Phase IIb/III Trial of Umbilical Cord-derived Mesenchymal Stem Cells for Patients with Acute Respiratory Distress Syndrome
(IIb/III UC-MSCs for ARDS)

REDUCTION OF NEUTROPHIL EXTRACELLULAR TRAP FORMATION BY MESENCHYMAL STEM CELLS AND THEIR EXOSOMES
Publication number: 20230107484

Abstract: Disclosed are methods of reducing lung inflammation in acute respiratory distress syndrome elicited by various factors such as COVID-19 infection by reduction of neutrophil extracellular trap formation through administration of mesenchymal stem cells and/or exosomes thereof. The invention provides means of inhibiting neutrophil release of extracellular traps by mesenchymal stem cells and/or exosomes derived from said mesenchymal stem cells. Additionally, synergies are provided between mesenchymal stem cells and/or exosomes derived from mesenchymal stem cells and agents approaches which reduce neutrophil extracellular trap formation.

Umbilical Cord-derived Mesenchymal Stem Cells for Patients with COVID-19 (“UC-MSC for COVID-19”) IND # 19757

The primary objective will be to assess effectiveness of UC-MSC treatment on proportion of patients alive and free of respiratory failure at Day 60 after randomization. The secondary objectives will be to assess all-cause mortality at Day 60, survival at day 31, number of subjects experiencing serious adverse events (SAEs) by day 31, SAE-free survival, time to recovery (evaluated until day 60), and time to oxygen requirement equal or below 40% oxygen.

METHODS AND COMPOSITIONS FOR THE CLINICAL DERIVATION OF AN ALLOGENIC CELL AND THERAPEUTIC USES
US 9,803,176 B2
Abstract: Various cells, stem cells, and stem cell components, includ­ing associated methods of generating and using such cells are provided. In one aspect, for example, an isolated cell that is capable of self-renewal and culture expansion and is obtained from a subepithelial layer of a mammalian umbili­cal cord tissue. Such an isolated cell expresses at least three cell markers selected from CD29, CD73, CD90, CD166, SSEA4, CD9, CD44, CD146, or CD105, and does not express at least three cell markers selected from CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD117, Stro-1, or HLA-DR.

Investigation of Umbilical Cord-derived Mesenchymal Stem Cells for the Treatment of Chronic Traumatic Encephalopathy Patients IND # 27377

To determine safety and efficacy of 100 million intravenously administered CTEcells™ allogeneic umbilical cord mesenchymal stem cells. Efficacy will be determined by behavioral scores, brain imaging, and reduction in inflammatory markers. Toxicity of treatment was evaluated for the duration of the study and will be graded according to the criteria of the World Health Organization.

PROTECTION AND REGENERATION OF NEUROLOGICAL FUNCTION BY USING STEM CELLS
Publication number: 20220125852
Abstract: Disclosed are therapeutic compounds, protocols, and compositions of matter useful for treatment of neurological conditions. In one embodiment the invention teaches the treatment of chronic traumatic encephalopathy (CTE) through protecting/regenerating the endothelial by administration of cells such as stem cells. In one embodiment stem cells are administered in order to protect the endothelium from apoptosis and to preserve the blood brain barrier. In another embodiment stem cells are administered together with endothelial progenitor cells in order to regenerate neural endothelium. In other embodiments preservation of brain integrity in conditions of degeneration is accomplished by administration of stem cells and/or endothelial cells.

JadiCell Therapy for COPD IND # 28508

To determine safety and efficacy of intravenously administered allogeneic JadiCell umbilical cord blood mesenchymal stem cells in patients with moderate-to-severe COPD. The Primary Endpoint, which is toxicity, will be assessed by number of adverse events (AEs). The Secondary Endpoint, which is efficacy will be evaluated at baseline and days 30, 60, and 90.

UMBILICAL CORD MESENCHYMAL STEM CELLS FOR TREATMENT OF CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND LUNG DEGENERATION
Publication number: 20230057957
Abstract: The invention discloses means of treating lung degenerative diseases including chronic obstructive pulmonary disease (CODP) using umbilical cord mesenchymal stem cells such as JadiCells alone, and/or using said cells under conditions that are activated in order to endow enhanced regenerative activity. In one embodiment said activation of said mesenchymal stem cells is performed through stimulation with a toll like receptor agonist at a concentration and duration sufficient to induce a >50% increase in keratinocyte growth factor expression from said stem cells. In another embodiment the invention provides the use of JadiCells as a means of producing exosomes, wherein said exosomes possess therapeutic properties capable of reducing inflammation, fibrosis and degeneration associated with COPD, as well as stimulation of regenerative activity. In some JadiCells are activated by a treatment with Activated Protein C.

Safety, Feasibility, and Immunomodulatory Activities of StemVacs in Patients with Advanced Solid Tumors IND # 17448

The Primary Objective is safety and feasibility of StemVacs administration at 12 months as assessed by lack of adverse medical events. The Secondary Objective is efficacy as judged by tumor response, time to progression, and immunological monitoring.

Stimulation of Immunity to Tumor Stem Cell Specific Proteins by Peptide Immunization
Application Number: 62/478532
Abstract: Treatment of cancer is disclosed through administration of proteins or specific peptides found on tumor stem cells in vivo, in a matter eliciting monocyte or dendritic cell migration in order to allow uptake of said administrated proteins or peptides, followed by administration of a maturation signal in vivo. The invention provides for treatment of cancer through induction of anticancer immunity and/or immunity towards tumor initiating stem cells.

Treatment of Metastatic Breast Cancer by StemVacs-V Cancer Immunotherapeutic IND transferred to Res Nova Bio,Inc.

The Primary Objective is safety and feasibility of StemVacs-V administration at 12 months as assessed by lack of adverse medical events. The Secondary Objective is efficacy as judged by tumor response, time to progression, and immunological monitoring.

Pluripotent Stem Cell Derived Dendritic Cells and Engineered Dendritic Cells for Cancer Immunotherapy
Publication number: 20220298491
Abstract: Disclosed are populations of dendritic cells generated from stem cells capable of inducing immunity towards cancer. In one embodiment said dendritic cells are generated from allogeneic inducible pluripotent stem cells, for some uses, said pluripotent stem cells are genetically engineered/edited to induce cancer specific immunity and/or resist immunosuppressive effect of tumor derived microenvironment. In one embodiment pluripotent stem cells are transfected with cancer stem cell antigens such as BORIS and/or NR2F6.

Mesenchymal Stem Cell Therapy of Epilepsy and Seizure Disorders
Application No.: 63418861
Disclosed are novel compositions of matter and treatment methods for reducing and/or reversing epilepsy through administration of mesenchymal stem cells in order to induce immune modulation and/or regenerative processes. In one embodiment umbilical cord mesenchymal stem cells are administered to a patient suffering from epilepsy at a concentration and frequency sufficient to inhibit neuronal hyperactivation and/or reduce neuroinflammatory status of the patient.

Protection and Regeneration of Neurological Function by Using Stem Cells
Publication number: 20220125852
Abstract: Disclosed are therapeutic compounds, protocols, and compositions of matter useful for treatment of neurological conditions. In one embodiment the invention teaches the treatment of chronic traumatic encephalopathy (CTE) through protecting/regenerating the endothelial by administration of cells such as stem cells. In one embodiment stem cells are administered in order to protect the endothelium from apoptosis and to preserve the blood brain barrier. In another embodiment stem cells are administered together with endothelial progenitor cells in order to regenerate neural endothelium. In other embodiments preservation of brain integrity in conditions of degeneration is accomplished by administration of stem cells and/or endothelial cells.

Examples

JadiCell Conditioned Media Inhibits Endothelial Death.

Injury associated with inflammation. Inflammation causes oxidative stress. Oxidative stress induces death of endothelial cells. Endothelial death exposes basement membrane, induces coagulopathy. Culture of HUVEC cells with upstream inflammatory agent TNF-alpha or downstream H2O2 results in death. Conditioned media (CM) from JadiCells decreases endothelial cell death.

JadiCell™ Reduces Immunogenicity of Endothelial Cells

Endothelial cells can act as antigen presenting cells. Stimulation of T cells by endothelial cells results in inflammation and breaking of blood brain barrier.  HLA expression on HUVEC was utilized to quantify one aspects of endothelial antigen presentation.  Mixed lymphocyte reaction used as a test of T cell activation.

JadiCell™ Reduces Thrombogenicity of Activated Endothelial Cells

In response to inflammation endothelial cells induce clotting by stimulation of extrinsic coagulation pathway. Tissue factor is activator of extrinsic pathway.  HUVEC cells were treated with endotoxin to stimulate activation. Assessment of Tissue Factor performed by flow cytometry

Chimeric Cells Comprising Dendritic Cells and Endothelial Cells Resembling Tumor Endothelium
Publication number: 20220306994
Abstract: Disclosed are means, methods and compositions of matter useful for induction of immunological responses towards tumor endothelial cells. In one embodiment the invention teaches fusion of dendritic cells and cells resembling tumor endothelial cells and administration of such chimeric cells as an immunotherapy for stimulation of tumor endothelial cell destruction. In other embodiments pluripotent stem cells are utilized to generate dendritic cells, wherein said dendritic cells are fused with pluripotent stem cell derived endothelial cells created in a manner to resemble tumor endothelial cells.

Treatment of Parkinson’s Disease by Immune Modulation and Regenerative Means
Application No.: 63/218,582
Disclosed are means, methods and compositions of matter for treatment Parkinson’s Disease through concurrent immune modulation and regenerative means. In one embodiment Parkinson’s Disease is treated by augmentation of T regulatory cell numbers and/or activity while concurrently providing regenerative cells such as mesenchymal stem cells, and/or dopamine secreting cells. In one embodiment administration of immunoglobulins such as IVIG together with low dose interleukin-2 and/or low dose naltrexone is disclosed as a preparatory means prior to administration of therapeutic cells such as stem cells. Other therapeutic means utilized in an adjuvant manner are also provided for hormonal rebalancing, transcranial magnetic stimulation, and deep brain stimulation.

Treatment of SARS-CoV-2 with Dendritic Cells for Innate and/or Adaptive Immunity
Publication number: 20210393681
Abstract: Disclosed are means, methods, and compositions of matter for prophylaxis and/or treatment of SARS-CoV-2 by administration of dendritic cells in a manner and frequency sufficient to induce activation of innate and/or adaptive immune responses. In one embodiment the invention teaches administration of dendritic cells pulsed with one or more innate immune stimulants in a manner endowing said dendritic cell with ability to induce augmentation of natural killer (NK) cell number and/or activity. In another embodiment the invention teaches the use of dendritic cells stimulated with innate immune activators in a manner to allow for uptake of viral particles and presentation of viral epitopes to T cells in order to stimulate immunological activation and/or memory responses.

Examples

Materials and Methods

Cell Lines

HeLa human cervical cancer cells were obtained from American Type Tissue Culture (ATCC: Manassas, VA) and grown under fully humidified 5% CO2 environment with MEM supplemented with 10% FBS, 2% sodium pyruvate, non-essential amino acids (2 mM), penicillin (100 units/ml), streptomycin (100 µg/ml), and glutamine (4 mM) (Gibco-BRL).  Cells were passaged by trypsinization twice weekly or as needed based on 75% confluency.

PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC)

PBMC were isolated from buffy coats by density-gradient centrifugation. Specifically, buffy coat cells were dispensed over five 50 ml falcon tubes, phosphate-buffered saline (PBS)/2% fetal calf serum (FCS) solution was added to reach a volume of 20 ml and 10 ml Ficoll-Paque® was gently added under the diluted buffy coat cells. Centrifugation was performed at 400 g for 20 min at room temperature (RT) and washing of PBMC was done three times with PBS/2% FCS. Culture of freshly isolated PBMC was performed in complete MEM media.

CELL TREATMENTS AND ANALYSIS

ACTIVEIMMUNE was diluted in complete MEM media prepared as described above.  Dilutions of 1:10, 1:100, 1;1000 and 1:10,000 were performed.  Negative controls were complete MEM media.  Positive controls were concanavalin A at a concentration of 2.5 ug/ml.  PBMC were plated at 1·5×10⁶ cells/ml in flat-bottom 96-well culture plates in a volume of 200 µl per well and incubated at 37° in a humidified 5% CO₂ atmosphere.  Conditioned media was then evaluated for IFN-gamma production using ELISA from R & D Systems (Quantikine ELISA).  Concentration was calculated by plotting against a standard curve generated with control cytokine.

HeLa cells were plated at a concentration of 10,000 cells per well in flat bottom plates and incubated with dilutions of ACTIVEIMMUNE at 1:10, 1:100, 1;1000 and 1:10,000.    The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed for assessment of proliferation.  In this assay soluble MTT is metabolized by mitochondrial enzyme activity of viable tumor cells, into an insoluble colored formazan product. Subsequently formazan were dissolved in DMSO and measured spectrophotometrically at 540 nm. Briefly, 200 μl of cell suspension  was seeded in 96-well microplates and incubated for 48 h (37°C, 5% CO₂ air humidified).

To evaluate cell survival, 20 μl of MTT solution (5 mg/ml in PBS) was added to each well and incubated for 3 h. Then gently 150 μl of old medium containing MTT was replaced by DMSO and pipetted to dissolve any formed formazan crystals. Absorbance was then determined at 540 nm by enzyme-linked immunosorbent assay (ELISA) plate reader. Each extract concentration was assayed in 4 wells and repeated 3-times.

ELISA

IFN-gamma, IL-4, IL-10 and IL-12 were assessed by ELISA (R and D Systems) utilizing supernatant from mitogen activated cultures and treated DC.

Dendritic Cells

DC were generated from PBMC resuspended in RPMI-10% FCS, and allowed to adhere to 6-well plates (Costar Corp., Cambridge, MA). After 2 h incubation at 37  Celsius, the nonadherent cells were removed and the adherent cells washed in phosphate buffered saline (PBS), followed by detachment by incubation with Mg 2+ and Ca 2+ free PBS containing 0.5 mM EDTA at 37 Celsius. The adherent fraction was subsequently cultured at 3 x 10(6)/ml in RPMI-10% FCS supplemented with 50 ng/ml GM-CSF and 1,000 U/ml IL-4. Media is changed every 2 days for a total of 8 days culture.  DC were  isolated by positive selection for CD83 and subsequently treated with ACTIVEIMMUNE on day 6 of culture.  Assessment of maturation was performed by flow cytometry for CD80 and CD86 expression.

Blockade of TLR-4 was performed using by culture in the presence of TLR4 antagonist LPS-RS (Invivogen (San Diego, CA), (5μg/mL), with pretreatment 4 hours before exposure to ACTIVEIMMUNE.

Results

ACTIVEIMMUNE Does Not Modulate Cellular Proliferation

ACTIVEIMMUNE has been reported to possess anticancer activity.  Accordingly, we conducted a series of experiments assessing ability of various concentrations of ACTIVEIMMUNE to inhibit proliferation of HeLa cells. We utilized the chemotherapeutic drug doxorubicin as a control. As seen in Figure 1a, various doses of ACTIVEIMMUNE did not affect proliferation of HeLa cells as assessed in the MTT assay after 48 hours of culture.  Importantly , supraphysiological doses of ACTIVEIMMUNE, as high as 1:10 diluted volume by volume in the tissue culture media did not result in inhibition of proliferation.  These data suggest that ACTIVEIMMUNE does not act through cytotoxic or cytostatic mechanisms.  These data were confirmed with other cell lines such as PC-3, DU-145, and non-malignant 3T3 fibroblasts (Figure 1b-d).

Figure 1: ACTIVEIMMUNE Does Not Alter Proliferation of Cancer Cells

ACTIVEIMMUNE Acts as a Cofactor for Cytokine Secretion from Immune Cells found in Peripheral Blood

To assess whether ACTIVEIMMUNE directly activates T cell production of cytokines, or whether it requires a costimulatory signal, such as concanavalin A (ConA), was examined.  ACTIVEIMMUNE did not affect viability of PBMC (data not shown). It appears that ACTIVEIMMUNE complements existing production of immune stimulatory molecules after a primary stimuli, but does not initiate immunity, at least based on IFN-gamma and IL-4 production (Figure 2a and 2b).  Given that different doses of ACTIVEIMMUNE possess different costimulatory profiles for the different cytokines, we questioned whether the effect was specific to conconavalin A stimulation, or whether other factors may be at play.  Accordingly, we substitute stimulation by conconavalin A to stimulation by phytohemagglutinin, a mitogen often used in studies stimulating human T cells.  As seen in Figures 2c and 2d, a similar pattern of IFN-gamma and IL-4 costimulation was observed with PHA acting as the primary stimulator.

Figure 2: ACTIVEIMMUNE Acts as a Costimulator of Lymphocyte Activation

 ACTIVEIMMUNE Induces Dendritic Cell Maturation in a TLR4 Dependent Manner

Given the contamination of antigen presenting cells in PBMC, and the fact that antigen presenting cells may be sending costimulatory signals to the T cells in response to ACTIVEIMMUNE treatment, a series of experiments were conducted to assess whether ACTIVEIMMUNE acts on the most potent antigen presenting cell, the dendritic cell.  Day 6 immature DC generated from monocytes by IL4 and GM-CSF treatment were used to assess maturation-inducing potential of ACTIVEIMMUNE.  Cells were treated with saline, lps positive control, and 3 concentrations of ACTIVEIMMUNE.  Additionally, blockade of TLR4 signalling was performed by cotreatment with LPS-RS, an antagonist of the TLR-4 receptor.  As seen in Figure 3a and 3b, ACTIVEIMMUNE was capable of upregulating expression of IL-12 and IL-10, respectively, suggesting from a functional perspective that DC activation was occurring.  Indeed the fact that IL-12 drives Th1 cytokine production and IL-10 drives Th2, these data are in agreement with the previous data suggesting that ACTIVEIMMUNE is capable of modulating immunity.  Definative evidence of maturation of DC was observed using flow cytometry, demonstrating that uprgulation of CD80 and CD86 was occurring as a result of ACTIVEIMMUNE treatment (Figures 3c and 3d).  In all experiments, blockade of TLR-4 by treatment with LPS-RS, an antagonist of TLR4, resulted in marked reduction of both LPS induced changes (positive control) as well as in activity of ACTIVEIMMUNE.

Figure 3: ACTIVEIMMUNE Activates Dendritic Cell Maturation

Molecular Characterization of ACTIVEIMMUNE

Two Dimensional Gel Electrophoresis

Two-dimensional electrophoresis was performed according to the carrier ampholine method of isoelectric focusing (O’Farrell, P.H., J. Biol. Chem. 250: 4007-4021, 1975, Burgess-Cassler, A., Johansen, J., Santek, D., Ide J., and Kendrick N., Clin. Chem. 35: 2297, 1989) by Kendrick Labs, Inc. (Madison, WI) as follows: Isoelectric focusing was carried out in a glass tube of inner diameter 2.3 mm using 2% pH 3-10 isodalt Servalytes (Serva, Heidelberg, Germany) for 9600 volt-hrs. One µg of an IEF internal standard, tropomyosin, was added to the sample.  This protein migrates as a doublet with lower polypeptide spot of MW 33,000 and pI 5.2. The enclosed tube gel pH gradient plot for this set of Servalytes was determined with a surface pH electrode.

For the 10% acrylamide gels, after equilibration for 10 min in Buffer ‘O’ (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8), each tube gel was sealed to the top of a stacking gel that overlaid a 10% acrylamide slab gel (0.75 mm thick).  SDS slab gel electrophoresis was carried out for about 4 hrs at 15 mA/gel. The following proteins (Sigma Chemical Co., St. Louis, MO and EMD Millipore, Billerica, MA) were used as molecular weight standards: myosin (220,000), phosphorylase A (94,000), catalase (60,000), actin (43,000), carbonic anhydrase (29,000) and lysozyme (14,000).  These standards appear along the basic edge of the silver-stained(Oakley, B.R., Kirsch, D.R. and Moris, N.R. Anal. Biochem. 105:361-363, 1980) 10% acrylamide slab gel. The gel was dried between sheets of cellophane with the acid edge to the left.

After equilibration for 15 min in Buffer “O” (10% glycerol, 50 mM dithiothreitol, 2.3% SDS and 0.0625 M tris, pH 6.8) each tube gel was sealed to the top of 10% acrylamide spacer gels which are on the top of 16.5% acrylamide peptide slab gels (Shagger, H. and Jagow, G. Anal. Biochem. 166: 368, 1987) (0.75 mm thick).  SDS slab gel electrophoresis was started at 15 mamp/gel for the first four hours and then carried out overnight at 12 mamp/gel as for the separation of peptides.  The slab gel electrophoresis was stopped after the bromophenol blue dye front had just started running off the gel. The following proteins (Sigma Chemical Co., St. Louis, MO and EMD Millipore, Billerica, MA) were added as molecular weight markers: phosphorylase A (94,000), catalase (60,000), actin (43,000) and lysozyme (14,000).  These standards appear as bands on the basic edge of the silver stained (Oakley, B.R., Kirsch, D.R. and Moris, N.R. Anal. Biochem. 105:361-363, 1980) 16.5% acrylamide slab gel. Low molecular weight markers from Sigma Chemical were also loaded  myoglobin (polypetide backbone) 1-153 16,950; Myoglobin (I+II, 1-131) 14,440; myoglobin (I+III, 56-153) 10,600; Myoglobin (I, 56-131) 8,160; myoglobin (II 1-55) 6,210; Glucagon 3,480; and Myoglobin (III, 132-153) 2,510.   The gel was silver-stained and dried between sheets of cellophane paper with the acid edge to the left.

Figure 4: Gel Run of ACTIVEIMMUNE under 10% Conditions

Figure 4 illustrates the gel run under 10% conditions The arrowhead illustrates the molecular weight spot indicative of the immune modulatory activity that was subsequently sequenced.

Proteomic Analysis/Sequencing

Protein digestion and peptide extraction. Proteins that were separated by SDS-PAGE/2D-PAGE and stained by Coomassie dye were excised, washed and the proteins from the gel were treated according to published protocols [157-159]. Briefly, the gel pieces were washed in high purity, high performance liquid chromatography HPLC grade water, dehydrated and cut into small pieces and destained by incubating in 50 mM ammonium bicarbonate, 50 mM ammonium bicarbonate/50% acetonitrile, and 100% acetonitrile under moderate shaking, followed by drying in a speed-vac concentrator. The gel bands were then rehydrated with 50 mM ammonium bicarbonate. The procedure was repeated twice. The gel bands were then rehydrated in 50 mM ammonium bicarbonate containing 10 mM DTT and incubated at 56^oC for 45 minutes. The DTT solution was then replaced by 50 mM ammonium bicarbonate containing 100 mM Iodoacetamide for 45 minutes in the dark, with occasional vortexing. The gel pieces were then re-incubated in 50 mM ammonium bicarbonate/50% acetonitrile, and 100% acetonitrile under moderate shaking, followed by drying in speed-vac concentrator. The dry gel pieces were then rehydrated using 50 mM ammonium bicarbonate containing 10 ng/L trypsin and incubated overnight at 37^oC under low shaking. The resulting peptides were extracted twice with 5% formic acid/50 mM ammonium bicarbonate/50% acetonitrile and once with 100% acetonitrile under moderate shaking. Peptide mixture was then dried in a speed-vac, solubilized in 20L of 0.1% formic acid/2% acetonitrile.

LC-MS/MS. The peptides mixture was analyzed by reversed phase liquid chromatography (LC) and MS (LC-MS/MS) using a NanoAcuity UPLC (Micromass/Waters, Milford, MA) coupled to a Q-TOF Ultima API MS (Micromass/Waters, Milford, MA), according to published procedures [157, 160-162]. Briefly, the peptides were loaded onto a 100 m x 10 mm NanoAquity BEH130 C18 1.7 m UPLC column (Waters, Milford, MA) and eluted over a 150 minute gradient of 2-80% organic solvent (ACN containing 0.1% FA) at a flow rate of 400 nL/min. The aqueous solvent was 0.1% FA in HPLC water. The column was coupled to a Picotip Emitter Silicatip nano-electrospray needle (New Objective, Woburn, MA). MS data acquisition involved survey MS scans and automatic data dependent analysis (DDA) of the top three ions with the highest intensity ions with the charge of 2+, 3+ or 4+ ions. The MS/MS was triggered when the MS signal intensity exceeded 10 counts/second. In survey MS scans, the three most intense peaks were selected for collision-induced dissociation (CID) and fragmented until the total MS/MS ion counts reached 10,000 or for up to 6 seconds each. The entire procedure used was previously described [157, 160, 161]. Calibration was performed for both precursor and product ions using 1 pmol GluFib (Glu1-Fibrinopeptide B) standard peptide with the sequence EGVNDNEEGFFSAR and the monoisotopic doubly-charged peak with m/z of 785.84.

Data processing and protein identification. The raw data were processed using ProteinLynx Global Server (PLGS, version 2.4) software as previously described [160]. The following parameters were used: background subtraction of polynomial order 5 adaptive with a threshold of 30%, two smoothings with a window of three channels in Savitzky-Golay mode and centroid calculation of top 80% of peaks based on a minimum peak width of 4 channels at half height. The resulting pkl files were submitted for database search and protein identification to the public Mascot database search (www.matrixscience.com, Matrix Science, London, UK) using the following parameters: databases from NCBI (all organisms, human proteis and rodent proteins for targeted identification of proteins), parent mass error of 1.3 Da, product ion error of 0.8 Da, enzyme used: trypsin, one missed cleavage, propionamide as cysteine fixed modification and Methionine oxidized as variable modification. To identify the false negative results, we used additional parameters such as different databases or organisms, a narrower error window for the parent mass error (1.2 and then 0.2 Da) and for the product ion error (0.6 Da), and up to two missed cleavage sites for trypsin. In addition, the pkl files were also searched against in-house PLGS database version 2.4 (www.waters.com) using searching parameters similar to the ones used for Mascot search. The Mascot and PLGS database search provided a list of proteins for each gel band. To eliminate false positive results, for the proteins identified by either one peptide or a mascot score lower than 25, we verified the MS/MS spectra that led to identification of a protein. The protein identified comprised of the amino acids:

EFDVILKAAGANKVAVIKAVRGATGLGLKEAKDLVESAPAALKEGVSKDDAEALKKALEEAGAEVEVK

ACTIVEIMMUNE Activated DC are Superior to LPS Activated DC in Suppressing B16 Melanoma

Mouse dendritic cells were generated by 6 day culture of bone marrow mononuclear cells in IL-4 and GM-CSF as previously described by us [163].  At day 6 DC were stimulated to mature by administration of 5 ug/ml LPS and 100 ng of TNF-alpha as a positive control.  In the experimental group DC were treated with leukocyte extract (LE), which was ACTIVEIMMUNE (ImmunoActive^TM) obtained from Argo Farma, Mexico, was added to dendritic cells at a concentration of 10 micrograms per ml.  C57/BL6 mice were inoculated with 500,000 B16 melanoma cells subcutaneously and tumors were allowed to grow for 7 days.  Dendritic cells (positive and experimental controls) or saline were administered subcutaneously to animals at a concentration of 1 million cells per animal in a volume of 200 microliters.  As seen in Figure 6, a significant reduction in tumor size was observed in mice receiving DC that were pretreated with leukocyte extract.  Tumor reduction efficacy was dependent on NK activity (8 mice per group).  The experiment was repeated however NK cells were depleted by administration of injections every 3 days of anti-NK1.1 (PK136 mouse IgG2, hybridoma HB191; ATCC) (200 μg/dose) antibody intraperitoneally after administration of tumors.  As seen in Figure 7, antitumor efficacy was diminished upon the loss of NK cells.

Figure 6 Suppression of Tumor Growth by Leukocyte Extract Treated Dendritic Cells

Figure 7 Suppression of Tumor Growth by Leukocyte Extract Treated Dendritic Cells in NK Dependent

Augmentation of Natural Killer Cell Activity and Induction of Cytotoxic Immunity Using Leukocyte Lysate Activated Allogeneic Dendritic Cells: StemVacs™
Application No.: 63/045863
Stimulation of immunity would be beneficial in various chronic conditions such as viral infections and neoplasia. Autologous dendritic cell therapy has been widely described in the immunotherapy literature and has been approved by the FDA for treatment of prostate cancer. Unfortunately, the need to generate individual doses is costly and limited by ability of the patients to have sufficient starting cell numbers available to generate sufficient dendritic cells. Here we describe the process of preparing allogeneic dendritic cells utilizing a leukocyte lysate based approach. These data support development of StemVacs for conditions that would benefit from NK activation such as cancer and COVID-19.

STIMULATION OF DENDRITIC CELL ACTIVITY BY HOMOTAURINE AND ANALOGUES THEREOF
Publication number: 20220235325
Abstract: Disclosed are means, methods, and compositions of matter useful for enhancement of dendritic cell activity. In one embodiment the invention provides the use of GABA agonists such as homotaurine for stimulation of dendritic cell activity. In one embodiment said dendritic cell activity is enhancement of natural killer cell activity and/or of T cell activity. In one embodiment NK cell activity is ability to induce cytotoxicity in neoplastically transformed cells, whereas T cell activity is either cytokine production for CD4 cells or cytotoxicity for CD8 cells.

Example 1 Homotaurine Increases Ability of StemVacs to Stimulate NK Activity

Peripheral blood mononuclear cells were obtained by ficoll centrifugation and plated with StemVacs umbilical cord derived dendritic cells.  StemVacs was generated by culture of umbilical blood adherent cells with interleukin 4 and GM-CSF for 7 days with maturation step induced by 24 hour culture TLR agonist.  Assessment of natural killer cell activity was performed subsequent to culture of cells with taurine (10 micrograms / ml) or homotaurine (10  micrograms /ml) for the indicated timepoints.  Cytotoxicity against NK target cell line was performed using the flow cytometry Promega assay.

Example 2 Homotaurine Increases Ability of StemVacs to Stimulate T cell Activity

Peripheral blood mononuclear cells were obtained by ficoll centrifugation and plated with StemVacs umbilical cord derived dendritic cells.  StemVacs was generated by culture of umbilical blood adherent cells with interleukin 4 and GM-CSF for 7 days with maturation step induced by 24 hour culture TLR agonist.  Assessment of T cell activty was performed subsequent to culture of cells with taurine (10 micrograms / ml) or homotaurine (10  micrograms /ml) for the indicated timepoints.  T cell activity was assessed by ELISA quantification of interferon gamma production after stimulation with 5 micrograms per ml of phytohemagglutinin.

Stimulation of Pulmonary Regenerative Exosomes by Mesenchymal Stem Cells and Derivatives Thereof
Application No.: 63/412861
Disclosed are therapeutic means for pulmonary degenerative conditions through the administration of mesenchymal stem cells in order to induce regenerative exosomes from dendritic cells expressing CD103. In one embodiment cultures of mesenchymal stem cells with dendritic cell progenitors are disclosed wherein said mesenchymal stem cells induce a modulation of STAT3 signaling in said dendritic cell endowing a regenerative property to said dendritic cells and exosomes derived from said cells.

ENHANCEMENT OF UMBILICAL CORD MESENCHYMAL STEM CELL THERAPEUTIC ACTIVITY BY STIMULATORS OF T REGULATORY CELLS AND/OR CELLS EXPRESSING CD73
Publication number: 20230057356
Abstract: Disclosed are means, compositions of matter and protocols useful for treatment of COVID-19 and/or other inflammatory pathologies through stimulation of T regulatory cells and/or T cells expressing CD73 using administration of umbilical cord derived mesenchymal stem cells such as JadiCells. In one embodiment dosage of JadiCells needed to treat a patient is determined by the increase of T regulatory cells and/or CD73 expressing cells that are increased in number and/or activity subsequent to a test dose of JadiCells. In another embodiment stimulators of T regulatory cells and/or CD73 expressing T cells are utilized together with JadiCells in order to augment therapeutic activity. In some embodiments administration of JadiCell is performed with low dose interleukin-2 as a treatment for COVID-19 or other inflammatory related pathologies.

Example 1: JadiCells Increase the Number of T regulatory Cells

BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and T regulatory cells were measured by FoxP3 staining (Promega kit, following manufacturers instructions).

Example 2: JadiCells Increase the Number of CD73 T Cells

BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and T regulatory cells were measured by CD73 staining (Promega kit, following manufacturers instructions).

Example 3: Depletion of Treg Cells Decreases Therapeutic Potential of JadiCells

BALB/c mice were depleted of CD25 T regulatory cells by administration once every two days of anti-CD25 antibody one week before initiation of experiment.  Subsequently animals were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and lung inflammation was assessed by neutrophils per viewing field.

Example 4: Low Dose IL-2 Increases Therapeutic Potential of JadiCells

BALB/c mice were treated with IL-2 (10 IU/mouse) once every two days one week before initiation of experiment.  Subsequently animals were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and lung inflammation was assessed by neutrophils per viewing field.

Enhancement of Umbilical Cord Mesenchymal Stem Cell Therapeutic Activity by Stimulators of T Regulatory Cells and/or Cells Expressing CD73
Publication number: 20230057356
Abstract: Disclosed are means, compositions of matter and protocols useful for treatment of COVID-19 and/or other inflammatory pathologies through stimulation of T regulatory cells and/or T cells expressing CD73 using administration of umbilical cord derived mesenchymal stem cells such as JadiCells. In one embodiment dosage of JadiCells needed to treat a patient is determined by the increase of T regulatory cells and/or CD73 expressing cells that are increased in number and/or activity subsequent to a test dose of JadiCells. In another embodiment stimulators of T regulatory cells and/or CD73 expressing T cells are utilized together with JadiCells in order to augment therapeutic activity. In some embodiments administration of JadiCell is performed with low dose interleukin-2 as a treatment for COVID-19 or other inflammatory related pathologies.

Example 1: JadiCells Increase the Number of T regulatory Cells

BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and T regulatory cells were measured by FoxP3 staining (Promega kit, following manufacturers instructions).

Example 2: JadiCells Increase the Number of CD73 T Cells

BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and T regulatory cells were measured by CD73 staining (Promega kit, following manufacturers instructions).

Example 3: Depletion of Treg Cells Decreases Therapeutic Potential of JadiCells

BALB/c mice were depleted of CD25 T regulatory cells by administration once every two days of anti-CD25 antibody one week before initiation of experiment.  Subsequently animals were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and lung inflammation was assessed by neutrophils per viewing field.

Example 4: Low Dose IL-2 Increases Therapeutic Potential of JadiCells

BALB/c mice were treated with IL-2 (10 IU/mouse) once every two days one week before initiation of experiment.  Subsequently animals were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were sacrificed and lung inflammation was assessed by neutrophils per viewing field.

Inhibition and Reversion of Chronic Obstructive Pulmonary Disease (COPD) by Endothelial Cell Regeneration
Application No.: May 12, 2022
Disclosed are means, treatment methods, and compositions of matter useful for prevention and/or reversion of chronic obstructive pulmonary disease (COPD). In one embodiment the invention provides the administration of mesenchymal stem cells and exosome thereof as a means of augmenting endogenous endothelial regeneration and/or endothelial regeneration stimulated by exogenous means. In some embodiments the invention provides administration of allogeneic mesenchymal stem cells together with autologous endothelial progenitor cells and/or mobilization of said autologous endothelial progenitor cells.

Lithium as a Monotherapy and/or Stem Cell Adjuvant Therapy for Pulmonary Fibrosis
Publication number: 20220370561
Abstract: Disclosed are compositions of matter, therapeutics, and protocols useful for reduction and/or reversion of pulmonary fibrosis. In one specific embodiment lithium chloride is administered together with a regenerative cell in a patient suffering from, or at risk of pulmonary fibrosis. In one embodiment said lithium chloride is administered as an adjuvant to a regenerative therapy, wherein said regenerative therapy is a gene therapy, a protein therapy, a cell therapy, or a tissue transplant. In one embodiment lithium chloride, or a salt thereof is utilized alone, or with a regenerative means, to evoke preservation and/or elongation of telomere length in pulmonary tissue.

Example 1: Lithium Enhances Ability of JadiCells to Increase T regulatory Cell Numbers in Mice.

10 week old Female BALB/c mice were administered 150 mg/kg of lithium chloride daily by the IP route.  Additionally some animals received Jadicells at 500,000.  Quantification of T regulatory cells was performed by intracellular staining for FoxP3 expression. 

Example 2: JadiCells are Superior to BM and Adipose MSC

• Bleomycin (BLM) administered intratracheally 3 mg/kg in female 10 week old C57/BL6 mice
• JadiCell™ or BM-MSC or adipose administered by tail vein at 5 days after BLM
• Animals sacrificed at day 12 and assessed for fibrosis (hydroxyproline content)

Example 3: Lithium Chloride Enhances Antifibrotic Activities of JadiCells

• Bleomycin (BLM) administered intratracheally 3 mg/kg in female 10 week old C57/BL6 mice
• JadiCell™ was injectedby tail vein at 5 days after BLM. Lithium was injected IP as described in example 1.

Animals sacrificed at day 12 and assessed for fibrosis (hydroxyproline content)

Induction of Neurogenesis using Umbilical Cord Derived Mesenchymal Stem Cells and Derivatives Thereof
Publication number: 20230047535
Abstract: Disclosed are means, compositions of matter and protocols useful for treatment of neurological dysfunctions through stimulation of adult neurogenesis using administration of umbilical cord derived mesenchymal stem cells such as JadiCells. In one embodiment viral induced neuropathy is reduced by administration of JadiCells to stimulate neurogenesis. In another embodiment the neurogenic activity of selective serotonin reuptake inhibitors is enhanced by administration of JadiCells. In some embodiments administration of JadiCell exosomes, conditioned media, microvesicles and/or apoptotic bodies is utilized to stimulate neurogenesis.

Example

JadiCell Administration is Superior to other Stem Cells for Stimulating Dentate Gyrus Neurogenesis

BALB/c mice were administered with 1 million of either bone marrow or adipose MSC or with JadiCells Intravenously at the same time as intraperitoneal injection of poly IC at the indicated concentrations.  After a period of 3 days animals were given BrdU and sacrificed the next day.  Proliferation was assessed based on BrdU incorporation. 10 animals per group were used.

Stimulation of Mesenchymal Stem Cell Therapeutic Activities by T Regulatory Cells
Application No.: 63/247016
Disclosed are novel means of enhancing mesenchymal stem cell regenerative activities including, intra alia, production from pulmonary leakage and suppression of scar tissue formation by co-administration with T regulatory cells. In some embodiments the invention provides an interaction between T regulatory cells and mesenchymal stem cells in which T regulatory cells stimulate upregulation of mesenchymal stem cell activity in a GITR dependent manner.

Immunotherapy of Schizophrenia and Schizophrenia Associated Suicidal Ideation/Suicide
Application No.: 63/077723
Disclosed are methods, means, and protocols of modifying the immune system so as to induce an immunologically tolerant state insofar as T regulatory cell number and/or activity is augmented in a patient suffering from schizophrenia. In one embodiment T regulatory cells are administered to the patient from exogenous sources, be they allogeneic or autologous. In other embodiments, T regulatory cells are generated endogenously through administration of immature dendritic cells, mesenchymal stem cells, and/or pharmaceutical means.

Induction of Concurrent Pulmonary Immune Modulation and Regeneration by Protein Mediated Conjugation of Immune Regulatory Cells with Endogenous Progenitor Cells
Application No.: 17/989588
Disclosed are means, methods and compositions of matter useful for treatment of inflammatory pulmonary diseases such as COVID-19 through administration of agents that facilitate interaction between immune modulatory cells and endogenous pulmonary progenitor cells. In one embodiment a bispecific antibody capable of facilitating the interaction between CD25 on T regulatory cells and CD47 on pulmonary epithelial stem cells is described.

Treatment of Trauma Associated Cognitive Dysfunction Using Mesenchymal Stem Cell Apoptotic Bodies and Compositions Thereof
Application No.: 63/317505
Disclosed are means, treatments and compositions of matter useful for treatment of chemotherapy/radiotherapy associated cognitive dysfunction. In one embodiment the invention provides the administration of mesenchymal stem cell apoptotic bodies alone or in combination with “regenerative adjuvants” to prevent and/or reverse cognitive dysfunction associated with chemotherapy and/or radiation therapy. In other embodiments the invention teaches the utilization of stem cell apoptotic bodies for induction of neuroregeneration directly or indirectly.

Treatment of Chronic Obstructive Pulmonary Disease by Mesenchymal Stem Cell Apoptotic Bodies and Compositions Thereof
Application No.: 63/397503
Disclosed are means, treatments and compositions of matter useful for treatment of chronic obstructive pulmonary disease (COPD). In one embodiment the invention provides the administration of mesenchymal stem cell apoptotic bodies alone or in combination with “regenerative adjuvants” to prevent and/or reverse reduction in lung function associated with COPD. In other embodiments the invention teaches the utilization of stem cell apoptotic bodies for induction of pulmonary regeneration directly or indirectly.

Activated Leukocyte Extract for Repair of Innate Immunity in Cancer Patients
Application No.: 62/478498
Disclosed are compositions, methods of use, and pharmaceutical preparations useful for modulation of immune responses. In one embodiment a composition is extracted polyvalently activated peripheral blood mononuclear cells through dialysis. Said immune modulator is useful for treatment of cancer and alleviation of cancer associated immune depression. In one embodiment, said immunomodulator acts as a costimulatory of T cell activation by modulation of cytokine production. In one embodiment said immune modulator is concentrated for miRNA species capable of activating innate immune cells.

Augmentation of Anti-Tumor Immunity by Mifepristone and Analogues Thereof
Application No.: 62/478507
The present invention relates to compositions of matter and methods useful for improving a treatment outcome and/or an alteration of immunity in a condition that benefits from immune stimulation. In particular, one embodiment of the invention teaches administration of sufficient doses of mifepristone or a derivative, alone, or in combination with an immunotherapeutic such as, but not limited to, an antibody, a vaccine, a cytokine, or a medicament whose therapeutic activity is associated with immune modulation.

Methods of Re-Activating Dormant Memory Cells with Anticancer Activity
Application No.: 62/478520
Disclosed are methods, protocols and compositions of matter useful for stimulation of anticancer immune responses. In one embodiment of the invention culture of buffy coat cells is performed in an environment resembling non-physiological conditions. Buffy coat derived products are subsequently harvested, concentrated, and added to a culture of monocytes and lymphocytes. Conditioned media from said second culture is subsequently utilized as an injectable solution for stimulation of anticancer immunity.

Pluripotent Stem Cell Derived Dendritic Cells and Engineered Dendritic Cells for Cancer Immunotherapy
Publication number: 20220298491
Abstract: Disclosed are populations of dendritic cells generated from stem cells capable of inducing immunity towards cancer. In one embodiment said dendritic cells are generated from allogeneic inducible pluripotent stem cells, for some uses, said pluripotent stem cells are genetically engineered/edited to induce cancer specific immunity and/or resist immunosuppressive effect of tumor derived microenvironment. In one embodiment pluripotent stem cells are transfected with cancer stem cell antigens such as BORIS and/or NR2F6.

Example 1: iPS Derived Dendritic Cells Induce Reduction of B16 Melanoma

iPSCs (ATCC-DYR0100 Human Induced Pluripotent Stem (IPS) Cells (ATCC® ACS-1011™) were cultured on feeder layers of OP9 cells for 6 to 7 days in α-MEM supplemented with 20% FBS. The mesodermally differentiated cells were then harvested, reseeded onto fresh OP9 cell layers, and cultured in α-MEM supplemented with 20% FBS, 20 ng/mL GM-CSF, and 50 μmol/L 2-ME. On day 13 to 14, floating cells were recovered by pipetting. These cells were considered to be iPSC-derived myeloid cells (iPS-MCs). The cells were infected with lentivirus vectors expressing the c-Myc  and the Brother of the Regulatory of Imprinted Sites (BORIS) gene, as well as shRNA encoding siRNA silencing VEGF-R in the presence of 8 ng/mL polybrene (Sigma-Aldrich), and were cultured in α-MEM supplemented with 20% FBS, 30 ng/mL GM-CSF, and 30 ng/mL M-CSF. After 5 to 6 days, proliferating cells appeared and were considered to be ESC- or iPSC-derived pMCs (ES-pMC or iPS-pMC, respectively). To induce the differentiation of these cells into DC-like cells (pMC-DC), they were cultured in RPMI-1640 supplemented with 20% FBS in the presence of 20 ng/mL IL4 plus 30 ng/mL GM-CSF for 3 days.

Mice were inoculated with 500,000 B16 melanoma.  Mice where injected with saline (Control), unmodified stem cell derived DC (Unmodified), stem cell derived DC expressing BORIS (BORIS) and BORIS transfected DC with VEGF-R silenced (BORIS VEGF R Silenced).  Tumor growth was assessed by calipers.

Compositions Capable of Stimulating Immunity Towards Tumor Blood Vessels
Publication number: 20220403330
Abstract: Disclosed are novel means, protocols, and compositions of matter for eliciting an immune response against blood vessels supplying neoplastic tissue. In one embodiment pluripotent stem cells are transfected with one or more genes capable of eliciting immunity. In some embodiments such genes are engineered under control of specific promoters to allow for various specificities of activity. In one specific embodiment pluripotent stem cells engineered to endow properties capable of inducing expression of the ?-Gal epitope (Gal?1,3Gal?1,4G1cNAc-R).

Example

iPSC expressing α-Gal epitope (Galα1,3Galα1,4GlcNAc-R) were differentiated into tumor endothelial like cells by culture in VEGF (10 ng/ml), PDGF-BB (5 ng/ml), PGE-2 (100 pg/ml).  Cells were incubated with human plasma and assessed for viability using MTT assay.

Amelioration and Treatment of Opioid Addiction
Publication number: 20220323547
Abstract: Disclosed are compositions of matter, protocols and treatment means for reducing and/or preventing opioid addiction. In one embodiment the invention teaches intranasal administration of umbilical cord blood plasma, or extracts thereof, together with pterostilbene or pterostilbene containing nanoparticles, and/or oxytocin, and/or human chorionic gonadotropin.

Example.

Neuroinflammation was induced by administration of LPS at 50 ng/mouse. Cord blood plasma and cord blood plasma with pterostilbene was administered intranasally.  Assessment of TLR expression in brain homogenates were performed by flow cytometry.

Immunotherapy for Opioid Addiction
Publication number: 20220193127
Abstract: Disclosed are means, methods and compositions of matter useful for reduction of brain inflammation and prevention of opioid addiction and/or tolerance. In one embodiment the invention provides utilization of platelet rich plasma (PRP), alone, or admixed with regenerative/anti-inflammatory adjuvants, for reduction of neural inflammation. In one embodiments PRP is admixed with oxytocin and administered intranasally in a patient at risk of opioid addiction. In another embodiment, PRP is admixed with fortified and non-fortified nigella sativa oil, and/or pterostilbene and administered intranasally. Other embodiments include utilization of autologous stromal vascular fraction cells alone and/or admixed with regenerative/anti-inflammatory adjuvants.

Example 1: Reduction of Brain Microglial Activation by PRP and Pterostilbene

BALB/c mice were anesthetized with isofluorane and administered pterstilbene (0.4mg/mouse) and/or platelet rich plasma (5 microliter per mouse) via intranasal route subsequent to induction of systemic inflammation by intraperitoneal administration of endotoxin. Mice were sacrificed after 24 hours of treatment and quantification of cytokine IL-18 was performed by ELISA from brain homogenate tissue.  As seen below, a synergistic reduction of TNF-alpha was observed.

Example 2: Reduction of Brain Microglial Activation by PRP and Oxytocin

BALB/c mice were anesthetized with isofluorane and administered oxytocin (0.1 IU/mouse) and/or platelet rich plasma (5 microliter per mouse) via intranasal route subsequent to induction of systemic inflammation by intraperitoneal administration of endotoxin. Mice were sacrificed after 24 hours of treatment and quantification of cytokine IL-18 was performed by ELISA from brain homogenate tissue.  As seen below, a synergistic reduction of TNF-alpha was observed.

Treatment of Bipolar Disorder Using Mesenchymal Stem Cells and Modification of Mesenchymal Stem Cells
Application No.: 63/408017
The invention discloses the utilization of mesenchymal stem cells, exosomes from mesenchymal stem cells, conditioned media from mesenchymal stem cells, apoptotic bodies from mesenchymal stem cells, and modified mesenchymal stem cells for treatment of bipolar disorder. In one embodiment mesenchymal stem cells isolated from umbilical cord tissue are treated carbon monoxide at a concentration sufficient to induce activation of heme-oxygenase I and infused into a patient at risk or suffering from bipolar disorder.

Gene Silencing Therapy of Acute Respiratory Disorder
Application No.: 63/402304
Disclosed are treatment means, compositions of matter and protocols useful for suppression of acute respiratory disorder (ARDS) through induction of RNA interference in the pulmonary microenvironment alone and/or in conjunction with mucolytic and/or DNA disrupting agents. In one embodiment short interfering RNA (siRNA) is prepared which targets complement receptors C3R and/or C5R together with TNF-receptor, IL-6 receptor and/or TLR4 and TLR9. In some embodiments nanostilbene is utilized as a delivery vehicle for siRNA delivery.

Treatment of Chronic Traumatic Encephalopathy via RNA Administration
Application No.: 62/775402
Disclosed are protocols, treatment means, and compositions of matter useful for treatment of Chronic Traumatic Encephalopathy through administration of RNA or modified RNA molecules. In one embodiment said RNA is generated to activate various toll like receptors (TLR), of which said activation leads to production of cytokines which paradoxically lead to protection from Chronic Traumatic Encephalopathy, wherein said protection constitutes a) reduction in glial cell activation, b) neuronal apoptosis due to excitotoxicity; and c) stimulation of endogenous regenerative processes including endothelial progenitor cell mobilization, proliferation of neuronal progenitor cells in the dentate gyrus and subventricular zones. In one particular embodiment targeting of RNA molecules is performed to specific brain cells including pyramidal neurons through the use of liposomes, exosomes, apoptotic bodies, nanoparticles and shark or cameloid antibodies is disclosed.

VasoSome’s product VSX-001 is a proprietary nanoparticle derived from specialized stem cells that can be utilized in a “universal donor” manner, meaning it does not have to be matched with the donor. The product possesses numerous properties of stem cells, including inhibition of inflammation, suppression of damage to blood vessels, and ability to regenerate damaged tissue. Advantages of administering VSX-001 compared to stem cells include: a) VSX-001 is substantially smaller in size than stem cells, allowing for superior distribution; b) VSX-001 does not multiply, thus possessing a superior long-term safety profile; and c) VSX-001 can be manufactured more economically as compared to stem cells.

Aneurysm Treatment by Exosomes which provides means of inhibition and/or treating aneurysms and other degenerated blood vessels through administration of regenerative cell derived exosomes, and/or regenerative cell derived apoptotic bodies. In one particular embodiment vessel regeneration is increased through administration of stem cell exosomes/or stem cell apoptotic bodies. Other embodiments include regeneration of vessels prone to aneurysms, repairing aneurysms of vessels, or acceleration of endothelialization after stent placement. Provided within the invention are methods of rejuvenating properties of said vessels associated with physiological health, examples of which include appropriate production of anti-coagulating/clotting factors, control of angiogenesis, and appropriate revascularization of injured tissue.