"Endothelial Antibody Factory" at the Blood Brain Barrier: Novel Approach to Therapy of Neurodegenerative Diseases.

The failures of anti-ß-amyloid immunotherapies suggested that the very low fraction of injected antibodies reaching the brain parenchyma due to the filtering effect of the BBB may be a reason for the lack of therapeutic effect. However, there is no treatment, as yet, for the amyotrophic lateral sclerosis (ALS) despite substantial evidence existing of the involvement of TDP-43 protein in the evolution of ALS. To circumvent this filtering effect, we have developed a novel approach to facilitate the penetration of antibody fragments (Fabs) into the brain parenchyma. Leveraging the homing properties of endothelial progenitor cells (EPCs), we transfected, ex vivo, such cells with vectors encoding anti-ß-amyloid and anti-TDP43 Fabs turning them into an "antibody fragment factory". When injected these cells integrate into the BBB, where they secrete anti-TDP43 Fabs. The results showed the formation of tight junctions between the injected engineered EPCs and the unlabeled resident endothelial cells. When the EPCs were further modified to express the anti-TDP43 Fab, we could observe integration of these cells into the vasculature and the secretion of Fabs. Results confirm that production and secretion of Fabs at the BBB level leads to their migration to the brain parenchyma where they might exert a therapeutic effect.

Secretion of proteins and antibody fragments from transiently transfected endothelial progenitor cells.

In neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and amyotrophic lateral sclerosis, neuroinflammation can lead to blood-brain barrier (BBB) breakdown. After intravenous or intra-arterial injection into mice, endothelial progenitor cells (EPCs) home to the damaged BBB to promote neurovascular repair. Autologous EPCs transfected to express specific therapeutic proteins offer an innovative therapeutic option. Here, we demonstrate that EPC transfection by electroporation with plasmids encoding the reporter protein GFP or an anti-ß-amyloid antibody fragment (Fab) leads to secretion of each protein. We also demonstrate the secreted anti-ß-amyloid Fab protein functions in ß-amyloid aggregate solubilization.

Myo-InositolTrisPyroPhosphate treatment leads to HIF-1α suppression and eradication of early hepatoma tumors in rats.

Myo-inositol trispyrophosphate (ITPP), a synthetic allosteric effector of hemoglobin, increases the regulated oxygen-releasing capacity of red blood cells (RBCs), leading to suppression of hypoxia-inducible factor 1α (HIF-1α) and to down-regulation of hypoxia-inducible genes such as vascular endothelial growth factor (VEGF). As a consequence, tumor growth is markedly affected. The effect of weekly intravenous injection of ITPP on an orthotopic, syngenic rat hepatocellular carcinoma (HCC) model was compared to that for untreated animals and animals subjected to conventional Doxorubicin chemotherapy. The longitudinal examination of HCC was performed by microCT imaging, and the cellular and molecular changes were evaluated by histology and Western blotting analysis of HIF-1α, VEGF, and caspase-3 gene expression in the tumor and in the surrounding liver. Hematologic impact was evaluated by blood cell-count measurement and determination of P50 (oxygen partial pressure for a 50 % oxygen saturation of hemoglobin). The HCC evaluation by microCT revealed a high potency of ITPP for tumor growth inhibition, thus allowing long-term survival and even cure of almost all the treated animals. The P50 value of hemoglobin in RBCs underwent a shift of 30 % following ITPP injection. Under these conditions, HIF-1α activity was strongly decreased, VEGF expression was down-regulated, and apoptosis was induced in HCC and surrounding liver cells, as indicated by Caspase-3 expression. ITPP did not affect hematologic parameters during treatment. The observations of in vivo tumor eradication suggest a significant clinical potential for ITPP in cancer therapy.

myo-Inositol trispyrophosphate: a novel allosteric effector of hemoglobin with high permeation selectivity across the red blood cell plasma membrane.

myo-Inositol trispyrophosphate (ITPP), a novel membrane-permeant allosteric effector of hemoglobin (Hb), enhances the regulated oxygen release capacity of red blood cells, thus counteracting the effects of hypoxia in diseases such as cancer and cardiovascular ailments. ITPP-induced shifting of the oxygen-hemoglobin equilibrium curve in red blood cells (RBCs) was inhibited by DIDS and NAP-taurine, indicating that band 3 protein, an anion transporter mainly localized on the RBC membrane, allows ITPP entry into RBCs. The maximum intracellular concentration of ITPP, determined by ion chromatography, was 5.5×10(-3) M, whereas a drop in concentration to the limit of detection was observed in NAP-taurine-treated RBCs. The dissociation constant of ITPP binding to RBC ghosts was found to be 1.72×10(-5) M. All data obtained indicate that ITPP uptake is mediated by band 3 protein and is thus highly tissue-selective towards RBCs, a feature of major importance for its potential therapeutic use.

Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo-inositol trispyrophosphate.

A major determinant of maximal exercise capacity is the delivery of oxygen to exercising muscles. myo-Inositol trispyrophosphate (ITPP) is a recently identified membrane-permeant molecule that causes allosteric regulation of Hb oxygen binding affinity. In normal mice, i.p. administration of ITPP (0.5-3 g/kg) caused a dose-related increase in the oxygen tension at which Hb is 50% saturated (p50), with a maximal increase of 31%. In parallel experiments, ITPP caused a dose-related increase in maximal exercise capacity, with a maximal increase of 57 +/- 13% (P = 0.002). In transgenic mice with severe heart failure caused by cardiac-specific overexpression of G alpha q, i.p. ITPP increased exercise capacity, with a maximal increase of 63 +/- 7% (P = 0.005). Oral administration of ITPP in drinking water increased Hb p50 and maximal exercise capacity (+34 +/- 10%; P < 0.002) in normal and failing mice. Consistent with increased tissue oxygen availability, ITPP decreased hypoxia inducible factor-1alpha mRNA expression in myocardium. It had no effect on myocardial contractility in isolated mouse cardiac myocytes and did not affect arterial blood pressure in vivo in mice. Thus, ITPP decreases the oxygen binding affinity of Hb, increases tissue oxygen delivery, and increases maximal exercise capacity in normal mice and mice with severe heart failure. ITPP is thus an attractive candidate for the therapy of patients with reduced exercise capacity caused by heart failure.

Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice.

We investigated the therapeutic effects of two different versions of Abeta(1-15 (16)) liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetra-palmitoylated amyloid 1-15 peptide (palmAbeta(1-15)), or with amyloid 1-16 peptide (PEG-Abeta(1-16)) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmAbeta(1-15) liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-Abeta(1-16) had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmAbeta(1-15), whereas those elicited by PEG-Abeta(1-16) were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly beta-sheet conformation of palmAbeta(1-15) and random coil of PEG-Abeta(1-16). We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a "conformational" disease, implying that antibodies against amyloid sequences in the beta-sheet conformation are preferred as potential therapeutic agents.

Suppression of hypoxia-induced HIF-1alpha and of angiogenesis in endothelial cells by myo-inositol trispyrophosphate-treated erythrocytes.

Allosteric regulation of oxygen delivery by RBCs may have significant effects on tumor growth. Indeed, angiogenesis, the formation of new blood vessels, is induced in growing tumors by low oxygen partial pressure. Hypoxia-inducible genes are switched on, among which are the VEGF gene and its receptors. Most important, under hypoxia, hypoxia-inducible factor 1alpha has a significantly prolonged half-life and up-regulates a number of hypoxia genes. Human microvascular endothelial cells (MECs), when subjected in vitro to hypoxia, align to form vessel-like structures as in the angiogenic process. We report here that, when cultured in hypoxic conditions in the presence of human RBCs loaded with a new membrane-permeant allosteric effector of Hb, myo-inositol trispyrophosphate (ITPP), endothelial cells (ECs) do not align, i.e., do not form "vessel"-like structures, because the "loaded" RBCs are capable of releasing under hypoxia more oxygen than their "normal" counterparts. Levels of VEGF and of hypoxia-inducible factor 1alpha, elevated in the human MECs under hypoxia, were dramatically reduced or even suppressed in the presence of the ITPP-loaded RBCs. Treatment of these ECs directly with free ITPP at different concentrations had no effect on their ability to undertake angiogenesis. Incubation with ITPP enhances the capacity of Hb to release bound oxygen, leading to higher oxygen tension in the hypoxic environment, thus inhibiting hypoxia-induced angiogenesis. These observations are suggestive of a potential in vivo role of ITPP-loaded, "low-O2-affinity" RBCs in cancer therapy.

Inositol tripyrophosphate: a new membrane permeant allosteric effector of haemoglobin.

Nine inositol tripyrophosphate (ITPP) salts have been synthesized. Their ability to act as allosteric effectors of haemoglobin (Hb) has been measured in vitro with free Hb and whole blood. All the synthesized compounds bound to free Hb and were also able to cross, to a certain extent, the plasma membrane of the red blood cells (RBCs) in whole blood samples, lowering the affinity of Hb for oxygen. The oxy-haemoglobin dissociation curves were significantly shifted towards higher values of oxygen partial pressures, both for free Hb and for intracellular Hb in whole blood.

A liposome-based therapeutic vaccine against beta -amyloid plaques on the pancreas of transgenic NORBA mice.

Immune tolerance to beta-amyloid (Abeta) was broken in NORBA transgenic mice presenting Abeta plaques on their pancreases. Vaccination of Black C57, BALB/c, and NORBA mice with the synthetic Abeta(1-16) sequence modified by covalently attaching two palmitoyl residues at each end of the peptide, subsequently reconstituted in liposomes-Lipid A elicited titers of 1:5,000 of anti-Abeta(1-16) antibodies within 10 weeks after the first inoculation. On direct interaction, sera with antibody titers of 1:5,000 solubilized in vitro up to 80% of preformed Abeta(1-42) aggregates. Cryosections of pancreases of unvaccinated NORBA mice show, on staining with Thioflavin T, extensive areas of high-intensity fluorescence in the acinar cell fields. Quantitation of the average fluorescence intensity in each section indicated that: (i) whereas nonvaccinated NORBA mice develop plaques within 45-60 days after birth, vaccinated 8-week-old NORBA mice did not develop amyloid plaques on their pancreases over a period of 7 months; (ii) cryosections from pancreases of 9- and 15-month-old vaccinated NORBA mice showed less than 50% of the fluorescence shown by cryosections from unvaccinated animals of the same age. The results indicate that palmitoylated Abeta peptides, reconstituted in liposomes-lipid A, are highly immunogenic, eliciting "therapeutic" antibody titers within 3 months of the first inoculation and preventing amyloid plaque formation in young animals or significantly reducing existing plaques in older transgenic mice. Possible implications for the therapy of Alzheimer's disease are discussed.