JUPITER, FL, United States
JUPITER, FL, United States

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Weissmann C.,Scripps Florida | Li J.,Scripps Florida | Mahal S.P.,Scripps Florida | Browning S.,Scripps Florida | Browning S.,Cytonics Corporation
EMBO Reports | Year: 2011

Prions consist mainly, if not entirely, of PrP Sc, an aggregated conformer of the host protein PrP C. Prions come in different strains, all based on the same PrP C sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected. © 2011 Euroopeean Moleolecullar Bioloology Organnization.


Browning S.R.,Cytonics Corporation | Weiser A.M.,Cytonics Corporation | Woolf N.,Cytonics Corporation | Golish S.R.,UNITED ORTHOPEDIC GROUP | And 4 more authors.
Journal of Bone and Joint Surgery - Series A | Year: 2012

Background: The effect of platelet-rich plasma on chondrocytes has been studied in cell and tissue culture. Less attention has been given to the effect of platelet-rich plasma on nonchondrocytic cell lineages within synovial joints, such as fibroblast-like synoviocytes, which produce cytokines and matrix metalloproteinases (MMPs) that mediate cartilage catabolism. The purpose of the present study was to determine the effect of platelet-rich plasma on cytokines and proteases produced by fibroblast-like synoviocytes. Methods: Platelet-rich plasma and platelet-poor plasma from harvested autologous blood were prepared with a commercially available system. Fibroblast-like synoviocytes were treated with platelet-rich plasma, platelet-poor plasma, recombinant PDGFββ (platelet-derived growth factor ββ), or phosphate-buffered saline solution and incubated at 37 °C for forty-eight hours. The concentrations of IL-1β (interleukin-1β), IL-1RA (IL-1 receptor antagonist), IL-6, IFN-γ (interferon-γ), IP-10 (interferon gamma-induced protein 10), MCP-1 (monocyte chemotactic protein-1), MIP-1β (macrophage inflammatory protein-1β), PDGFββ, RANTES, TNF-α (tumor necrosis factor-α), VEGF (vascular endothelial growth factor), MMP-1, MMP-3, and MMP-9 in the culture medium were determined by multiplex immunoassay. Results: Platelet-rich plasma cultured in medium contained multiple catabolic mediators in substantial concentrations, including MMP-9 (15.8 ± 2.3 ng/mL) and MMP-1 (2.5 ± 0.8 ng/mL), as well as proinflammatory mediators IL-1β, IL-6, IFN-γ, IP-10, MCP-1, MIP-1β, RANTES, and TNF-α in concentrations between 20 pg/mL and 20 ng/mL. Platelet-poor plasma contained significantly lower concentrations of these compounds. Platelet-rich plasma was used to treat human fibroblast-like synoviocytes, and the resulting concentrations of mediators were corrected for the concentrations in the platelet-rich plasma alone. Compared with untreated fibroblast-like synoviocytes, synoviocytes treated with platelet-rich plasma exhibited significantly greater levels of MMP-1 (363 ± 94.0 ng/mL, p = 0.018) and MMP-3 (278 ± 90.0 ng/mL, p = 0.018). In contrast, platelet-poor plasma had little effect on mediators secreted by the synoviocytes. PDGFββ-treated fibroblast-like synoviocytes exhibited a broad proinflammatory cytokine response at four and forty-eight hours. Conclusions: Platelet-rich plasma was shown to contain a mixture of anabolic and catabolic mediators. Synoviocytes treated with platelet-rich plasma responded with substantial MMP secretion, which may increase cartilage catabolism. Synoviocytes responded to PDGF with a substantial proinflammatory response. Clinical Relevance: The multiple catabolic mediators in platelet-rich plasma and the secretion of MMPs by fibroblast-like synoviocytes treated with platelet-rich plasma could potentially accelerate cartilage catabolism, and this warrants further investigation. Copyright © 2012 by The Journal of Bone and Joint Surgery, Incorporated.


Scuderi G.J.,Stanford University | Golish S.R.,Stanford University | Cook F.F.,Jupiter Medical Center | Cuellar J.M.,New York University | And 2 more authors.
Journal of Bone and Joint Surgery - Series A | Year: 2011

Background: Molecular biomarkers associated with knee pain may be useful as diagnostic modalities, prognostic indicators, and surrogate end points for therapeutic trials. The present study describes a novel complex of fibronectin and aggrecan that is present in the affected knee of patients with pain and meniscal abnormality. Methods: The present prospective study included thirty patients with knee pain, mechanical symptoms, and magnetic resonance imaging findings that were positive for a meniscal tear who chose arthroscopic partial meniscectomy after unsuccessful nonoperative management. Synovial fluid was aspirated at the time of surgery and was assayed for the fibronectinaggrecan complex with use of a heterogeneous enzyme-linked immunosorbent assay (ELISA). The results were compared with knee aspirates from ten asymptomatic volunteers with no pain who underwent magnetic resonance imaging of the knee. Results: The mean optical density (and standard deviation) of the fibronectin-aggrecan complex was significantly greater in synovial fluid from knees undergoing arthroscopic surgery as compared with fluid from asymptomatic controls (13.29 ± 8.48 compared with 0.03 ± 0.09; p < 0.001). The mean age in the study group was significantly greater than in control group (46.0 ± 12.6 compared with 38.5 ± 6.0 years; p = 0.02), but controlling for age did not affect the results. Post hoc, an optical density cutoff value of 0.3 distinguished the study group from the control group with 100% accuracy. Conclusions: A novel fibronectin-aggrecan complex is present in the synovial fluid of painful knees with meniscal abnormality. The fibronectin-aggrecan complex may prove to be useful as a clinical biomarker or therapeutic target. Further research is warranted to correlate functional outcome after surgery with the fibronectin-aggrecan complex and other cartilage biomarkers. Level of Evidence: Diagnostic Level IV. See Instructions to Authors for a complete description of levels of evidence. Copyright © 2011 by The Journal of Bone and Joint Surgery, Incorporated.


Abrams G.D.,Stanford University | Abrams G.D.,Hines Veterans Administration Hospital | Safran M.R.,Stanford University | Shapiro L.M.,Stanford University | And 5 more authors.
Knee Surgery, Sports Traumatology, Arthroscopy | Year: 2014

Purpose: To report hip synovial fluid cytokine concentrations in hips with and without radiographic arthritis. Methods: Patients with no arthritis (Tonnis grade 0) and patients with Tonnis grade 2 or greater hip osteoarthritis (OA) were identified from patients undergoing either hip arthroscopy or arthroplasty. Synovial fluid was collected at the time of portal establishment for those undergoing hip arthroscopy and prior to arthrotomy for the arthroplasty group. Analytes included fibronectin-aggrecan complex (FAC) as well as a standard 12 cytokine array. Variables recorded were Tonnis grade, centre-edge angle of Wiberg, as well as labrum and cartilage pathology for the hip arthroscopy cohort. A priori power analysis was conducted, and a Mann-Whitney U test and regression analyses were used with an alpha value of 0.05 set as significant. Results: Thirty-four patients were included (17 arthroplasty, 17 arthroscopy). FAC was the only analyte to show a significant difference between those with and without OA (p < 0.001). FAC had significantly higher concentration in those without radiographic evidence of OA undergoing microfracture versus those not receiving microfracture (p < 0.05). Conclusion: There was a significantly higher FAC concentration in patients without radiographic OA. Additionally, those undergoing microfracture had increased levels of FAC. As FAC is a cartilage breakdown product, no significant amounts may be present in those with OA. In contrast, those undergoing microfracture have focal area(s) of cartilage breakdown. These data suggest that FAC may be useful in predicting cartilage pathology in those patients with hip pain but without radiographic evidence of arthritis. Level of evidence: Diagnostic, Level III. © 2014 Springer-Verlag Berlin Heidelberg.


Patent
Cytonics Corporation | Date: 2013-02-21

Systems and methods for purification and concentration of autologous alpha-2-macroglobulin (A2M) from whole blood are provided. Also provided are diagnostic methods for identifying sites in the synovial joints, spine, tendons or ligaments for treatment of pain, degeneration, or inflammation with autologous A2M. Methods for utilizing autologous A2M in combination with other autologous treatments (e.g. platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half life of the protein in joints and spine disc or epidural space.


Systems and methods for purification and concentration of autologous alpha-2 macroglobulin (A2M) from whole blood and or recombinant A2M are provided. Also provided are methods of treating wounds with A2M. Methods for utilizing A2M in combination with other treatments (e.g., platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half-life of the protein for treating wounds.


Systems and methods for purification and concentration of autologous alpha-2 macroglobulin (A2M) from whole blood and or recombinant A2M are provided. Also provided are methods of treating wounds with A2M. Methods for utilizing A2M in combination with other treatments (e.g., platelets and other growth factors) are provided in addition to combinations with exogenous drugs or carriers. Also provided is a method of producing recombinant A2M wild type or variants thereof where the bait region was modified to enhance the inhibition characteristics of A2M and/or to prolong the half-life of the protein for treating wounds.


Browning S.R.,Cytonics Corporation
The Journal of bone and joint surgery. American volume | Year: 2012

The effect of platelet-rich plasma on chondrocytes has been studied in cell and tissue culture. Less attention has been given to the effect of platelet-rich plasma on nonchondrocytic cell lineages within synovial joints, such as fibroblast-like synoviocytes, which produce cytokines and matrix metalloproteinases (MMPs) that mediate cartilage catabolism. The purpose of the present study was to determine the effect of platelet-rich plasma on cytokines and proteases produced by fibroblast-like synoviocytes. Platelet-rich plasma and platelet-poor plasma from harvested autologous blood were prepared with a commercially available system. Fibroblast-like synoviocytes were treated with platelet-rich plasma, platelet-poor plasma, recombinant PDGFββ (platelet-derived growth factor ββ), or phosphate-buffered saline solution and incubated at 37°C for forty-eight hours. The concentrations of IL-1β (interleukin-1β), IL-1RA (IL-1 receptor antagonist), IL-6, IFN-γ (interferon-γ), IP-10 (interferon gamma-induced protein 10), MCP-1 (monocyte chemotactic protein-1), MIP-1β (macrophage inflammatory protein-1β), PDGFββ, RANTES, TNF-α (tumor necrosis factor-α), VEGF (vascular endothelial growth factor), MMP-1, MMP-3, and MMP-9 in the culture medium were determined by multiplex immunoassay. Platelet-rich plasma cultured in medium contained multiple catabolic mediators in substantial concentrations, including MMP-9 (15.8 ± 2.3 ng/mL) and MMP-1 (2.5 ± 0.8 ng/mL), as well as proinflammatory mediators IL-1β, IL-6, IFN-γ, IP-10, MCP-1, MIP-1β, RANTES, and TNF-α in concentrations between 20 pg/mL and 20 ng/mL. Platelet-poor plasma contained significantly lower concentrations of these compounds. Platelet-rich plasma was used to treat human fibroblast-like synoviocytes, and the resulting concentrations of mediators were corrected for the concentrations in the platelet-rich plasma alone. Compared with untreated fibroblast-like synoviocytes, synoviocytes treated with platelet-rich plasma exhibited significantly greater levels of MMP-1 (363 ± 94.0 ng/mL, p = 0.018) and MMP-3 (278 ± 90.0 ng/mL, p = 0.018). In contrast, platelet-poor plasma had little effect on mediators secreted by the synoviocytes. PDGFββ-treated fibroblast-like synoviocytes exhibited a broad proinflammatory cytokine response at four and forty-eight hours. Platelet-rich plasma was shown to contain a mixture of anabolic and catabolic mediators. Synoviocytes treated with platelet-rich plasma responded with substantial MMP secretion, which may increase cartilage catabolism. Synoviocytes responded to PDGF with a substantial proinflammatory response.


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 192.30K | Year: 2010

DESCRIPTION (provided by applicant): Spinal related complaints are the most common reason for physician visits and healthcare expenditures. Conditions related to back pain account for more hospitalizations than any other musculoskeletal condition and the back is the body part most often involved in work-related disabilities. The spine market is the fastest growing market in orthopedics, creating a unique opportunity to commercialize innovative diagnostic and treatment technologies that are tailored towards specific clinical challenges. One of the greatest challenges for physicians is reaching an accurate diagnosis when faced with certain orthopedic problems. Spinal related sciatic pain can have multiple etiologies, creating diagnostic challenges. Various technologies exist to assist physicians to accurately diagnose these patients, but many are expensive, time consuming and require multiple trips to care providers. A rapid, less costly and accurate diagnosis would greatly benefit the patient. Cytonics Corporation is a privately held biotechnology company that has discovered a novel protein biomarker that not only identifies the source of radicular pain but also specifies an effective treatment course. Objectives during this SBIR Phase I funding period are to further characterize and validate a novel diagnostic biomarker for back pain syndromes that cause radiculopathy. Two Specific Aims are proposed to accomplish these objectives. The first Aim further characterizes a novel protein complex identified by Cytonics as a biomarker of pain caused by low back pain (LBP) syndromes. A standardized antibody based assay will be developed that specifically recognizes this new protein complex biomarker. The second Aim performs a prospective clinical research study to validate the diagnostic assay. Efficient and specific diagnostic tests for neurological root irritation caused by LBP syndromes that also indicate effective treatment strategies will fill an unmet medical need and establish Cytonics as a leader within a large and expanding marketplace. In addition, Cytonics will obtain invaluable information on disease pathogenesis and biochemical targets for the expeditious development of a next generation diagnostic assay and pharmacotherapeutic(s). PUBLIC HEALTH RELEVANCE: Low back pain (LBP) syndromes are one of the most common forms of pain and causes of disability. Americans spend at least 90 billion each year on low back pain, the most common cause of job-related disability and a leading contributor to missed work with an estimated economic burden in the US in hundreds of billions per year. There are many causes of back pain, and proper determination of the source of pain helps guide treatment strategies. Common causes of back pain include disc herniation, spinal stenosis, back muscle strain, and disc degeneration. Pathology of one or more lumbar intervertebral discs is thought to result in severe pain and abnormal sensations radiating from the nerve root down the leg (radiculopathy). Current methods to diagnose radiculopathy and determine proper treatment strategies (steroid injections, surgery, or rest and hope for pain resolution) are inadequate and therefore a very large market and unmet medical need exists. The overall objectives of this clinical proteomics study are to characterize and validate a novel protein complex as a biomarker for radicular pain that also identify patients that will respond to epidural steroid treatment. The objectives of Cytonics Corporation during this SBIR Phase I funding period is to address the following specific aims related to the diagnosis and treatment of LBP syndromes that cause neurological irritation: Aim 1: Further characterize our recently-identified biomarker protein complex and standardize the Cytonics diagnostic assay prototype for neurological root irritation caused by LBP syndromes. A) Additional mass spectrometry and antibody-based approaches will be used to further characterize the novel biomarker protein complex identified in our new preliminary studies. B) Reproducibility, sensitivity and accuracy are essential for robust biomarker detection. Thus, antibody reagents and assay reference standards must be tested to generate the best protocols to maximize diagnostic signal reproducibility and accuracy. These protocols and reagents will be used in all subsequent product development steps and for sample collection kits to be marketed to clinics. Aim 2: Perform a prospective clinical validation study for the Cytonics diagnostic assay. A) Collect patient samples in a multi-center clinical research study. B) Measure levels of the novel protein complex by ELISA generated in Aim 1. Completion of the proposed Specific Aims will enable Cytonics Corporation to create novel and proprietary diagnostic tests that can be rapidly brought to market during a subsequent SBIR Phase II protocol. Efficient and specific diagnostic tests for neurological root irritation caused by LBP syndromes that also indicate effective treatment strategies will fill an unmet medical need and establish Cytonics as a leader within a large and expanding marketplace. In addition, Cytonics will obtain invaluable information on disease pathogenesis and biochemical targets for the expeditious development of a next generation diagnostic assay and pharmacotherapeutic(s).


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 1.60M | Year: 2011

DESCRIPTION (provided by applicant): This is a proposal to collect the necessary data for FDA regulatory approval of the Cytonics assay as a diagnostic tool to identify patients who will respond to steroid injection therapy for LBP due to radiculopathy. Low back pain (LBP) syndromes include spinal degenerative conditions with and without nerve root or cauda equina irritation. These syndromes represent some of the most common reasons for patients to seek medical attention. LBP syndromes associated with neurological irritation are often effectively treated, whereas those not primarily associated with neurological irritation have proven resistant. The greatest current challenge in the clinical care of patients with LBP syndromes is identifying those who sufferfrom active neurological irritation and therefore may benefit from interventional therapies, such as corticosteroid injection (ESI). However, there is currently no validated test to discriminate between patients who will or will not respond to ESI. For example, only ~50% of the gt10 million yearly epidural steroid injections successfully relieve pain. Many patients that don't respond to initial steroid injections will receive follow-up injections at the same or a different spinal location. These unnecessaryinjections are costly and have side effects. In Phase I, we developed and validated an assay that can prospectively identify patients who will respond to steroid treatment. Our Specific Aim in Phase II is to collect the necessary data for FDA regulatory approval of the Cytonics assay as a diagnostic tool to identify patients who will respond to steroid injection therapy for LBP due to radiculopathy. To achieve this Objective, we will carry out the following Tasks: Task #1: Validate our assay to meet FDA manufacturing guidelines. Task #2: Carry out a large prospective study appropriate for obtaining PMA clearance using our validated assay. Task #3: Prepare and submit a PMA to the FDA. The assay will be sold as a kit, complete with all of the reagents required for the assay. We will provide the diagnostic kit to CLIA labs that are interested in providing the testing service, many of which will be located in surgical hospitals or outpatient centers. We will utilize a network of medical product distributors to provide information on the diagnostic to spine surgeons, neurologist, and pain management specialists in an effort to encourage the use of the test for their patients. We will also publish and present extensively on results of using our product. Phase II will result in a diagnostic assay to be submitted for FDA approval. PUBLIC HEALTH RELEVANCE: This is a project to develop a diagnostic assay that helps physicians know when to use steroid injections for treating back pain. Currently, 10 million such injections are made each year at a cost of about 2000 per injection. Half of the injections are ineffective. Our assay will allow physicians to know when a patient will not respond to an injection, thereby avoiding the needless injections and saving about10 billion dollars per year in ineffective treatments, as well as allowing the physician to focus on treating the patient more effectively.

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