblastoma: A Multi-Slice MRSI-Based Volumetric Analysis for Noninvasive Assessment of Temozolomide Treatment.

Glioblastoma (GBM) is the most common aggressive primary brain tumor in adults, with a short survival time even after aggressive therapy. Non-invasive surrogate biomarkers of therapy response may be relevant for improving patient survival. Previous work produced such biomarkers in preclinical GBM using semi-supervised source extraction and single-slice Magnetic Resonance Spectroscopic Imaging (MRSI). Nevertheless, GBMs are heterogeneous and single-slice studies could prevent obtaining relevant information. The purpose of this work was to evaluate whether a multi-slice MRSI approach, acquiring consecutive grids across the tumor, is feasible for preclinical models and may produce additional insight into therapy response. Nosological images were analyzed pixel-by-pixel and a relative responding volume, the Tumor Responding Index (TRI), was defined to quantify response. Heterogeneous response levels were observed and treated animals were ascribed to three arbitrary predefined groups: high response (HR, n = 2), TRI = 68.2 ± 2.8%, intermediate response (IR, n = 6), TRI = 41.1 ± 4.2% and low response (LR, n = 2), TRI = 13.4 ± 14.3%, producing therapy response categorization which had not been fully registered in single-slice studies. Results agreed with the multi-slice approach being feasible and producing an inverse correlation between TRI and Ki67 immunostaining. Additionally, ca. 7-day oscillations of TRI were observed, suggesting that host immune system activation in response to treatment could contribute to the responding patterns detected.

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study.

BACKGROUND: Magnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs. RESULTS: The post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP. CONCLUSION: We have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.

Efficient γ-amino-proline-derived cell penetrating peptide-superparamagnetic iron oxide nanoparticle conjugates via aniline-catalyzed oxime chemistry as bimodal imaging nanoagents.

Aniline-catalyzed oxime chemistry was employed to conjugate a γ-amino-proline-derived cell penetrating peptide to superparamagnetic iron oxide nanoparticles (SPIONs). Internalization of the novel nanoconjugate into HeLa cells was found to be remarkably higher compared to the analogous TAT-SPION conjugate.

Self-assembled multifunctional Fe/MgO nanospheres for magnetic resonance imaging and hyperthermia.

A one-step process for the production of nanoparticles presenting advanced magnetic properties can be achieved using vapor condensation. In this article, we report on the fabrication of Fe particles covered by a uniform MgO epitaxial shell. MgO has a lower surface energy than Fe, which results in a core-shell crystal formation. The particles satisfy a few of technical requirements for the practical use in real clinics, such as a high biocompatibility in living cells in-vitro, an injection through blood vessels without any clothing problems in murine model, a high absorption rate for magnetic hyperthermia at small particle concentration, and the potential to be used as contrast agent in the field of diagnostic magnetic imaging. They are also able to be used in drug delivery and magnetic-activated cell sorting. FROM THE CLINICAL EDITOR: In this paper, the authors report on the synthesis of Fe particles covered by a uniform MgO epitaxial shell resulting in a core-shell crystal formation. The particles are proven to be useful as contrast agents for magnetic resonance imaging and have the potential to be useful as heating mediators for cancer therapy through hyperthermia. They also might be used in drug delivery and magnetic-activated cell sorting.

Longitudinal diffusion tensor imaging in a rat brain glioma model.

In order to investigate the properties of water motion within and around brain tumors as a function of tumor growth, longitudinal diffusion tensor imaging (DTI) was carried out in a rat brain glioma (C6) model. As tumors grew in size, significant anisotropy of water diffusion was seen both within and around the tumor. The tissue water surrounding the tumor exhibited high planar anisotropy, as opposed to the linear anisotropy normally seen in white matter, indicating that cells were experiencing stress in a direction normal to the tumor border. When tumors were sufficiently large, significant anisotropy was also seen within the tumor because of longer-range organization of cancer cells within the tumor borders. These findings have important implications for diffusion-weighted MRI experiments examining tumor growth and response to therapy.

Allopregnanolone treatment, both as a single injection or repetitively, delays demyelination and enhances survival of Niemann-Pick C mice.

Niemann-Pick C disease (NPC) is an irreversible neurodegenerative disorder without current treatment. It is thought to result from deficient intracellular cholesterol and/or ganglioside trafficking. We have investigated the effects of allopregnanolone treatments on survival, weight loss, motor function, magnetic resonance imaging (MRI), and neuropathology in the mouse model of NPC (Npc1(-/-) mice). We confirmed previous results showing that a single injection of 250 microg of allopregnanolone on postnatal day 7 significantly extended the life span of Npc1(-/-) mice. This caused a marked difference in the weight curves of the treated mice but no statistical difference in the Rota-Rod performance. T2-weighted MRI and diffusion tensor imaging (DTI) of treated mice showed values of signal intensity and fractional anisotropy closer to those of wild-type mice than those of untreated Npc1(-/-) mice. Neuropathology showed that day-7 treatment markedly suppressed astrocyte reaction and significantly reduced microglial activation. Furthermore, the steroid treatment also increased myelination in brains of Npc1(-/-) mice. Similar effects of allopregnanolone treatment were observed in Npc1(-/-), mdr1a(-/-) double-mutant mice, which have a deficient blood-brain barrier, resulting in increased steroid uptake. The effects on survival and weight loss of a single injection on day 7 followed by injections every 2 weeks were also evaluated in Npc1(-/-) mice, and the beneficial effects were found to be greater than with the single injection at day 7. We conclude that allopregnanolone treatment significantly ameliorates several symptoms of NPC in Npc1(-/-) mice, presumably by effects on myelination or neuronal connectivity.

Uncovering of intracellular water in cultured cells.

The complexity of biologic tissues, with multiple compartments each with its own diffusion and relaxation properties, requires complex formalisms to model water signal in most magnetic resonance imaging or magnetic resonance spectroscopy experiments. In this article, we describe a magnetic susceptibility-induced shift in the resonance frequency of extracellular water by the introduction of a gadolinium contrast agent to medium perfusing a hollow fiber bioreactor. The frequency shift of the extracellular water (+185 Hz at 9.4 T) uncovers the intracellular water and allows direct measurement of motional and relaxation properties of the intracellular space. The proposed method provides a unique tool for understanding the mechanisms underlining diffusion and relaxation in the intracellular space.