Co-localization and confinement of ecto-nucleotidases modulate extracellular adenosine nucleotide distributions.

Nucleotides comprise small molecules that perform critical signaling roles in biological systems. Adenosine-based nucleotides, including adenosine tri-, di-, and mono-phosphate, are controlled through their rapid degradation by diphosphohydrolases and ecto-nucleotidases (NDAs). The interplay between nucleotide signaling and degradation is especially important in synapses formed between cells, which create signaling 'nanodomains'. Within these 'nanodomains', charged nucleotides interact with densely-packed membranes and biomolecules. While the contributions of electrostatic and steric interactions within such nanodomains are known to shape diffusion-limited reaction rates, less is understood about how these factors control the kinetics of nucleotidase activity. To quantify these factors, we utilized reaction-diffusion numerical simulations of 1) adenosine triphosphate (ATP) hydrolysis into adenosine monophosphate (AMP) and 2) AMP into adenosine (Ado) via two representative nucleotidases, CD39 and CD73. We evaluate these sequentially-coupled reactions in nanodomain geometries representative of extracellular synapses, within which we localize the nucleotidases. With this model, we find that 1) nucleotidase confinement reduces reaction rates relative to an open (bulk) system, 2) the rates of AMP and ADO formation are accelerated by restricting the diffusion of substrates away from the enzymes, and 3) nucleotidase co-localization and the presence of complementary (positive) charges to ATP enhance reaction rates, though the impact of these contributions on nucleotide pools depends on the degree to which the membrane competes for substrates. As a result, these contributions integratively control the relative concentrations and distributions of ATP and its metabolites within the junctional space. Altogether, our studies suggest that CD39 and CD73 nucleotidase activity within junctional spaces can exploit their confinement and favorable electrostatic interactions to finely control nucleotide signaling.

Iron nanoparticle-labeled murine mesenchymal stromal cells in an osteoarthritic model persists and suggests anti-inflammatory mechanism of action.

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA, but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages, which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™, iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints, and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery, mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1, 2 and 4weeks]). 4 weeks after injection, mice were imaged by MRI, and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS, CD206, F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory, homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively, and iii) Masson's Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs, although some ferumoxytol label was detected in proximity to MSCs in macrophages, likely due to phagocytosis of apoptotic MSCs, increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time, we show that MSC-treated mice had increased ratios of %CD206+: %F4/80+ (homeostatic macrophages) (p<0.05), and decreased ratios of %iNOS+: %F4/80+ macrophages (p<0.01), supporting our hypothesis that MSCs may modulate synovial inflammation.

Bone Marrow Mesenchymal Stromal Cell Treatment in Patients with Osteoarthritis Results in Overall Improvement in Pain and Symptoms and Reduces Synovial Inflammation.

Patients with late-stage Kellgren-Lawrence knee osteoarthritis received a single intra-articular injection of 1, 10, or 50 million bone marrow mesenchymal stromal cells (BM-MSCs) in a phase I/IIa trial to assess safety and efficacy using a broad toolset of analytical methods. Besides safety, outcomes included patient-reported outcome measures (PROMs): Knee Injury and Osteoarthritis Outcome Score (KOOS) and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC); contrast-enhanced magnetic resonance imaging (MRI) for cartilage morphology (Whole Organ MRI Scores [WORMS]), collagen content (T2 scores), and synovitis; and inflammation and cartilage turnover biomarkers, all over 12 months. BM-MSCs were characterized by a panel of anti-inflammatory markers to predict clinical efficacy. There were no serious adverse events, although four patients had minor, transient adverse events. There were significant overall improvements in KOOS pain, symptoms, quality of life, and WOMAC stiffness relative to baseline; the 50 million dose achieved clinically relevant improvements across most PROMs. WORMS and T2 scores did not change relative to baseline. However, cartilage catabolic biomarkers and MRI synovitis were significantly lower at higher doses. Pro-inflammatory monocytes/macrophages and interleukin 12 levels decreased in the synovial fluid after MSC injection. The panel of BM-MSC anti-inflammatory markers was strongly predictive of PROMs over 12 months. Autologous BM-MSCs are safe and result in significant improvements in PROMs at 12 months. Our analytical tools provide important insights into BM-MSC dosing and BM-MSC reduction of synovial inflammation and cartilage degradation and provide a highly predictive donor selection criterion that will be critical in translating MSC therapy for osteoarthritis. Stem Cells Translational Medicine 2019;8:746&757.

19F-perfluorocarbon-labeled human peripheral blood mononuclear cells can be detected in vivo using clinical MRI parameters in a therapeutic cell setting.

A 19Fluorine (19F) perfluorocarbon cell labeling agent, when employed with an appropriate cellular MRI protocol, allows for in vivo cell tracking. 19F cellular MRI can be used to non-invasively assess the location and persistence of cell-based cancer vaccines and other cell-based therapies. This study was designed to determine the feasibility of labeling and tracking peripheral blood mononuclear cells (PBMC), a heterogeneous cell population. Under GMP-compliant conditions human PBMC were labeled with a 19F-based MRI cell-labeling agent in a manner safe for autologous re-injection. Greater than 99% of PBMC labeled with the 19F cell-labeling agent without affecting functionality or affecting viability. The 19F-labeled PBMC were detected in vivo in a mouse model at the injection site and in a draining lymph node. A clinical cellular MR protocol was optimized for the detection of PBMC injected both at the surface of a porcine shank and at a depth of 1.2 cm, equivalent to depth of a human lymph node, using a dual 1H/19F dual switchable surface radio frequency coil. This study demonstrates it is feasible to label and track 19F-labeled PBMC using clinical MRI protocols. Thus, 19F cellular MRI represents a non-invasive imaging technique suitable to assess the effectiveness of cell-based cancer vaccines.

Strategy for an abbreviated in-house qualification of a commercially available Rapid Microbiology Method (RMM) for canadian regulatory approval.

BACKGROUND AIMS: Cell therapy products (CTP) typically require full sterility, endotoxin and Mycoplasma testing before product release. Often this is not feasible with fresh cells, and sponsors may rely on rapid microbiological methods (RMM). RMM must be qualified in-house using the sponsor's facilities, equipment, consumables, cells and matrices to meet regulatory approval. Herein, we present a cost-effective strategy to conduct an in-house abbreviated qualification of a commercially available RMM kit to meet Health Canada regulatory requirements. METHODS: We performed an abbreviated qualification using a polymerase chain reaction (PCR)-based Mycoplasma testing method involving assay sensitivity and ruggedness, based on an experimental plan that was pre-approved by Health Canada. Briefly, investigational CTPs were tested in-house using a PCR-based Mycoplasma detection kit. Assay sensitivity was determined using a 10-fold dilution series of genomic DNA of only two Mycoplasma species, Mycoplasma arginini and Mycoplasma hominis in the absence of CTP-matrix as the kit had been previously validated against nine species. Matrix interference was measured by testing independent CTP samples. Testing by different operators on different days measured ruggedness. RESULTS: The RMM Mycoplasma qualification exceeded sensitivity (4 genome copies per reaction for M. arginini and 0.12 genome copies per reaction for M. hominis) and met ruggedness requirements without matrix interference, as required by the Pharmacopoeial guidelines (Ph. Eur. 2.6.7 and USP <1223>). DISCUSSION: Our approach represents a minimal qualification that can be performed by an academic institution while ensuring regulatory compliance for implementing RMM testing for in-process and product-release testing of CTPs.

A method to obtain mean organ doses in a RANDO phantom.

A quantitative method of obtaining average organ dose from point measurements made in the male RANDO phantom is described for 24 compact organs of interest in patient dosimetry. A three-dimensional Cartesian coordinate system was created by considering each of the 36 RANDO phantom sections as the z coordinate, and using a rectangular frame to assign x and y coordinates relative to the center of each section. Anatomical atlases and clinical experience were used to identify the location and extent of each organ and tissue in the RANDO phantom. This proposed scheme is comparable to one used in a commercial phantom and offers investigators a comprehensive protocol for obtaining mean organ doses in the RANDO phantom.