Targeting PKCθ promotes satellite cell self-renewal

Skeletal muscle regeneration following injury depends on the ability of satellite cells (SCs) to proliferate, self-renew, and eventually differentiate. The factors that regulate the process of self-renewal are poorly understood. In this study we examined the role of PKCθ in SC self-renewal and differentiation. We show that PKCθ is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKCθ promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKCθ or its pharmacological inhibition in vivo did not affect SC number in healthy muscle. By contrast, after induction of muscle injury, lack or inhibition of PKCθ resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKCθ does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKCθ-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKCθ plays an important role in SC self-renewal by stimulating their expansion through symmetric division, and it may represent a promising target to manipulate satellite cell self-renewal in pathological conditions

Strategies to improve muscle repair and the outcome of cell mediated therapies in muscular dystrophy

The goal of this project was to establish whether lack of PKCΘ in dystrophic muscle preserves regenerative ability of resident cell populations and improves the survival and the ability of transplanted stem cells to generate new muscle fibers and correct the genetic defect of the recipient. Duchenne muscular dystrophy (DMD) is a genetic disease characterized by muscle wasting and chronic inflammation. Moreover, the constant cycles of degeneration and regeneration and the hostile microenvironment affect the satellite cells function and exhaust their regenerative capacity. Previous work done in our laboratory showed that lack of PKC in mdx mice, the mouse model of DMD, improves muscle maintenance, regeneration and performance and reduces muscle inflammation. The observed phenotype was primarily due to lack of PKCΘ in hemopoietic cells, as demonstrated by BM transplantation experiments. Indeed, PKCΘ plays a unique role in T cell activation, and represents an attractive molecular target for the treatment of immune disorders. Current results obtained in the laboratory show that lack of PKC in mdx prevents early lymphocytes recruitment in muscle, altering the inflammatory response (Lozanoska-Ochser et al. ms in prep). In the present study, I show that the lack of PKC in mdx mouse boosts muscle regeneration even at advanced stages of the disease and prevents fibrosis. This phenotype is associated to a both increased maintenance of the satellite cells pool, through up-regulation of the Notch pathway, and increased myogenic and pro-myogenic activity of FAPs. Fibroadipogenic progenits (FAPs), a population of muscle interstitial cells, are known to either contribute to muscle regeneration or to fibroadipogenic degeneration generating myofibroblasts and adipose cells, depending on the environmental stimuli. Moreover, I show in this study that lack of PKC in dystrophic muscle prolongs survival of transplanted mesoangioblasts (MABs) within muscle, compared to mdx, and favours their ability to form myofibers. Together, these results suggest that lack of PKC in dystrophic muscle makes a more favourable environment for both endogenous and exogenous cell populations to contribute to muscle maintenance and repair. This phenotype is probably dependent on alterations in the immune response. The underlying mechanisms are under investigation and may contribute to the identification of additional molecular/ cellular target to ameliorate the disease and to improve cell mediated therapeutic strategies

Pharmacological inhibition of PKCθ counteracts muscle disease in a mouse model of duchenne muscular dystrophy

Inflammation plays a considerable role in the progression of Duchenne Muscular Dystrophy (DMD), a severe muscle disease caused by a mutation in the dystrophin gene. We previously showed that genetic ablation of Protein Kinase C θ (PKCθ) in mdx, the mouse model of DMD, improves muscle healing and regeneration, preventing massive inflammation. To establish whether pharmacological targeting of PKCθ in DMD can be proposed as a therapeutic option, in this study we treated young mdx mice with the PKCθ inhibitor Compound 20 (C20). We show that C20 treatment led to a significant reduction in muscle damage associated with reduced immune cells infiltration, reduced inflammatory pathways activation, and maintained muscle regeneration. Importantly, C20 treatment is efficient in recovering muscle performance in mdx mice, by preserving muscle integrity. Together, these results provide proof of principle that pharmacological inhibition of PKCθ in DMD can be considered an attractive strategy to modulate immune response and prevent the progression of the disease

Analysis of a Spanish energy scenario with Generation IV nuclear reactors

The advantages of fast-spectrum reactors consist not only of an efficient use of fuel through the breeding of fissile material and the use of natural or depleted uranium, but also of the potential reduction of the amount of actinides such as americium and neptunium contained in the irradiated fuel. The first aspect means a guaranteed future nuclear fuel supply. The second fact is key for high-level radioactive waste management, because these elements are the main responsible for the radioactivity of the irradiated fuel in the long term. The present study aims to analyze the hypothetical deployment of a Gen-IV Sodium Fast Reactor (SFR) fleet in Spain. A nuclear fleet of fast reactors would enable a fuel cycle strategy different than the open cycle, currently adopted by most of the countries with nuclear power. A transition from the current Gen-II to Gen-IV fleet is envisaged through an intermediate deployment of Gen-III reactors. Fuel reprocessing from the Gen-II and Gen-III Light Water Reactors (LWR) has been considered. In the so-called advanced fuel cycle, the reprocessed fuel used to produce energy will breed new fissile fuel and transmute minor actinides at the same time. A reference case scenario has been postulated and further sensitivity studies have been performed to analyze the impact of the different parameters on the required reactor fleet. The potential capability of Spain to supply the required fleet for the reference scenario using national resources has been verified. Finally, some consequences on irradiated final fuel inventory are assessed. Calculations are performed with the Monte Carlo transport-coupled depletion code SERPENT together with post-processing tools

The roles of different forms of IL-15 in human melanoma progression

BackgroundMelanoma is a lethal skin cancer, and the risk of developing it is increased by exposure to ultraviolet (UV) radiation. The production of cytokines such as interleukin-15 (IL-15), induced by the exposure of skin cells to UV rays, could also promote melanoma development. The aim of this study is to investigate the possible role of Interleukin-15/Interleukin-15 Receptor α (IL-15/IL-15Rα) complexes in melanoma development.MethodsThe expression of IL-15/IL-15Rα complexes by melanoma cells was evaluated both ex vivo and in vitro by tissue microarray, PCR, and flow cytometry. The presence of the soluble complex (sIL-15/IL-15Rα) in the plasma of metastatic melanoma patients was detected using an ELISA assay. Subsequently, we investigated the impact of natural killer (NK) cell activation after rIL-2 starvation followed by exposure to the sIL-15/IL-15Rα complex. Finally, by analyzing public datasets, we studied the correlation between IL-15 and IL-15Rα expressions and melanoma stage, NK and T-cell markers, and overall survival (OS).ResultsAnalysis of a melanoma tissue microarray shows a significant increase in the number of IL-15+ tumor cells from the benign nevi to metastatic melanoma stages. Metastatic melanoma cell lines express a phorbol-12-myristate-13-acetate (PMA)-cleavable membrane-bound IL-15 (mbIL-15), whereas cultures from primary melanomas express a PMA-resistant isoform. Further analysis revealed that 26% of metastatic patients present with consistently high plasmatic levels of sIL-15/IL-15Rα. When the recombinant soluble human IL-15/IL-15Rα complex is added to briefly starved rIL-2-expanded NK cells, these cells exhibit strongly reduced proliferation and levels of cytotoxic activity against K-562 and NALM-18 target cells. The analysis of public gene expression datasets revealed that high IL-15 and IL-15Rα intra-tumoral production correlates with the high levels of expression of CD5+ and NKp46+ (T and NK markers) and significantly correlates with a better OS in stages II and III, but not in stage IV.ConclusionsMembrane-bound and secreted IL-15/IL-15Rα complexes are continuously present during progression in melanoma. It is notable that, although IL-15/IL-15Rα initially promoted the production of cytotoxic T and NK cells, at stage IV promotion of the development of anergic and dysfunctional cytotoxic NK cells was observed. In a subgroup of melanoma metastatic patients, the continuous secretion of high amounts of the soluble complex could represent a novel NK cell immune escape mechanism

MicroRNA Expression Profiling in Behçet’s Disease

Background. Behçet’s disease (BD) is a chronic inflammatory multisystem disease characterized by oral and genital ulcers, uveitis, and skin lesions. MicroRNAs (miRNAs) are key regulators of immune responses. Differential expression of miRNAs has been reported in several inflammatory autoimmune diseases; however, their role in BD is not fully elucidated. We aimed to identify miRNA expression signatures associated with BD and to investigate their potential implication in the disease pathogenesis. Methods. miRNA microarray analysis was performed in blood cells of BD patients and healthy controls. miRNA expression profiles were analyzed using Affymetrix arrays with a comprehensive coverage of miRNA sequences. Pathway analyses were performed, and the global miRNA profiling was combined with transcriptoma data in BD. Deregulation of selected miRNAs was validated by real-time PCR. Results. We identified specific miRNA signatures associated with BD patients with active disease. These miRNAs target pathways relevant in BD, such as TNF, IFN gamma, and VEGF-VEGFR signaling cascades. Network analysis revealed several miRNAs regulating highly connected genes within the BD transcriptoma. Conclusions. The combined analysis of deregulated miRNAs and BD transcriptome sheds light on some epigenetic aspects of BD identifying specific miRNAs, which may represent promising candidates as biomarkers and/or for the design of novel therapeutic strategies in BD

Targeting PKCθ in skeletal muscle and muscle diseases: good or bad?

Protein kinase C-theta (PKCθ) is a member of the novel calcium-indipendent protein kinase C (PKC) family, with a relatively selective tissue distribution. Most studies have focussed on its unique role in T lymphocyte activation and suggest that inhibition of PKC could represent a novel therapeutic approach in the treatment of chronic inflammation, autoimmunity and allograft rejection. However, considering that PKC is also expressed in other cell types, including skeletal muscle cells, it is important to understand its function in different tissues before proposing it as a molecular target for the treatment of immune mediated diseases. A number of studies have highlighted the role of PKC in mediating several intracellular pathways regulating muscle cell development, homeostasis and remodelling, although a comprehensive picture is still lacking. Moreover, we recently showed that lack of PKC in a mouse model of Duchenne Muscular Dystrophy ameliorates the progression of the disease. Here, we review new developments in our understanding of the involvement of PKC in intracellular mechanisms regulating skeletal muscle development, growth and maintenance under physiological conditions, and recent advances showing a hitherto unrecognized role of PKC in promoting muscular dystrophy

Adrenocortical oncocytoma: Case report and review of the literature

Only 9 oncocytic neoplasms of true adrenal origin have been described to date. It therefore seemed of interest to study the histochemical and ultrastructural features of a non-functioning monolateral adrenocortical oncocytoma which was incidentally detected by ultrasonography and magnetic resonance imaging in a 28-year-old woman. The tumor was round, well encapsulated and weighed 73 g. It consisted of islets of eosinophilic cells, and did not display any sign of necrosis. The proliferation rate (as evaluated by mitotic index and percentage of MIB-1 Ki67 positive cells) was low, and atypic mitoses were absent; some rare cells with nuclear atypias were observed and the capsule was focally invaded by oncocytes. Immunocytochemistry did not show expression of vimentin or cytokeratin. The oncocytes had an abundant cytoplasm packed with mitochondria containing plate-like cristae. Smooth endoplasmic reticulum was virtually absent, while rough endoplasmic reticulum cisternae and free ribosomes were abundant. Although the classic histological approach clearly indicates the benign nature of the tumor, the immunocytochemical and ultrastructural features of oncocytes may suggest their potential for malignant behavior. </jats:p

Interleukin-15 and cancer: some solved and many unsolved questions

Soluble interleukin (IL)-15 exists under two forms: as monomer (sIL-15) or as heterodimeric complex in association with sIL-15Rα (sIL-15/IL-15Rα). Both forms have been successfully tested in experimental tumor murine models and are currently undergoing investigation in phase I/II clinical trials. Despite more than 20 years research on IL-15, some controversial issues remain to be addressed. A first point concerns the detection of the sIL-15/IL-15Rα in plasma of healthy donors or patients with cancer and its biological significance. The second and third unsolved question regards the protumorigenic role of the IL-15/IL-15Rα complex in human cancer and the detrimental immunological consequences associated to prolonged exposure of natural killer (NK) cells to both forms of soluble IL-15, respectively. Data suggest that in vivo prolonged or repeated exposure to monomeric sIL-15 or the soluble complex may lead to NK hypo-responsiveness through the expansion of the CD8+/CD44+ T cell subset that would suppress NK cell functions. In vitro experiments indicate that soluble complex and monomeric IL-15 may cause NK hyporesponsiveness through a direct effect caused by their prolonged stimulation, suggesting that this mechanism could also be effective in vivo. Therefore, a better knowledge of IL-15 and a more appropriate use of both its soluble forms, in terms of concentrations and time of exposure, are essential in order to improve their therapeutic use. In cancer, the overproduction of sIL-15/IL-15Rα could represent a novel mechanism of immune escape. The soluble complex may act as a decoy cytokine unable to efficiently foster NK cells, or could induce NK hyporesponsiveness through an excessive and prolonged stimulation depending on the type of IL-15Rα isoforms associated. All these unsolved questions are not merely limited to the knowledge of IL-15 pathophysiology, but are crucial also for the therapeutic use of this cytokine. Therefore, in this review, we will discuss key unanswered issues on the heterogeneity and biological significance of IL-15 isoforms, analyzing both their cancer-related biological functions and their therapeutic implications

Reduction of volumetric bone mineral density in postmenopausal women with hepatitis C virus-correlated chronic liver disease: a peripheral quantitative computed tomography (pQCT) study

BACKGROUND: The prevalence of osteoporosis in chronic liver disease (CLD) varies considerably among the studies, depending on patient selection and diagnostic criteria. We aimed to measure bone turnover markers and volumetric bone mineral density (BMD) in a group of postmenopausal women with CLD using both dual energy X-ray absorptiometry (DXA) and peripheral quantitative computed tomography (pQCT), in comparison with age-matched healthy subjects. METHODS: Thirty-five postmenopausal patients with HCV-correlated CLD and 35 healthy postmenopausal women, as controls, underwent a DXA scan at lumbar and femoral level and a pQCT measurement of the nondominant forearm. Serum concentrations of biochemical markers relevant to bone turnover were also measured. RESULTS: Patients showed no differences in DXA values either at lumbar or femoral level compared to controls. On the contrary, pQCT geometrical (cortical cross-sectional area) and volumetric (total and trabecular volumetric BMD) parameters were significantly reduced in the CLD women. Also the Strength-Strain Index (SSI), an estimate of diaphyseal bone resistance to bending and torsion, was significantly lower in patients than in controls. Patients with CLD presented an unbalanced bone turnover, with increased bone resorption markers. CONCLUSIONS: The bone geometrical and volumetric parameters measured in our CLD postmenopausal women, by pQCT, show a reduced bone mineral quality and stiffness. Conversely, DXA-measurements seem unable to appreciate the bone alterations in these patients. This would encourage further studies to validate pQCT analysis as a diagnostic tool for a correct estimate of bone involvement in CLD