Federica Colleoni

Skin-derived stem cells transplanted into resorbable guides provide functional nerve regeneration after sciatic nerve resection

The regeneration in the peripheral nervous system is often incomplete and the treatment of severe lesions with nerve tissue loss is primarily aimed at recreating nerve continuity. Guide tubes of various types, filled with Schwann cells, stem cells, or nerve growth factors are attractive as an alternative therapy to nerve grafts. In this study, we evaluated whether skin‐derived stem cells (SDSCs) can improve peripheral nerve regeneration after transplantation into nerve guides. We compared peripheral nerve regeneration in adult rats with sciatic nerve gaps of 16 mm after autologous transplantation of GFP‐labeled SDSCs into two different types of guides: a synthetic guide, obtained by dip coating with a L‐lactide and trimethylene carbonate (PLA‐TMC) copolymer and a collagen‐based guide. The sciatic function index and the recovery rates of the compound muscle action potential were significantly higher in the animals that received SDSCs transplantation, in particular, into the collagen guide, compared to the control guides filled only with PBS. For these guides the morphological and immunohistochemical analysis demonstrated an increased number of myelinated axons expressing S100 and Neurofilament 70, suggesting the presence of regenerating nerve fibers along the gap. GFP positive cells were found around regenerating nerve fibers and few of them were positive for the expression of glial markers as S‐100 and glial fibrillary acidic protein. RT‐PCR analysis confirmed the expression of S100 and myelin basic protein in the animals treated with the collagen guide filled with SDSCs. These data support the hypothesis that SDSCs could represent a tool for future cell therapy applications in peripheral nerve regeneration.

Combinatorial administration of molecules that simultaneously inhibit angiogenesis and invasion leads to increased therapeutic efficacy in mouse models of malignant glioma.

Purpose: We investigated the ability of the combinatorial administration of different inhibitors with activities on glioma angiogenesis, migration, and proliferation to produce a prolonged inhibition of glioma growth. Experimental Design: We combined inhibitors affecting solely tumor angiogenesis (PF-4/CTF, cyclo-VEGI) or inhibitors affecting both angiogenesis and invasion together (PEX, PF-4/DLR). Results: When administered in combination, these drugs produced a prolonged and increased inhibition of glioma growth independently from the type of inhibitor used. The combinatory administration was more effective than the administration of a single inhibitor alone, and a strong therapeutic response was reached with a significantly lower amount of protein. The strongest inhibition was observed when human PEX and PF-4/DLR, which affect both glioma angiogenesis and invasion by separate mechanisms, were combined. Conclusions: This supports the concept that prolonged glioma growth inhibition can be achieved by simultaneous delivery of molecules that target both tumor and endothelial cells and acting by separate mechanisms.

Panax ginseng C.A. Mayer G115 modulates pro-inflammatory cytokine production in mice throughout the increase of macrophage toll-like receptor 4 expression during physical stress

The effect of Panax ginseng C.A. Meyer G115 on inflammatory cytokine production and toll-like receptor 4 (TLR4) RNA expression was examined in mice during 4 weeks of swimming stress. Mice were assigned to four groups: (1) control (no exercise); (2) control-G115 (25 mg/kg/day p.o.); (3) stress (kept swimming for 60 min daily); and (4) stress-G115 (25 mg/kg/day p.o. and kept swimming for 60 min daily). Peritoneal macrophages were collected at rest each week. RNA was extracted and processed for real-time PCR. An aliquot of macrophages was lipopolysaccharide (LPS)-stimulated for TNF-alpha and IL-1beta production. Different expression patterns between untreated and treated groups, and between TLR2 and TLR4 were found. High levels of TLR4 expression in the control-G115 group were detectable significantly at the first, and at the second week (P<.01 and P<.001, respectively). In the stress group, TLR4 showed a peak at the first week (P<.001 vs. controls) and then decreased gradually. In the stress-G115 group, the levels of TLR4 expression increased gradually at the second week (P<.001 vs. controls) with a peak at the third week (P<.001). Levels of TLR4 expression at the fourth week had returned to the basal level. Levels of TLR2 expression were not affected by treatment in all groups. A significant increase of LPS-stimulated IL-1beta and TNF-alpha concentrations was present in trained animals with similar patterns of TLR4 expression. These results support the hypothesis that enhancement of the production of pro-inflammatory cytokine can be linked to an increased expression of TLR4 on macrophages. Moreover, G115 increases the expression of TLR4 and the release of cytokines with a different pattern compared to the stressed alone group.

The Role of Stem Cells in Muscular Dystrophies

Muscular dystrophies are heterogeneous neuromuscular disorders of inherited origin, including Duchenne muscular dystrophy (DMD). Cell-based therapies were used to promote muscle regeneration with the hope that the host cells repopulated the muscle and improved muscle function and pathology. Stem cells were preferable for therapeutic applications, due to their capacity of self-renewal and differentiative potential. In the last years, encouraging results were obtained with adult stem cells to treat muscular dystrophies. Adult stem cells were found into various tissues of the body and they were able to maintain, generate, and replace terminally differentiated cells within their own specific tissue because of cell turnover or tissue injury. Moreover, it became clear that these cells could participate into regeneration of more than just their resident organ. Here, we described multiple types of muscle and non muscle-derived myogenic stem cells, their characterization and their possible use to treat muscular dystrophies. We also underlined that most promising possibility for the management and therapy of DMD is a combination of different approaches, such as gene and stem cell therapy.

Inositol 1,4,5-trisphosphate (IP3)-dependent Ca2+ signaling mediates delayed myogenesis in Duchenne muscular dystrophy fetal muscle.

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder characterized by muscle wasting and premature death. The defective gene is dystrophin, a structural protein, absence of which causes membrane fragility and myofiber necrosis. Several lines of evidence showed that in adult DMD patients dystrophin is involved in signaling pathways that regulate calcium homeostasis and differentiation programs. However, secondary aspects of the disease, such as inflammation and fibrosis development, might represent a bias in the analysis. Because fetal muscle is not influenced by gravity and does not suffer from mechanical load and/or inflammation, we investigated 12-week-old fetal DMD skeletal muscles, highlighting for the first time early alterations in signaling pathways mediated by the absence of dystrophin itself. We found that PLC/IP3/IP3R/Ryr1/Ca2+ signaling is widely active in fetal DMD skeletal muscles and, through the calcium-dependent PKCα protein, exerts a fundamental regulatory role in delaying myogenesis and in myofiber commitment. These data provide new insights into the origin of DMD pathology during muscle development. Summary: The absence of dystrophin in human fetal muscle alters IP3 signaling pathways and consequently causes a delay in myogenesis and myofiber commitment.

Stem cell salvage of injured peripheral nerve

We previously developed a collagen tube filled with autologous skin-derived stem cells (SDSCs) for bridging long rat sciatic nerve gaps. Here we present a case report describing a compassionate use of this graft for repairing the polyinjured motor and sensory nerves of the upper arms of a patient. Preclinical assessment was performed with collagen/SDSC implantation in rats after sectioning the sciatic nerve. For the patient, during the 3-year follow-up period, functional recovery of injured median and ulnar nerves was assessed by pinch gauge test and static two-point discrimination and touch test with monofilaments, along with electophysiological and MRI examinations. Preclinical experiments in rats revealed rescue of sciatic nerve and no side effects of patient-derived SDSC transplantation (30 and 180 days of treatment). In the patient treatment, motor and sensory functions of the median nerve demonstrated ongoing recovery postimplantation during the follow-up period. The results indicate that the collagen/SDSC artificial nerve graft could be used for surgical repair of larger defects in major lesions of peripheral nerves, increasing patient quality of life by saving the upper arms from amputation.

Therapeutic Potential of Immunoproteasome Inhibition in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy is an inherited fatal genetic disease characterized by mutations in dystrophin gene, causing membrane fragility leading to myofiber necrosis and inflammatory cell recruitment in dystrophic muscles. The resulting environment enriched in proinflammatory cytokines, like IFN-γ and TNF-α, determines the transformation of myofiber constitutive proteasome into the immunoproteasome, a multisubunit complex involved in the activation of cell-mediate immunity. This event has a fundamental role in producing peptides for antigen presentation by MHC class I, for the immune response and also for cytokine production and T-cell differentiation. Here, we characterized for the first time the presence of T-lymphocytes activated against revertant dystrophin epitopes, in the animal model of Duchenne muscular dystrophy, the mdx mice. Moreover, we specifically blocked i-proteasome subunit LMP7, which was up-regulated in dystrophic skeletal muscles, and we demonstrated the rescue of the dystrophin expression and the amelioration of the dystrophic phenotype. The i-proteasome blocking lowered myofiber MHC class I expression and self-antigen presentation to T cells, thus reducing the specific antidystrophin T cell response, the muscular cell infiltrate, and proinflammatory cytokine production, together with muscle force recovery. We suggest that i-proteasome inhibition should be considered as new promising therapeutic approach for Duchenne muscular dystrophy pathology.

Glucocorticoids Increase In Vitro and In Vivo Activities of Antibiotics against Chlamydophila pneumoniae

ABSTRACT The in vitro and in vivo antichlamydial activities of dexamethasone and beclomethasone alone and in combination with an antibiotic were tested. In vitro, dexamethasone and beclomethasone decreased the number of inclusion-forming units versus the control number (P < 0.001). The combination of glucocorticoids with azithromycin, telithromycin, or levofloxacin was more active than antibiotics used alone (P < 0.001). The combination, tested in a murine Chlamydophila pneumoniae infection model, produced similar results.

Expression of CD20 reveals a new store-operated calcium entry modulator in skeletal muscle

Among the scarce available data about the biological role of the membrane protein CD20, there is some evidence that this protein functions as a store-operated Ca(2+) channel and/or regulates transmembrane Ca(2+) trafficking. Recent findings indicate that store-operated Ca(2+) entry (SOCE) plays a central role in skeletal muscle function and development, but there remain a number of unresolved issues relating to SOCE modulation in this tissue. Here we describe CD20 expression in skeletal muscle, verifying its membrane localization in myoblasts and adult muscle fibers. Additionally, we show that inhibition of CD20 through antibody binding or gene silencing resulted in specific impairment of SOCE in C2C12 myoblasts. Our results provide novel insights into the CD20 expression pattern, and suggest that functional CD20 is required for SOCE to consistently occur in C2C12 myoblasts. These findings may contribute to future identification of mechanisms and molecules involved in the fine regulation of store-operated Ca(2+) entry in skeletal muscle.

Improvement of endurance of DMD animal model using natural polyphenols

Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is characterized by muscular wasting caused by dystrophin deficiency that ultimately ends in force reduction and premature death. In addition to primary genetic defect, several mechanisms contribute to DMD pathogenesis. Recently, antioxidant supplementation was shown to be effective in the treatment of multiple diseases including muscular dystrophy. Different mechanisms were hypothesized such as reduced hydroxyl radicals, nuclear factor-κB deactivation, and NO protection from inactivation. Following these promising evidences, we investigated the effect of the administration of a mix of dietary natural polyphenols (ProAbe) on dystrophic mdx mice in terms of muscular architecture and functionality. We observed a reduction of muscle fibrosis deposition and myofiber necrosis together with an amelioration of vascularization. More importantly, the recovery of the morphological features of dystrophic muscle leads to an improvement of the endurance of treated dystrophic mice. Our data confirmed that ProAbe-based diet may represent a strategy to coadjuvate the treatment of DMD.