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Amyotrophic lateral sclerosis (ALS) is a motor neuron disease of animals and man with life-altering consequences. The defining pathology is destruction of neurons resulting in loss of neuromuscular connections. Genetic predisposition to develop ALS is a factor in a small number of cases, about 10%. The most commonly recognized presentation is idiopathic (no identified cause) and results in spontaneous disease (sALS). Spontaneous ALS may be due to multiple factors and that means treating disease would require multiple therapies in a targeted, individual approach.

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Our hypothesis is that sALS initiates innate immune mechanisms that become dysfunctional at the cellular level. We suggest that a bystander mechanism could initiate innate immune responses that become dysregulated and the dysfunctional systems vary with the site of the pathology. Therefore, it becomes important to identify individual disease mechanisms with biomarkers and have targeted treatments for each individual.

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The mission of Neurodegenerative Disease Research Inc., NDR, is to identify and validate biomarkers for determining drug effectiveness in neurodegenerative diseases and to disseminate research findings to interested parties via peer reviewed publications.

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Our areas of interest are in vitro cell testing to identify disease pathogenesis in response to drug molecules, facilitate modeling to test treatments for specific immune pathways, and evaluate bioassays that could potentially assist in determining a response to treatment.

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In 1978 Dr. Gideon Goldstein observed that the molecule levamisole, an effective antihelminthic agent as well as an immunoregulatory agent, exerted its putative immuno-regulatory action by mimicry of the thymic hormone thymopoietin.

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He suggested that levamisole formed a thymopoietin-mimetic tertiary structure and stimulated lymphocytes by its imidazole component or possibly metabolized to OMPI, a reducing compound which affects radical scavenging in activated lymphocytes.

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The body of work produced by Dr. Goldstein demonstrated the physiological effects of thymopoietin on the immune system including neutrophils, macrophages, and lymphocytes and most importantly on regulatory T cells that restore homeostasis in a dysregulated immune system.

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Thymopoietin is a polypeptide hormone produced by the thymus and skin. An active region of thymopoietin is a pentapeptide, thymopentin, that has all the biological activities of the native hormone. The skin produces a regulatory molecule with one base modification in the structure that eliminates the neuromuscular action.

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Thymopentin was developed into a therapy for chronic hepatitis B, chronic hepatitis C, an adjuvant therapy for chemotherapy-induced immune depression, immune insufficiency and immune suppression in patients with non-small cell lung carcinoma, malignant melanoma, hepatocellular carcinoma, breast cancer, non-Hodgkin’s lymphoma, colorectal cancer, head and neck cancer, leukemia’s, pancreatic carcinoma, and renal cell carcinoma.

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Biological experiments had determined that the thymus secreted a hormone that regulated neuromuscular transmission and the pentapeptide affected the neuromuscular regulatory function. The mechanism of action of thymopentin is not completely understood, but thymopoietin promotes T-cell differentiation and maturation, increases production of INF-γ, IL2, IL3, and expression of IL2 receptors following activation by mitogens or antigens, increase NK cell activity, and increases the production of migratory inhibitory factor, MIF.

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The similarity of the structure of thymopentin (above, left) and levamisole (above, right) are illustrated. The chemical levamisole phosphate is used in ruminants as an antihelminthic and levamisole HCl, a less toxic molecule (below, left), is use as an immunomodulatory agent in horses and people. Steroid-sensitive idiopathic nephrotic syndrome is a frequent disorder in children in which levamisole HCL was shown to be effective in maintaining a steroid-free remission. It is possible that there is an age-related difference in levamisole metabolism that needs investigation. The metabolites of levamisole is shown below, right.

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There is an increased incidence of agranulocytosis associated with levamisole in some adult patients, about 5% of the population. There may be an increased risk of agranulocytosis in patients carrying the leukocyte antigen B27 genotype.

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One of NDRs interests is to identify the agranulocytic stimulating aspect of levamisole HCl, or a metabolite, allowing us to modify the chemical structure. Maintaining the pluripotent immunomodulating activities of a substituted levamisole HCl molecule, while preventing toxicity, may be a benefit to patients with neuromuscular degenerative diseases.

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Amyotrophic lateral sclerosis is a heterogeneous neurodegenerative disorder with a variable site of disease initiation and rate of progression. There are no established blood or CSF-based markers that outperform clinical observation to provide an early diagnosis, accurate prediction of disease progression, or clinical stratification of ALS phenotypic variants. (Leoni E, 2019) Proinflammatory cytokines, reactive oxygen species (ROS), and pro-inflammatory lipid-derived compounds result in neuronal damage and supply a positive feedback loop of neuroinflammation. A multiplicity of proinflammatory cytokines can compensate in the absence of any single factor and it is unlikely that continuing efforts to target a single factor in humans will provide significant therapeutic benefit in patients with ALS. (Trostchansky, 2019)

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Since many factors affect the onset and progression of the disease, it is important to identify biomarkers to aid the identification of drugs that influence and mitigate the damage, prevent motor neuron loss and neuromuscular denervation. Analyzing signals from adipose tissue cells (ATC’s) and how the population of endothelial cells from ATC change over time may be useful. The individual manifestation of the disease including a variable site of disease initiation and rate of progression will require patient-specific treatments. An in vitro analysis of an individual’s current disease state to direct treatment is a worthy goal. Small molecules and stem cells are active areas of investigation in ALS therapies that aren’t based on patient-staged disease.

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Stem cells are used as a restorative therapy in ALS. The risk of damaging the nervous system and exacerbating disease with stem cells is apparent. In addition to physical damage the transplanted cells are not homogenous across patients or appropriate at all stages of disease. Autologous stem cells are sensitive to the microenvironment and consideration of the microenvironment is appropriate to both the source and destination of the transplanted cells.

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Locally, motor neuron oxidative stress as it occurs in ALS is exacerbated by induction of uncoupling proteins (UCP). What is interesting is that in disease there is increased aerobic glycolysis, mitochondrial cell stress, low ATP and UCP upregulation that results in intracellular toxicity while diabetes and mild obesity is associated with increased blood glucose and may be protective against ALS. There is some indication that the pathogenesis of ALS may be due to a “crosstalk“ between vascular endothelium in the central nervous system (CNS) and cells in adipose tissue. Theoretically, as subclinical disease progresses to a clinical state, the crosstalk could signal and alter the adipose tissue, an effect in the literature called “browning”, increasing the beige/brown fat population. A local outcome (at the vascular endothelium) of browning would be an “uncoupling of ATP production from oxygen consumption in the electron transport chain. Mitochondrial uncoupling leads to futile cycling of ATP synthase, consuming oxygen, expending calories and producing heat” (Yang 2017). Metabolic dysfunctions in ALS were discussed by Tefera (2017). We propose it is beneficial to evaluate ATC from ALS patients for browning.

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It is known that paracrine secretions from stem cells are beneficial in animal models of ALS. (Walker 2019) It is therefore valuable to study stem cell secretions provided in secretome/conditioned media from in vitro culture of stem cells to modulate the immune and inflammatory component ALS and effect neuroprotection for motor neurons. In this way, only the beneficial components are administered to the patient once the correct consortium of agents are recognized. Stem cells are sourced from multiple tissues, ATC may prove uniquely beneficial.

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Critical information to understand the application of stem cell secretome/conditioned media is adequately characterizing the effective components mediating therapeutic benefits and optimizing the medium for clinical application. A proposed source of conditioned media is from ASC recognizing the possible communication between the central nervous system and ATC. We anticipate that the diseased environment may influence the phenotype of ASC’s and propose to evaluate the frequency (# per gram of tissue) of phenotype by identifying three cell surface markers using flow cytometry. In addition, mitochondrial aging and secretion of antioxidants will be evaluated by several markers (including TMRM, ABA ELISA, FGF2 ELISA, qRT-PCR for senescence genes, secretome). The cultured ATC’s can serve in an in vitro system (confidential information) to test drug effects on the characterized ATC population.

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The likelihood for progress toward a stem cell based, cell-free therapy in humans is that there already is a precedent. The dysfunction and function of endothelial cells determined by cell surface antibodies by flow cytometry as well as the ability to self-heal after stress can be measured. Because there is a phenotypic specific molecular footprint suggesting that treatments may only be effective if directed against the disease when the pathology can be pinned down to a defined molecular profile, analysis of ASC could provide a snapshot of the disease and indicate an appropriate therapy.