[Show abstract][Hide abstract] ABSTRACT: Monosodium urate (MSU) and calcium pyrophosphate dihydrate (CPPD) crystal-induced interleukin 1 beta (IL1beta) release contributes to inflammation in subcutaneous air pouch and peritoneal models of acute gout and pseudogout. However, consequences of IL1 inhibition have not been explored in more clinically relevant models of crystal-induced arthritis.
To develop a novel mouse model of acute gouty ankle arthritis and use it to assess the effects of genetic deletion of IL1 receptor type (IL1R1) and of exogenous mIL1 Trap (a high-affinity blocker of mouse IL1alpha and IL1beta) on pain, synovitis and systemic inflammatory biomarkers.
MSU crystals were injected into the mouse ankle joint and pain and ankle swelling were measured over 4 days. The effects of IL1 inhibition were determined in this model, and in the comparator models of crystal-induced peritonitis and subcutaneous air pouch inflammation.
Both IL1R1-null mice and mice pretreated with mIL1 Trap showed reduced neutrophil influx in MSU and CPPD crystal-induced peritonitis and air pouch models (p<0.05). In the ankle joint model, both IL1R1 knockout mice and pretreatment with mIL1 Trap were associated with significant reductions in MSU crystal-induced elevations in hyperalgesia, inflammation, serum amyloid A and the levels of multiple inflammatory cytokines and chemokines (p<0.05). Additionally, it was found that administration of mIL1 Trap after MSU crystal injection reduced established hyperalgesia and ankle swelling.
IL1 inhibition both prevented and relieved pain and ankle joint inflammation in response to intra-articular MSU crystals in mice. Results suggested that IL1 Trap has the potential to both prevent and treat gouty arthritis.
Annals of the rheumatic diseases 07/2009; 68(10):1602-8. DOI:10.1136/ard.2009.109355 · 10.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The cytokine cascade in pain and inflammatory processes is a tremendously complex system, involving glial, immune, and neuronal cell interactions. IL-1beta is a pro-inflammatory cytokine that has been implicated in pain, inflammation and autoimmune conditions. This review will focus on studies that shed light on the critical role of IL-1beta in various pain states, including the role of the intracellular complex, the inflammasome, which regulates IL-1beta production. Evidence will be presented demonstrating the importance of IL-1beta in both the induction of pain and in the maintenance of pain in chronic states, such as after nerve injury. Additionally, the involvement of IL-1beta as a key mediator in the interaction between glia and neurons in pain states will be discussed. Taken together, the evidence presented in the current review showing the importance of IL-1beta in animal and human pain states, suggests that blockade of IL-1beta be considered as a therapeutic opportunity.
Brain Research Reviews 04/2009; 60(1):57-64. DOI:10.1016/j.brainresrev.2008.12.020 · 5.93 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Adult mammalian testis is a source of pluripotent stem cells. However, the lack of specific surface markers has hampered identification and tracking of the unrecognized subset of germ cells that gives rise to multipotent cells. Although embryonic-like cells can be derived from adult testis cultures after only several weeks in vitro, it is not known whether adult self-renewing spermatogonia in long-term culture can generate such stem cells as well. Here, we show that highly proliferative adult spermatogonial progenitor cells (SPCs) can be efficiently obtained by cultivation on mitotically inactivated testicular feeders containing CD34+ stromal cells. SPCs exhibit testicular repopulating activity in vivo and maintain the ability in long-term culture to give rise to multipotent adult spermatogonial-derived stem cells (MASCs). Furthermore, both SPCs and MASCs express GPR125, an orphan adhesion-type G-protein-coupled receptor. In knock-in mice bearing a GPR125-beta-galactosidase (LacZ) fusion protein under control of the native Gpr125 promoter (GPR125-LacZ), expression in the testis was detected exclusively in spermatogonia and not in differentiated germ cells. Primary GPR125-LacZ SPC lines retained GPR125 expression, underwent clonal expansion, maintained the phenotype of germline stem cells, and reconstituted spermatogenesis in busulphan-treated mice. Long-term cultures of GPR125+ SPCs (GSPCs) also converted into GPR125+ MASC colonies. GPR125+ MASCs generated derivatives of the three germ layers and contributed to chimaeric embryos, with concomitant downregulation of GPR125 during differentiation into GPR125- cells. MASCs also differentiated into contractile cardiac tissue in vitro and formed functional blood vessels in vivo. Molecular bookmarking by GPR125 in the adult mouse and, ultimately, in the human testis could enrich for a population of SPCs for derivation of GPR125+ MASCs, which may be employed for genetic manipulation, tissue regeneration and revascularization of ischaemic organs.
[Show abstract][Hide abstract] ABSTRACT: Neuromedin U (NMU) has recently been reported to have a role in nociception and inflammation. To clarify the function of the two known NMU receptors, NMU receptor 1 (NMUR1) and NMU receptor 2 (NMUR2), during nociception and inflammation in vivo, we generated mice in which the genes for each receptor were independently deleted. Compared to wild type littermates, mice deficient in NMUR2 showed a reduced thermal nociceptive response in the hot plate, but not in the tail flick, test. In addition, the NMUR2 mutant mice showed a reduced behavioral response and a marked reduction in thermal hyperalgesia following capsaicin injection. NMUR2-deficient mice also showed an impaired pain response during the chronic, but not acute, phase of the formalin test. In contrast, NMUR1-deficient mice did not show any nociceptive differences compared to their wild type littermates in any of the behavioral tests used. We observed the same magnitude of inflammation in both lines of NMU receptor mutant mice compared to their wild type littermates after injection with complete Freund's adjuvant (CFA), suggesting no requirement for either receptor in this response. Thus, the pro-nociceptive effects of NMU in mice appear to be mediated through NMUR2, not NMUR1.
[Show abstract][Hide abstract] ABSTRACT: One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive-negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5-1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.
[Show abstract][Hide abstract] ABSTRACT: Discoidin domain receptor 2 (DDR2) is an unusual receptor tyrosine kinase in that its ligand is fibrillar collagen rather
than a growth factor-like peptide. We examined signal transduction pathways of DDR2. Here we show that DDR2 is also unusual
in that it requires Src activity to be maximally tyrosine-phosphorylated, and that Src activity also promotes association
of DDR2 with Shc. The interaction with Shc involves a portion of Shc not previously implicated in interaction with receptor
tyrosine kinases. These results identify Src kinase and the adaptor protein Shc as key signaling intermediates in DDR2 signal
transduction. Furthermore, Src is required for DDR2-mediated transactivation of the matrix metalloproteinase-2 promoter. The
data support a model in which Src and the DDR2 receptor cooperate in a regulated fashion to direct the phosphorylation of
both the receptor and its targets.
[Show abstract][Hide abstract] ABSTRACT: Localizing cell surface receptors to specific subcellular positions can be critical for their proper functioning, as most notably demonstrated at neuronal synapses. PDZ proteins apparently play critical roles in such protein localizations. Receptor tyrosine kinases have not been previously shown to interact with PDZ proteins in vertebrates. We report that Eph receptors and their membrane-linked ligands all contain PDZ recognition motifs and can bind and be clustered by PDZ proteins. In addition, we find that Eph receptors and ligands colocalize with PDZ proteins at synapses. Thus, PDZ proteins may play critical roles in localizing vertebrate receptor tyrosine kinases and/or their ligands and may be particularly important for Eph function in guidance or patterning or at the synapse.