Neutrophils infiltrate sensory ganglia and mediate chronic widespread pain in fibromyalgia.

Fibromyalgia is a debilitating widespread chronic pain syndrome that occurs in 2 to 4% of the population. The prevailing view that fibromyalgia results from central nervous system dysfunction has recently been challenged with data showing changes in peripheral nervous system activity. Using a mouse model of chronic widespread pain through hyperalgesic priming of muscle, we show that neutrophils invade sensory ganglia and confer mechanical hypersensitivity on recipient mice, while adoptive transfer of immunoglobulin, serum, lymphocytes, or monocytes has no effect on pain behavior. Neutrophil depletion abolishes the establishment of chronic widespread pain in mice. Neutrophils from patients with fibromyalgia also confer pain on mice. A link between neutrophil-derived mediators and peripheral nerve sensitization is already established. Our observations suggest approaches for targeting fibromyalgia pain via mechanisms that cause altered neutrophil activity and interactions with sensory neurons.

Gate control of sensory neurotransmission in peripheral ganglia by proprioceptive sensory neurons.

Melzak and Wall's gate control theory proposed that innocuous input into the dorsal horn of the spinal cord represses pain-inducing nociceptive input. Here we show that input from proprioceptive parvalbumin-expressing sensory neurons tonically represses nociceptor activation within dorsal root ganglia. Deletion of parvalbumin-positive sensory neurons leads to enhanced nociceptor activity measured with GCaMP3, increased input into wide dynamic range neurons of the spinal cord and increased acute and spontaneous pain behaviour, as well as potentiated innocuous sensation. Parvalbumin-positive sensory neurons express the enzymes and transporters necessary to produce vesicular GABA that is known to be released from depolarized somata. These observations support the view that gate control mechanisms occur peripherally within dorsal root ganglia.

Mesenchymal stromal cell secretory molecules improve the functional survival of human islets.

AIMS: Human islet transplantation as a therapy for type 1 diabetes is compromised by the loss of functional beta cells in the immediate post-transplantation period. Mesenchymal stromal cells (MSCs) and MSC-derived secretory peptides improve the outcomes of islet transplantation in rodent models of diabetes. Here, we utilized a mouse model for human islet transplantation and assessed the effects of a cocktail of MSC-secreted peptides (screened by MSC-secretome for human islet GPCRs) on the functional survival of human islets. METHODS: Human islets from nine donors (Age: 36-57; BMI: 20-35) were treated with a cocktail of human recombinant annexin A1 (ANXA1), stromal cell-derived factor-1 (SDF-1/CXCL12) and complement component C3 (C3a). Glucose-stimulated insulin secretion (GSIS) was assessed in static incubation, and cytokine-induced apoptosis was assessed by measuring caspase 3/7 activity. mRNA expression levels were determined by qPCR. Human islet function in vivo was assessed using a novel model for human islet transplantation into a T1D mouse model. Human islet function in vivo was assessed using islet transplantation under the kidney capsule of immunodeficient mice prior to STZ destruction of endogenous mouse beta cells to model T1DM. RESULTS: Pretreatment with a cocktail of MSC-secreted peptides increased GSIS in vitro and protected against cytokine-induced apoptosis in human islets isolated from nine donors. Animals transplanted with either treated or untreated human islets remained normoglycaemic for up to 28 days after STZ-administration to ablate the endogenous mouse beta cells, whereas non-transplanted animals showed significantly increased blood glucose immediately after STZ administration. Removal of the human islet graft by nephrectomy resulted in rapid increases in blood glucose to similar levels as the non-transplanted controls. Pretreating human islets with the MSC-derived cocktail significantly improved glucose tolerance in graft recipients, consistent with enhanced functional survival of the treated islets in vivo. CONCLUSION: Pretreating human islets before transplantation with a defined cocktail of MSC-derived molecules could be employed to improve the quality of human islets for transplantation therapy for type 1 diabetes.

Disease modification and symptom relief in osteoarthritis using a mutated GCP-2/CXCL6 chemokine.

We showed that the chemokine receptor C-X-C Motif Chemokine Receptor 2 (CXCR2) is essential for cartilage homeostasis. Here, we reveal that the CXCR2 ligand granulocyte chemotactic protein 2 (GCP-2) was expressed, during embryonic development, within the prospective permanent articular cartilage, but not in the epiphyseal cartilage destined to be replaced by bone. GCP-2 expression was retained in adult articular cartilage. GCP-2 loss-of-function inhibited extracellular matrix production. GCP-2 treatment promoted chondrogenesis in vitro and in human cartilage organoids implanted in nude mice in vivo. To exploit the chondrogenic activity of GCP-2, we disrupted its chemotactic activity, by mutagenizing a glycosaminoglycan binding sequence, which we hypothesized to be required for the formation of a GCP-2 haptotactic gradient on endothelia. This mutated version (GCP-2-T) had reduced capacity to induce transendothelial migration in vitro and in vivo, without affecting downstream receptor signaling through AKT, and chondrogenic activity. Intra-articular adenoviral overexpression of GCP-2-T, but not wild-type GCP-2, reduced pain and cartilage loss in instability-induced osteoarthritis in mice. We suggest that GCP-2-T may be used for disease modification in osteoarthritis.

In vivo potency assay for the screening of bioactive molecules on cartilage formation.

Cartilage regeneration is a priority in medicine for the treatment of osteoarthritis and isolated cartilage defects. Several molecules with potential for cartilage regeneration are under investigation. Unfortunately, in vitro chondrogenesis assays do not always predict the stability of the newly formed cartilage in vivo. Therefore, there is a need for a stringent, quantifiable assay to assess in vivo the capacity of molecules to promote the stable formation of cartilage that is resistant to calcification and endochondral bone formation. We developed an ectopic cartilage formation assay (ECFA) that enables one to assess the capacity of bioactive molecules to support cartilage formation in vivo using cartilage organoids. The ECFA predicted good clinical outcomes when used as a quality control for efficacy of chondrocyte preparations before implantation in patients with cartilage defects. In this assay, articular chondrocytes from human donors or animals are injected either intramuscularly or subcutaneously in nude mice. As early as 2 weeks later, cartilage organoids can be retrieved. The size of the implants and their degree of differentiation can be assessed by histomorphometry, immunostainings of molecular markers and real-time PCR. Mineralization can be assessed by micro-computed tomography or by staining. The effects of molecules on cartilage formation can be tested following the systemic administration of the molecule in mice previously injected with chondrocytes, or after co-injection of chondrocytes with cell lines overexpressing and secreting the protein of interest. Here we describe the ECFA procedure, including steps for harvesting human and bovine articular cartilage, isolating primary chondrocytes, preparing overexpression cell lines, injecting the cells intramuscularly and retrieving the implants. This assay can be performed by technicians and researchers with appropriate animal training within 3 weeks.

Update on pain in arthritis.

PURPOSE OF REVIEW: Osteoarthritis is a degenerative joint disease that features pain as a hallmark symptom. This review summarises progress and obstacles in our understanding of pain mechanisms in arthritis. RECENT FINDINGS: Pain phenotypes in osteoarthritis are poorly characterized in clinical studies and animal studies are largely carti-centric. Different animal models incur variable disease progression patterns and activation of distinct pain pathways, but studies reporting both structural and pain outcomes permit better translational insights. In patients, classification of osteoarthritis disease severity is only based on structural integrity of the joint, but pain outcomes do not consistently correlate with joint damage. The complexity of this relationship underlines the need for pain detection in criteria for osteoarthritis classification and patient-reported outcome measures. SUMMARY: Variable inflammatory and neuropathic components and spatiotemporal evolution underlie the heterogeneity of osteoarthritis pain phenotypes, which must be considered to adequately stratify patients. Revised classification of osteoarthritis at different stages encompassing both structural and pain outcomes would significantly improve detection and diagnosis at both early and late stages of disease. These are necessary advancements in the field that would also improve trial design and provide better understanding of basic mechanisms of disease progression and pain in osteoarthritis.

WNT3A-loaded exosomes enable cartilage repair.

Cartilage defects repair poorly. Recent genetic studies suggest that WNT3a may contribute to cartilage regeneration, however the dense, avascular cartilage extracellular matrix limits its penetration and signalling to chondrocytes. Extracellular vesicles actively penetrate intact cartilage. This study investigates the effect of delivering WNT3a into large cartilage defects in vivo using exosomes as a delivery vehicle. Exosomes were purified by ultracentrifugation from conditioned medium of either L-cells overexpressing WNT3a or control un-transduced L-cells, and characterized by electron microscopy, nanoparticle tracking analysis and marker profiling. WNT3a loaded on exosomes was quantified by western blotting and functionally characterized in vitro using the SUPER8TOPFlash reporter assay and other established readouts including proliferation and proteoglycan content. In vivo pathway activation was assessed using TCF/Lef:H2B-GFP reporter mice. Wnt3a loaded exosomes were injected into the knees of mice, in which large osteochondral defects were surgically generated. The degree of repair was histologically scored after 8 weeks. WNT3a was successfully loaded on exosomes and resulted in activation of WNT signalling in vitro. In vivo, recombinant WNT3a failed to activate WNT signalling in cartilage, whereas a single administration of WNT3a loaded exosomes activated canonical WNT signalling for at least one week, and eight weeks later, improved the repair of osteochondral defects. WNT3a assembled on exosomes, is efficiently delivered into cartilage and contributes to the healing of osteochondral defects.

ROR2 blockade as a therapy for osteoarthritis.

Osteoarthritis is characterized by the loss of the articular cartilage, bone remodeling, pain, and disability. No pharmacological intervention can currently halt progression of osteoarthritis. Here, we show that blocking receptor tyrosine kinase-like orphan receptor 2 (ROR2) improves cartilage integrity and pain in osteoarthritis models by inhibiting yes-associated protein (YAP) signaling. ROR2 was up-regulated in the cartilage in response to inflammatory cytokines and mechanical stress. The main ligand for ROR2, WNT5A, and the targets YAP and connective tissue growth factor were up-regulated in osteoarthritis in humans. In vitro, ROR2 overexpression inhibited chondrocytic differentiation. Conversely, ROR2 blockade triggered chondrogenic differentiation of C3H10T1/2 cells and suppressed the expression of the cartilage-degrading enzymes a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5. The chondrogenic effect of ROR2 blockade in the cartilage was independent of WNT signaling and was mediated by down-regulation of YAP signaling. ROR2 signaling induced G protein and Rho-dependent nuclear accumulation of YAP, and YAP inhibition was required but not sufficient for ROR2 blockade-induced chondrogenesis. ROR2 silencing protected mice from instability-induced osteoarthritis with improved structural outcomes, sustained pain relief, and without apparent side effects or organ toxicity. Last, ROR2 silencing in human articular chondrocytes transplanted in nude mice led to the formation of cartilage organoids with more and better differentiated extracellular matrix, suggesting that the anabolic effect of ROR2 blockade is conserved in humans. Thus, ROR2 blockade is efficacious and well tolerated in preclinical animal models of osteoarthritis.

Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis.

Cartilage loss leads to osteoarthritis, the most common cause of disability for which there is no cure. Cartilage regeneration, therefore, is a priority in medicine. We report that agrin is a potent chondrogenic factor and that a single intraarticular administration of agrin induced long-lasting regeneration of critical-size osteochondral defects in mice, with restoration of tissue architecture and bone-cartilage interface. Agrin attracted joint resident progenitor cells to the site of injury and, through simultaneous activation of CREB and suppression of canonical WNT signaling downstream of ß-catenin, induced expression of the chondrogenic stem cell marker GDF5 and differentiation into stable articular chondrocytes, forming stable articular cartilage. In sheep, an agrin-containing collagen gel resulted in long-lasting regeneration of bone and cartilage, which promoted increased ambulatory activity. Our findings support the therapeutic use of agrin for joint surface regeneration.

Generation of Integration-Free Patient Specific iPS Cells Using Episomal Plasmids Under Feeder Free Conditions.

Reprogramming somatic cells into a pluripotent state involves the overexpression of transcription factors leading to a series of changes that end in the formation of induced pluripotent stem cells (iPSCs). These iPSCs have a wide range of potential uses from drug testing and in vitro disease modelling to personalized cell therapies for patients. While viral methods for reprogramming factor delivery have been traditionally preferred due to their high efficiency, it is now possible to generate iPSCs using nonviral methods at similar efficiencies. We developed a robust reprogramming strategy that combines episomal plasmids and the use of commercially available animal free reagents that can be easily adapted for the GMP manufacture of clinical grade cells.

A systematic evaluation of integration free reprogramming methods for deriving clinically relevant patient specific induced pluripotent stem (iPS) cells.

A systematic evaluation of three different methods for generating induced pluripotent stem (iPS) cells was performed using the same set of parental cells in our quest to develop a feeder independent and xeno-free method for somatic cell reprogramming that could be transferred into a GMP environment. When using the BJ fibroblast cell line, the highest reprogramming efficiency (1.89% of starting cells) was observed with the mRNA based method which was almost 20 fold higher than that observed with the retrovirus (0.2%) and episomal plasmid (0.10%) methods. Standard characterisation tests did not reveal any differences in an array of pluripotency markers between the iPS lines derived using the various methods. However, when the same methods were used to reprogram three different primary fibroblasts lines, two derived from patients with rapid onset parkinsonism dystonia and one from an elderly healthy volunteer, we consistently observed higher reprogramming efficiencies with the episomal plasmid method, which was 4 fold higher when compared to the retroviral method and over 50 fold higher than the mRNA method. Additionally, with the plasmid reprogramming protocol, recombinant vitronectin and synthemax® could be used together with commercially available, fully defined, xeno-free essential 8 medium without significantly impacting the reprogramming efficiency. To demonstrate the robustness of this protocol, we reprogrammed a further 2 primary patient cell lines, one with retinosa pigmentosa and the other with Parkinsons disease. We believe that we have optimised a simple and reproducible method which could be used as a starting point for developing GMP protocols, a prerequisite for generating clinically relevant patient specific iPS cells.