Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models

Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.

Initial Liver Copper Status in Finishing Beef Steers Fed Three Dietary Concentrations of Copper Affects Beta Agonist Performance, Carcass Characteristics, Lipolysis Response, and Muscle Inflammation Markers

Ninety-three Angus-crossbred steers (470 ± 35 kg) were assigned to a 3 × 2 factorial to determine the effects of Cu status and beta agonist (BA) on performance, carcass characteristics, lipolytic rate, and muscle inflammation. Factors included Cu supplementation (mg Cu/kg dry matter (DM)) at: 0 (LO), 10 (MED), or 20 (HI) from Cu amino acid complex (Availa Cu; Zinpro) with no BA (NoRAC) or 300 mg·steer−1·day−1 of ractopamine hydrochloride (RAC; Optaflexx; Elanco) for final 28 days of 88-day trial. Linear and quadratic effects of Cu status within BA treatment were tested. Pre-BA gain was not affected by Cu supplementation (p ≥ 0.57), although day 53 liver Cu quadratically increased (p = 0.01). Average daily gain and muscle IL-8 gene expression quadratically increased (p ≤ 0.01), with MED having greatest gain and gene expression. Ribeye area tended to quadratically increase with Cu supplementation within RAC (p = 0.08). In vitro basal lipolytic rate tended to quadratically increase with Cu supplementation within RAC (p = 0.11), while stimulated lipolytic rate tended to linearly increase within NoRAC (p = 0.10). These data suggest lipolysis and the BA response of steers are influenced by dietary and liver Cu concentrations.

Clinical manfestation of dermatomyositis and difficulties in its differential diagnosis

Dermatomyositis (DM) is an idiopathic inflammatory myopathy with muscle weakness and specific cutaneous findings. The proximal weakness of the limb muscles is present in the majority of the patients, in a proportion of them however the muscle inflammation is preceded by the skin symptoms with several months, and in 40%, skin symptoms are the only manifestations of the disease. While, there may have many difficulties in differential diagnosis of DM without myositis, dermatologists have a major role in the early recognition of the disorder and in the initiation of the proper therapy

Pharmacological and Genetic Inhibition of PD-1 Demonstrate an Important Role of PD-1 in Ischemia-Induced Skeletal Muscle Inflammation, Oxidative Stress, and Angiogenesis

Angiogenesis is an important process under both physiological and pathophysiological conditions. Here we investigated the role and the underlying mechanism of PD-1 in hindlimb ischemia-induced inflammation and angiogenesis in mice. We found that inhibition of PD-1 by genetic PD-1 knockout or pharmacological PD-1 blocking antibodies dramatically attenuated hindlimb blood perfusion, angiogenesis, and exercise capacity in mice after femoral artery ligation. Mechanistically, we found that PD-1 knockout significantly exacerbated ischemia-induced muscle oxidative stress, leukocyte infiltration and IFN-γ production before abnormal angiogenesis in these mice. In addition, we found that the percentages of IFN-γ positive macrophages and CD8 T cells were significantly increased in P-1 knockout mice after hindlimb ischemia. Macrophages were the major leukocyte subset infiltrated in skeletal muscle, which were responsible for the enhanced muscle leukocyte-derived IFN-γ production in PD-1 knockout mice after hindlimb ischemia. Moreover, we demonstrated that IFN-γ significantly attenuated vascular endothelial cell proliferation, tube formation and migration in vitro. IFN-γ also significantly enhanced vascular endothelial cell apoptosis. In addition, the total number of TNF-α positive leukocytes/muscle weight were significantly increased in PD-1-/- mice after hindlimb ischemia. These data indicate that PD-1 exerts an important role in ischemia-induced muscle inflammation and angiogenesis.

Orange peel extract reduces the inflammatory state of skeletal muscle after downhill running via an increase in IL-1RA

It has been reported that orange peel extract (OPE) and the four major polymethoxyflavones (PMFs) in OPE have a protective effect against downhill running (DR)-induced skeletal muscle inflammation. However, the mechanism is not well understood. We investigated the potential of OPE and PMF compounds for increasing anti-inflammatory cytokine levels. The plasma interleukin-1 receptor antagonist (IL-1RA) level was increased 1 and 8 h after OPE administration in rats. Nobiletin induced the secretion of IL-1RA from C2C12 myotubes. In the inflammatory state of skeletal muscle after DR, OPE administration reduced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, NF-κB-DNA binding, and monocyte chemotactic protein-1 mRNA levels, but these effects were all abrogated by the intravenous administration of IL-1RA neutralizing antibody. These results indicated that OPE reduces skeletal muscle inflammatory state after DR via an increase in IL-1RA, and that IL-1 receptor signaling is important for skeletal muscle inflammation after DR.