Human decellularized dermal matrix seeded with adipose-derived stem cells enhances wound healing in a murine model: Experimental study.

OBJECTIVE: Full-thickness cutaneous wounds treated with split-thickness skin grafts often result in unaesthetic and hypertrophic scars. Dermal substitutes are currently used together with skin grafts in a single treatment to reconstruct the dermal layer of the skin, resulting in improved quality of scars. Adipose-derived stem cells (ASCs) have been described to enhance wound healing through structural and humoral mechanisms. In this study, we investigate the compatibility of xenogen-free isolated human ASCs seeded on human acellular dermal matrix (Glyaderm®) in a murine immunodeficient wound model. METHODS: Adipose tissue was obtained from abdominal liposuction, and stromal cells were isolated mechanically and cultured xenogen-free in autologous plasma-supplemented medium. Glyaderm® discs were seeded with EGFP-transduced ASCs, and implanted on 8 mm full-thickness dorsal wounds in an immunodeficient murine model, in comparison to standard Glyaderm® discs. Re-epithelialization rate, granulation thickness and vascularity were assessed by histology on days 3, 7 and 12. Statistical analysis was conducted using the Wilcoxon signed-rank test. EGFP-staining allowed for tracking of the ASCs in vivo. Hypoxic culture of the ASCs was performed to evaluate cytokine production. RESULTS: ASCs were characterized with flowcytometric analysis and differentiation assay. EGFP-tranduction resulted in 95% positive cells after sorting. Re-epithelialization in the ASC-seeded Glyaderm® side was significantly increased, resulting in complete wound healing in 12 days. Granulation thickness and vascularization were significantly increased during early wound healing. EGFP-ASCs could be retrieved by immunohistochemistry in the granulation tissue in early wound healing, and lining vascular structures in later stages. CONCLUSION: Glyaderm® is an effective carrier to deliver ASCs in full-thickness wounds. ASC-seeded Glyaderm® significantly enhances wound healing compared to standard Glyaderm®. The results of this study encourage clinical trials for treatment of full-thickness skin defects. Furthermore, xenogen-free isolation and autologous plasma-augmented culture expansion of ASCs, combined with the existing clinical experience with Glyaderm®, aid in simplifying the necessary procedures in a GMP-laboratory setting.

Xenogen-free isolation and culture of human adipose mesenchymal stem cells.

BACKGROUND: Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. METHODS: Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flowcytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. RESULTS: Mechanical isolation is more successful in isolating CD34+/CD31-/CD45-/CD13+/CD73+/CD146- ASCs from lipo-aspirate than isolation via collagenase digestion (p <0 .05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. CONCLUSION: A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications.

Inhibition of MALT1 Decreases Neuroinflammation and Pathogenicity of Virulent Rabies Virus in Mice.

Rabies virus is a neurovirulent RNA virus, which causes about 59,000 human deaths each year. Treatment for rabies does not exist due to incomplete understanding of the pathogenesis. MALT1 mediates activation of several immune cell types and is involved in the proliferation and survival of cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, leading to the expression of immunoregulatory genes. Here, we examined the impact of genetic or pharmacological MALT1 inhibition in mice on disease development after infection with the virulent rabies virus strain CVS-11. Morbidity and mortality were significantly delayed in Malt1-/- compared to Malt1+/+ mice, and this effect was associated with lower viral load, proinflammatory gene expression, and infiltration and activation of immune cells in the brain. Specific deletion of Malt1 in T cells also delayed disease development, while deletion in myeloid cells, neuronal cells, or NK cells had no effect. Disease development was also delayed in mice treated with the MALT1 protease inhibitor mepazine and in knock-in mice expressing a catalytically inactive MALT1 mutant protein, showing an important role of MALT1 proteolytic activity. The described protective effect of MALT1 inhibition against infection with a virulent rabies virus is the precise opposite of the sensitizing effect of MALT1 inhibition that we previously observed in the case of infection with an attenuated rabies virus strain. Together, these data demonstrate that the role of immunoregulatory responses in rabies pathogenicity is dependent on virus virulence and reveal the potential of MALT1 inhibition for therapeutic intervention.IMPORTANCE Rabies virus is a neurotropic RNA virus that causes encephalitis and still poses an enormous challenge to animal and public health. Efforts to establish reliable therapeutic strategies have been unsuccessful and are hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protease that mediates the activation of several innate and adaptive immune cells in response to multiple receptors, and therapeutic MALT1 targeting is believed to be a valid approach for autoimmunity and MALT1-addicted cancers. Here, we study the impact of MALT1 deficiency on brain inflammation and disease development in response to infection of mice with the highly virulent CVS-11 rabies virus. We demonstrate that pharmacological or genetic MALT1 inhibition decreases neuroinflammation and extends the survival of CVS-11-infected mice, providing new insights in the biology of MALT1 and rabies virus infection.

MALT1 Controls Attenuated Rabies Virus by Inducing Early Inflammation and T Cell Activation in the Brain.

MALT1 is involved in the activation of immune responses, as well as in the proliferation and survival of certain cancer cells. MALT1 acts as a scaffold protein for NF-κB signaling and a cysteine protease that cleaves substrates, further promoting the expression of immunoregulatory genes. Deregulated MALT1 activity has been associated with autoimmunity and cancer, implicating MALT1 as a new therapeutic target. Although MALT1 deficiency has been shown to protect against experimental autoimmune encephalomyelitis, nothing is known about the impact of MALT1 on virus infection in the central nervous system. Here, we studied infection with an attenuated rabies virus, Evelyn-Rotnycki-Abelseth (ERA) virus, and observed increased susceptibility with ERA virus in MALT1-/- mice. Indeed, after intranasal infection with ERA virus, wild-type mice developed mild transient clinical signs with recovery at 35 days postinoculation (dpi). Interestingly, MALT1-/- mice developed severe disease requiring euthanasia at around 17 dpi. A decreased induction of inflammatory gene expression and cell infiltration and activation was observed in MALT1-/- mice at 10 dpi compared to MALT1+/+ infected mice. At 17 dpi, however, the level of inflammatory cell activation was comparable to that observed in MALT1+/+ mice. Moreover, MALT1-/- mice failed to produce virus-neutralizing antibodies. Similar results were obtained with specific inactivation of MALT1 in T cells. Finally, treatment of wild-type mice with mepazine, a MALT1 protease inhibitor, also led to mortality upon ERA virus infection. These data emphasize the importance of early inflammation and activation of T cells through MALT1 for controlling the virulence of an attenuated rabies virus in the brain.IMPORTANCE Rabies virus is a neurotropic virus which can infect any mammal. Annually, 59,000 people die from rabies. Effective therapy is lacking and hampered by gaps in the understanding of virus pathogenicity. MALT1 is an intracellular protein involved in innate and adaptive immunity and is an interesting therapeutic target because MALT1-deregulated activity has been associated with autoimmunity and cancers. The role of MALT1 in viral infection is, however, largely unknown. Here, we study the impact of MALT1 on virus infection in the brain, using the attenuated ERA rabies virus in different models of MALT1-deficient mice. We reveal the importance of MALT1-mediated inflammation and T cell activation to control ERA virus, providing new insights in the biology of MALT1 and rabies virus infection.

Identification of a ZEB2-MITF-ZEB1 transcriptional network that controls melanogenesis and melanoma progression.

Deregulation of signaling pathways that control differentiation, expansion and migration of neural crest-derived melanoblasts during normal development contributes also to melanoma progression and metastasis. Although several epithelial-to-mesenchymal (EMT) transcription factors, such as zinc finger E-box binding protein 1 (ZEB1) and ZEB2, have been implicated in neural crest cell biology, little is known about their role in melanocyte homeostasis and melanoma. Here we show that mice lacking Zeb2 in the melanocyte lineage exhibit a melanoblast migration defect and, unexpectedly, a severe melanocyte differentiation defect. Loss of Zeb2 in the melanocyte lineage results in a downregulation of the Microphthalmia-associated transcription factor (Mitf) and melanocyte differentiation markers concomitant with an upregulation of Zeb1. We identify a transcriptional signaling network in which the EMT transcription factor ZEB2 regulates MITF levels to control melanocyte differentiation. Moreover, our data are also relevant for human melanomagenesis as loss of ZEB2 expression is associated with reduced patient survival.

The antibiotic streptomycin assessed in a battery of in vitro tests for reproductive toxicology.

Streptomycin is one of the most widely used antibiotics and is frequently added to cell culture media to prevent bacterial growth. We tested streptomycin in a battery of in vitro assays for assessment of reproductive toxicity. The follicle bio-assay (FBA) is a multiparametric long-term follicle culture system mimicking ovarian function; in vitro fertilisation (IVF) of exposed oocytes enables gamete quality determination through fecundability; the mouse embryo assay (MEA) analyses pre-implantation embryo development whereas the embryonic stem cell test (EST) studies post-implantation embryotoxicity. The FBA revealed a concentration-dependent decrease in oocyte nuclear maturation during continuous exposure from 50 microg/ml streptomycin onwards, characterised by a significantly reduced polar body-rate (40% vs. 92% in the control group). Oocytes that remained arrested in metaphase I (germinal vesicle breakdown) had aberrant spindle formation. IVF of long-term exposed oocytes in the FBA to 50 microg/ml streptomycin resulted in a significantly lower fertilisation rate of 23% vs. 74% in the control group and were unable to develop to the blastocyst stage. The MEA revealed no effect at pre-implantation embryo development and quality. Furthermore, no embryo-toxic effects of streptomycin were observed in the EST. In conclusion, oocytes are vulnerable to streptomycin treatment. Long-term exposure might cause fertility problems in the female and caution should be taken using streptomycin in cell culture media for assisted reproductive technology (ART).