Regulatory T Cells Contribute to Resistance against Lyme Arthritis.

The symptoms of Lyme disease are caused by inflammation induced by species of the Borrelia burgdorferisensu lato complex. The various presentations of Lyme disease in the population suggest that differences exist in the intensity and regulation of the host response to the spirochete. Previous work has described correlations between the presence of regulatory T cells and recovery from Lyme arthritis. However, the effects of Foxp3-expressing CD4+ T cells existing prior to, and during, B. burgdorferi infection have not been well characterized. Here, we used C57BL/6 "depletion of regulatory T cell" mice to assess the effects these cells have on the arthritis-resistant phenotype characteristic of this mouse strain. We showed that depletion of regulatory T cells prior to infection with B. burgdorferi resulted in sustained swelling, as well as histopathological changes, of the tibiotarsal joints that were not observed in infected control mice. Additionally, in vitro stimulation of splenocytes from these regulatory T cell-depleted mice resulted in increases in gamma interferon and interleukin-17 production and decreases in interleukin-10 production that were not evident among splenocytes of infected mice in which Treg cells were not depleted. Depletion of regulatory T cells at various times after infection also induced rapid joint swelling. Collectively, these findings provide evidence that regulatory T cells existing at the time of, and possibly after, B. burgdorferi infection may play an important role in limiting the development of arthritis.

Myostatin inhibition using mRK35 produces skeletal muscle growth and tubular aggregate formation in wild type and TgACTA1D286G nemaline myopathy mice.

Nemaline myopathy (NM) is a heterogeneous congenital skeletal muscle disease with cytoplasmic rod-like structures (nemaline bodies) in muscle tissue. While weakness in NM is related to contractile abnormalities, myofiber smallness is an additional abnormality in NM that may be treatable. We evaluated the effects of mRK35 (a myostatin inhibitor developed by Pfizer) treatment in the TgACTA1D286G mouse model of NM. mRK35 induced skeletal muscle growth that led to significant increases in animal bodyweight, forelimb grip strength and muscle fiber force, although it should be noted that animal weight and forelimb grip strength in untreated TgACTA1D286G mice was not different from controls. Treatment was also associated with an increase in the number of tubular aggregates found in skeletal muscle. These findings suggest that myostatin inhibition may be useful in promoting muscle growth and strength in Acta1-mutant muscle, while also further establishing the relationship between low levels of myostatin and tubular aggregate formation.

Use Of Ankle Immobilization In Evaluating Treatments To Promote Longitudinal Muscle Growth In Mice.

INTRODUCTION: Difficulty in modeling congenital contractures (deformities of muscle-tendon unit development that include shortened muscles and lengthened tendons) has limited research of new treatments. METHODS: Early immobilization of the ankle in prepuberal mice was used to produce deformities similar to congenital contractures. Stretch treatment, electrostimulation, and local intramuscular injection of a follistatin analog (FST-288) were assessed as therapeutic interventions for these deformities. RESULTS: Ankle immobilization at full plantarflexion and 90 ° created tendon lengthening and muscle shortening in the tibialis anterior and soleus. Stretch treatment produced minimal evidence for longitudinal muscle growth and electrostimulation provided no additional benefit. Stretch treatment with FST-288 produced greater longitudinal muscle growth and less tendon lengthening, constituting the best treatment response. DISCUSSION: Ankle immobilization recapitulates key morphologic features of congenital contracture, and these features can be mitigated by a combination of stretch and pharmacological approaches that may be useful in patients. Muscle Nerve 58: 718-725, 2018.

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), isolated from Plumbago zeylanica, inhibits ultraviolet radiation-induced development of squamous cell carcinomas.

Plumbagin (PL) (5-hydroxy-2-methyl-1,4-napthoquinone), a medicinal plant-derived naphthoquinone, was isolated from the roots of the Plumbago zeylanica L. (also known as Chitrak). The roots of P. zeylanica L. have been used in Indian medicine for >2500 years as an anti-atherogenic, cardiotonic, hepatoprotective and neuroprotective agent. We present here that topical application of non-toxic doses (100-500 nmol) of PL to skin elicits dose-dependent inhibition of ultraviolet radiation (UVR)-induced development of squamous cell carcinomas (SCC). In this experiment, FVB/N mice were exposed to UVR (2 kJ/m(2)) three times weekly from a bank of six Kodacel-filtered FS40 sunlamps (∼ 60% UVB and 40% UVA). Carcinoma incidence in mice treated with vehicle, 100, 200 or 500 nmol PL, at 44 weeks post-UVR, were 86, 80 (P = 0.67), 53 (P = 0.12) and 7% (P = 0.0075), respectively. Both vehicle and PL-treated mice gained weight and did not exhibit any signs of toxicity during the entire period of the experiment. Molecular mechanisms associated with inhibition of UVR-induced development of SCC involved induction of apoptosis and inhibition of cell proliferation. Specific findings are that PL treatment (i) inhibited UVR-induced DNA binding of activating protein-1, nuclear factor-kappaB, Stat3 transcription factors and Stat3-regulated molecules (cdc25A and Survivin); (ii) inhibited protein levels of pERK1/2, PI3K85, pAKTSer473, Bcl(2), BclxL, proliferating cell nuclear antigen and cell cycle inhibitory proteins p27 and p21 and (iii) increased UVR-induced Fas-associated death domain expression, poly (ADP-ribose) polymerase protein cleavage and Bax/Bcl(2) ratio. Taken together, our findings suggest that PL may be a novel agent for the prevention of skin cancer.

Ultraviolet radiation and 12-O-tetradecanoylphorbol-13-acetate-induced interaction of mouse epidermal protein kinase Cε with Stat3 involve integration with ERK1/2.

We have reported that protein kinase C epsilon (PKCε) expression level in epidermis dictates the susceptibility of mice to the development of squamous cell carcinomas (SCC) elicited either by repeated exposure to ultraviolet radiation (UVR) or by the DMBA-TPA tumor promotion protocol. To find clues about the mechanism by which PKCε mediates susceptibility to UVR-induced development of SCC, we found that PKCε-over-expressing transgenic mice, as compared to their wild-type littermates, when exposed to UVR, elicit enhanced phosphorylation of Stat3 at Ser727 residues. Stat3 is constitutively activated in SCC and UVR fails to induce SCC in Stat3 mutant mice. Stat3Ser727 phosphorylation is essential for Stat3 transcriptional activity (Cancer Res. 67: 1385, 2007). We now present several novel findings including that PKCε integrates with its downstream partner ERK1/2 to phosphorylate Stat3Ser727. In these experiments, mice were either exposed to UVR (2 kJ/m(2)/dose) emitted by Kodacel-filtered FS-40 sun lamps or treated with TPA (5 nmol). Both UVR and TPA treatment stimulated PKCε-Stat3 interaction, Stat3Ser727 phosphorylation and Stat3-regulated gene COX-2 expression. PKCε-Stat3 interaction and Stat3Ser727 phosphorylation was also observed in SCC elicited by repeated UVR exposures of mice. PKCε-Stat3 interaction was PKCε specific. UVR or TPA-stimulated Stat3Ser727 phosphorylation accompanied interaction of PKCε with ERK1/2 in intact mouse skin in vivo. Deletion of PKCε in wild-type mice attenuated both TPA and UVR-induced expression of phosphoforms of ERK1/2 and Stat3Ser727. These results indicate that PKCε integrates with ERK1/2 to mediate both TPA and UVR-induced epidermal Stat3Ser727 phosphorylation. PKCε and Stat3 may be potential molecular targets for SCC prevention.

Pathological Issues in Dystrophinopathy in the Age of Genetic Therapies.

Dystrophinopathy is a class of genetic skeletal muscle disease characterized by myofiber degeneration and regeneration due to insufficient levels or functioning of dystrophin. Pathological evaluation for dystrophinopathy includes the identification of dystrophic skeletal muscle pathology and the immunohistochemical evaluation of dystrophin epitopes, but biopsies have become rare in recent years. However, the evaluation of dystrophin expression in the research setting has become critically important due to recent advances in genetic therapies, including exon skipping and gene therapy. Given the number of these therapies under evaluation in patients, it is likely that the traditional methods of evaluating dystrophinopathy will need to evolve in the near future. This review discusses current muscle biopsy diagnostic practices in dystrophinopathy and further focuses on how these practices have evolved in the context of therapeutic interventions for dystrophinopathy.

Tissue Response and Biodistribution of Injectable Cellulose Nanocrystal Composite Hydrogels.

Interest in cellulose nanocrystal (CNC)-based hydrogels for drug delivery, tissue engineering, and other biomedical applications has rapidly expanded despite the minimal in vivo research reported to date. Herein, we assess both in vitro protein adsorption and cell adhesion as well as in vivo subcutaneous tissue responses and CNC biodistribution of injectable CNC-poly(oligoethylene glycol methacrylate) (POEGMA) hydrogels. Hydrogels with different PEG side chain lengths, CNC loadings, and with or without in situ magnetic alignment of the CNCs are compared. CNC loading has a minimal impact on protein adsorption but significantly increases cell adhesion. In vivo, both CNC-only and CNC-POEGMA injections largely stay at their subcutaneous injection site over one month, with minimal bioaccumulation of CNCs in any typical clearance organ. CNC-POEGMA hydrogels exhibit mild acute and chronic inflammatory responses, although significant fibroblast penetration was observed with the magnetically aligned hydrogels. Collectively, these results suggest that CNC-POEGMA hydrogels offer promise in practical biomedical applications.

Myostatin Inhibition Using ActRIIB-mFc Does Not Produce Weight Gain or Strength in the Nebulin Conditional KO Mouse.

Mutations in at least 12 genes are responsible for a group of congenital skeletal muscle diseases known as nemaline myopathies (NMs). NMs are associated with a range of clinical symptoms and pathological changes often including the presence of cytoplasmic rod-like structures (nemaline bodies) and myofiber hypotrophy. Our recent work has identified a variable degree of behavioral benefit when treating 2 NM mouse models due to mutations in Acta1 with myostatin inhibition. This study is focused on the effects of delivering ActRIIB-mFc (Acceleron; a myostatin inhibitor) to the nebulin conditional knockout KO (Neb cKO) mouse model of NM. Treatment of Neb cKO mice with ActRIIB-mFc did not produce increases in weight gain, strength, myofiber size, or hypertrophic pathway signaling. Overall, our studies demonstrate a lack of response in Neb cKO mice to myostatin inhibition, which differs from the response observed when treating other NM models.

Injectable and Degradable Poly(Oligoethylene glycol methacrylate) Hydrogels with Tunable Charge Densities as Adhesive Peptide-Free Cell Scaffolds.

Injectable, dual-responsive, and degradable poly(oligo ethylene glycol methacrylate) (POEGMA) hydrogels are demonstrated to offer potential for cell delivery. Charged groups were incorporated into hydrazide and aldehyde-functionalized thermoresponsive POEGMA gel precursor polymers via the copolymerization of N,N'-dimethylaminoethyl methacrylate (DMAEMA) or acrylic acid (AA) to create dual-temperature/pH-responsive in situ gelling hydrogels that can be injected via narrow gauge needles. The incorporation of charge significantly broadens the swelling, degradation, and rheological profiles achievable with injectable POEGMA hydrogels without significantly increasing nonspecific protein adsorption or chronic inflammatory responses following in vivo subcutaneous injection. However, significantly different cell responses are observed upon charge incorporation, with charged gels significantly improving 3T3 mouse fibroblast cell adhesion in 2D and successfully delivering viable and proliferating ARPE-19 human retinal epithelial cells via an "all-synthetic" matrix that does not require the incorporation of cell-adhesive peptides.

Sdha+/- Rats Display Minimal Muscle Pathology Without Significant Behavioral or Biochemical Abnormalities.

Mitochondrial diseases (MDs) result from alteration of the mitochondrial respiratory chain (MRC) function. Despite the prevalence of MDs in the population, the paucity of animal models available limits the understanding of these disorders. Mutations in SDHA, a gene that codes for the alpha subunit of succinate dehydrogenase (SDH), can cause some forms of MD. SDHA is a crucial contributor to MRC function. In order to expand the range of MD animal models available, we attempted to generate a Sdha knockout rat. Since homozygous Sdha-/- rats could neither be identified in newborn litters, nor as early as embryonic day 14, we evaluated wild-type (WT) and heterozygous Sdha+/- genotypes. No differences in behavioral, biochemical, or molecular evaluations were observed between WT and Sdha+/- rats at 6 weeks or 6 months of age. However, 30% of Sdha+/- rats displayed mild muscle fiber atrophy with rare fibers negative for cytochrome oxidase and SDH on histochemical staining. Collectively, our data provide additional evidence that modeling SDH mutations in rodents may be challenging due to animal viability, and heterozygous rats are insufficiently symptomatic at a phenotypic and molecular level to be of significant use in the study of SDH deficiency.

Host interleukin 6 production regulates inflammation but not tryptophan metabolism in the brain during murine GVHD.

Graft-versus-host disease (GVHD) induces pathological damage in peripheral target organs leading to well-characterized, organ-specific clinical manifestations. Patients with GVHD, however, can also have behavioral alterations that affect overall cognitive function, but the extent to which GVHD alters inflammatory and biochemical pathways in the brain remain poorly understood. In the current study, we employed complementary murine GVHD models to demonstrate that alloreactive donor T cells accumulate in the brain and affect a proinflammatory cytokine milieu that is associated with specific behavioral abnormalities. Host IL-6 was identified as a pivotal cytokine mediator, as was host indoleamine 2,3-dioxygenase (IDO-1), which was upregulated in GVHD in an IL-6-dependent manner in microglial cells and was accompanied by dysregulated tryptophan metabolism in the dorsal raphe nucleus and prefrontal cortex. Blockade of the IL-6 signaling pathway significantly reduced donor T cell accumulation, inflammatory cytokine gene expression, and host microglial cell expansion, but did not reverse GVHD-induced tryptophan metabolite dysregulation. Thus, these results indicate that inhibition of IL-6 signaling attenuates neuroinflammation, but does not reverse all of the metabolic abnormalities in the brain during GVHD, which may also have implications for the treatment of neurotoxicity occurring after other T cell-based immune therapies with IL-6-directed approaches.