Multidrug-Loaded Lipid Nanoemulsions for the Combinatorial Treatment of Cerebral Cavernous Malformation Disease

Cerebral cavernous malformation (CCM) or cavernoma is a major vascular disease of genetic origin, whose main phenotypes occur in the central nervous system, and is currently devoid of pharmacological therapeutic strategies. Cavernomas can remain asymptomatic during a lifetime or manifest with a wide range of symptoms, including recurrent headaches, seizures, strokes, and intracerebral hemorrhages. Loss-of-function mutations in KRIT1/CCM1 are responsible for more than 50% of all familial cases, and have been clearly shown to affect cellular junctions, redox homeostasis, inflammatory responses, and angiogenesis. In this study, we investigated the therapeutic effects of multidrug-loaded lipid nanoemulsions in rescuing the pathological phenotype of CCM disease. The pro-autophagic rapamycin, antioxidant avenanthramide, and antiangiogenic bevacizumab were loaded into nanoemulsions, with the aim of reducing the major molecular dysfunctions associated with cavernomas. Through Western blot analysis of biomarkers in an in vitro CCM model, we demonstrated that drug-loaded lipid nanoemulsions rescue antioxidant responses, reactivate autophagy, and reduce the effect of pro-angiogenic factors better than the free drugs. Our results show the importance of developing a combinatorial preventive and therapeutic approach to reduce the risk of lesion formation and inhibit or completely revert the multiple hallmarks that characterize the pathogenesis and progression of cavernomas

Multidrug-Loaded Lipid Nanoemulsions for the Combinatorial Treatment of Cerebral Cavernous Malformation Disease

Cerebral cavernous malformation (CCM) or cavernoma is a major vascular disease of genetic origin, whose main phenotypes occur in the central nervous system, and is currently devoid of pharmacological therapeutic strategies. Cavernomas can remain asymptomatic during a lifetime or manifest with a wide range of symptoms, including recurrent headaches, seizures, strokes, and intracerebral hemorrhages. Loss-of-function mutations in KRIT1/CCM1 are responsible for more than 50% of all familial cases, and have been clearly shown to affect cellular junctions, redox homeostasis, inflammatory responses, and angiogenesis. In this study, we investigated the therapeutic effects of multidrug-loaded lipid nanoemulsions in rescuing the pathological phenotype of CCM disease. The pro-autophagic rapamycin, antioxidant avenanthramide, and antiangiogenic bevacizumab were loaded into nanoemulsions, with the aim of reducing the major molecular dysfunctions associated with cavernomas. Through Western blot analysis of biomarkers in an in vitro CCM model, we demonstrated that drug-loaded lipid nanoemulsions rescue antioxidant responses, reactivate autophagy, and reduce the effect of pro-angiogenic factors better than the free drugs. Our results show the importance of developing a combinatorial preventive and therapeutic approach to reduce the risk of lesion formation and inhibit or completely revert the multiple hallmarks that characterize the pathogenesis and progression of cavernomas

Alendronate-Grafted Nanoemulsions for Bone-Targeted Vincristine Delivery: Preliminary Studies on Cell and Animal Models

Bone is a site of distant metastases, which are a common cause of morbidity and mortality with a high socio-economic impact, for many malignant tumours. In order to engineer pharmacological therapies that are suitable for this debilitating disease, this experimental work presents injectable lipid nanoemulsions, which are endowed with a long history of safe clinical usage in parenteral nutrition, their loading with vincristine and their grafting with alendronate, with a dual purpose: merging the anticancer activity of bisphosphonates and vincristine, and enhancing bone-targeted delivery. In cell studies, alendronate synergised with the anti-migration activity of vincristine, which is important as migration plays a key role in the metastatisation process. In preliminary animal studies, carried out thanks to IVIS technology, alendronate conjugation enhanced the bone targeting of fluorescently labelled nanoemulsions. These encouraging results will drive further studies on suitable animal models of the disease

Parenteral Nanoemulsions Loaded with Combined Immuno- and Chemo-Therapy for Melanoma Treatment

High-grade melanoma remains a major life-threatening illness despite the improvement in therapeutic control that has been achieved by means of targeted therapies and immunotherapies in recent years. This work presents a preclinical-level test of a multi-pronged approach that includes the loading of immunotherapeutic (ICOS-Fc), targeted (sorafenib), and chemotherapeutic (temozolomide) agents within Intralipid®, which is a biocompatible nanoemulsion with a long history of safe clinical use for total parenteral nutrition. This drug combination has been shown to inhibit tumor growth and angiogenesis with the involvement of the immune system, and a key role is played by ICOS-Fc. The inhibition of tumor growth in subcutaneous melanoma mouse models has been achieved using sub-therapeutic drug doses, which is most likely the result of the nanoemulsion’s targeting properties. If translated to the human setting, this approach should therefore allow therapeutic efficacy to be achieved without increasing the risk of toxic effects

Characterization of Specific Immune Responses to Different Aspergillus Antigens during the Course of Invasive Aspergillosis in Hematologic Patients

Several studies in mouse model of invasive aspergillosis (IA) and in healthy donors have shown that different Aspergillus antigens may stimulate different adaptive immune responses. However, the occurrence of Aspergillus-specific T cells have not yet been reported in patients with the disease. In patients with IA, we have investigated during the infection: a) whether and how specific T-cell responses to different Aspergillus antigens occur and develop; b) which antigens elicit the highest frequencies of protective immune responses and, c) whether such protective T cells could be expanded ex-vivo. Forty hematologic patients have been studied, including 22 patients with IA and 18 controls. Specific T cells producing IL-10, IFN-\u3b3, IL-4 and IL-17A have been characterized through enzyme linked immunospot and cytokine secretion assays on 88 peripheral blood (PB) samples, by using the following recombinant antigens: GEL1p, CRF1p, PEP1p, SOD1p, \u3b11-3glucan, \u3b21-3glucan, galactomannan. Specific T cells were expanded through short term culture. Aspergillus-specific T cells producing non-protective interleukin-10 (IL-10) and protective interferon-gamma (IFN-\u3b3) have been detected to all the antigens only in IA patients. Lower numbers of specific T cells producing IL-4 and IL-17A have also been shown. Protective T cells targeted predominantly Aspergillus cell wall antigens, tended to increase during the IA course and to be associated with a better clinical outcome. Aspergillus-specific T cells could be successfully generated from the PB of 8 out of 8 patients with IA and included cytotoxic subsets able to lyse Aspergillus hyphae. Aspergillus specific T-cell responses contribute to the clearance of the pathogen in immunosuppressed patients with IA and Aspergillus cell wall antigens are those mainly targeted by protective immune responses. Cytotoxic specific T cells can be expanded from immunosuppressed patients even during the infection by using the above mentioned antigens. These findings may be exploited for immunotherapeutic purposes in patients with I

An Isolated Stellar-mass Black Hole Detected through Astrometric Microlensing

We report the first unambiguous detection and mass measurement of an isolated stellar-mass black hole (BH). We used the Hubble Space Telescope (HST) to carry out precise astrometry of the source star of the long-duration (tE ≃ 270 days), high-magnification microlensing event MOA-2011-BLG-191/OGLE-2011-BLG-0462 (hereafter designated as MOA-11-191/OGLE-11-462), in the direction of the Galactic bulge. HST imaging, conducted at eight epochs over an interval of 6 yr, reveals a clear relativistic astrometric deflection of the background star's apparent position. Ground-based photometry of MOA-11-191/OGLE-11-462 shows a parallactic signature of the effect of Earth's motion on the microlensing light curve. Combining the HST astrometry with the ground-based light curve and the derived parallax, we obtain a lens mass of 7.1 ± 1.3 M⊙ and a distance of 1.58 ± 0.18 kpc. We show that the lens emits no detectable light, which, along with having a mass higher than is possible for a white dwarf or neutron star, confirms its BH nature. Our analysis also provides an absolute proper motion for the BH. The proper motion is offset from the mean motion of Galactic disk stars at similar distances by an amount corresponding to a transverse space velocity of ∼45 km s−1, suggesting that the BH received a "natal kick" from its supernova explosion. Previous mass determinations for stellar-mass BHs have come from radial velocity measurements of Galactic X-ray binaries and from gravitational radiation emitted by merging BHs in binary systems in external galaxies. Our mass measurement is the first for an isolated stellar-mass BH using any technique

An Isolated Stellar-mass Black Hole Detected through Astrometric Microlensing*

We report the first unambiguous detection and mass measurement of an isolated stellar-mass black hole (BH). We used the Hubble Space Telescope (HST) to carry out precise astrometry of the source star of the long-duration (t (E) similar or equal to 270 days), high-magnification microlensing event MOA-2011-BLG-191/OGLE-2011-BLG-0462 (hereafter designated as MOA-11-191/OGLE-11-462), in the direction of the Galactic bulge. HST imaging, conducted at eight epochs over an interval of 6 yr, reveals a clear relativistic astrometric deflection of the background star's apparent position. Ground-based photometry of MOA-11-191/OGLE-11-462 shows a parallactic signature of the effect of Earth's motion on the microlensing light curve. Combining the HST astrometry with the ground-based light curve and the derived parallax, we obtain a lens mass of 7.1 +/- 1.3 M (circle dot) and a distance of 1.58 +/- 0.18 kpc. We show that the lens emits no detectable light, which, along with having a mass higher than is possible for a white dwarf or neutron star, confirms its BH nature. Our analysis also provides an absolute proper motion for the BH. The proper motion is offset from the mean motion of Galactic disk stars at similar distances by an amount corresponding to a transverse space velocity of similar to 45 km s(-1), suggesting that the BH received a "natal kick" from its supernova explosion. Previous mass determinations for stellar-mass BHs have come from radial velocity measurements of Galactic X-ray binaries and from gravitational radiation emitted by merging BHs in binary systems in external galaxies. Our mass measurement is the first for an isolated stellar-mass BH using any technique