Development of a Murine model to dissect the CpG-oligonucleotide-enhancement of the killing of human B Cells by rituximab.

International audienceAs a model to dissect the effects of CpG-oligonucleotides (CpG) on rituximab (RTX)-mediated therapeutic killing of autoimmune or malignant B lymphocytes, nude mice were grafted with Daudi human B cells. These mice were then injected with RTX alone or together with CpG. The human B cell aggregate was measured, and the reactive infiltrate analyzed after selective depletion of murine circulating cells. Macrophages (MØ) were identified in infiltrates, but not polymorphonuclear neutrophils (PMN), as confirmed by the failure of quantitative polymerase chain reaction to detect transcripts for PMN-specific myeloperoxidase in graft extracts. Evidence that MØ predominate over PMN in the anti-B cell RTX-induced immune mechanisms, include the presence of MØ-derived cytokines, and the lack of consequences of depletion of NK cells or B lymphocytes on the CpG-mediated effects on RTX. Interestingly however, removal of circulating PMN reduced the number of MØ attracted by the Daudi B cells. Our interpretation that CpG-induced complement activation is required for PMN to influence MØ was first based on overproduction of C5a in treated mice. This excess was due to the binding of the inhibitor of the alternative pathway of complement to CpG, as demonstrated by the elution of factor H from CpG-affinity-chromatography columns. Thus MØ are recruited to the tissue in the presence of C5a, and exploited locally by RTX

Two-photon microscopy of dermal innervation in a human re-innervated model of skin.

International audienceWhen skin is injured, innervation can be severely disrupted. The subsequent re-innervation processes are poorly understood notably because of the inability to image the full meandering course of nerves with their ramifications and endings from histological slices. In this letter, we report on two-photon excitation fluorescence (TPEF) microscopy of entire human skin explants re-innervated by rodent sensory neurons labelled with the styryl dye FM1-43. TPEF imaging of nerve fibres to a depth up to roughly 300 μm within the dermis was demonstrated, allowing three-dimensional reconstruction of the neural tree structure. Endogenous second-harmonic imaging of type I fibrillar collagen was performed in parallel to TPEF imaging using the same nonlinear microscope, revealing the path of the nerves through the dermis

Image Processing Methods for the Automated Assessment of Neuronal Outgrowth

International audienceNeuronal outgrowth assessment is useful to understand the development of peripheral or centralneurons and their regeneration after wounding. It consists in the determination of the length of the cellextensions (neurite length) using photos of neuron cultures. As the manual determination of neurite length istime-consuming and operator-dependent, many semi- or fully-automated methods have been developed. Most ofthem have been designed to analyze fluorescence microscopy images which allow clear delineation of cellbodies and neurites from the background. In this paper, we propose a new easy-to-use fully automated computervision methodbased on denoising, background subtraction, edge and envelope detection, and designed toanalyze compressed images (JPEG format) of non-fluorescent living neurons. A statistical tool was alsointegrated in the program to provide turnkey data to biologists. The reliability of our program was tested usingimages of differentiated PC-12 cell culture. Statistical analysis showed non-significant difference between themanual determination and our automated metho

Self-maintenance of neurogenic inflammation contributes to a vicious cycle in skin

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Biotechnological Management of Skin Burn Injuries: Challenges and Perspectives in Wound Healing and Sensory Recovery

International audienceMany wound management protocols have been developed to improve wound healing after burn with the primordial aim to restore the barrier function of the skin and also provide a better esthetic outcome. Autologous skin grafts remain the gold standard in the treatment of skin burn, but this treatment has its limitation especially for patients presenting limited donor sites due to extensive burn areas. Deep burn injuries also alter the integrity of skin-sensitive innervation and have an impact on patient's quality of life by compromising perceptions of touch, temperature, and pain. Thus, patients can suffer from long-term disabilities ranging from cutaneous sensibility loss to chronic pain. The cellular mechanisms involved in skin reinnervation following injury are not elucidated yet. Depending on the depth of the burn, nerve sprouting can occur from the wound bed or the surrounding healthy tissue, but somehow this process fails to provide correct reinnervation of the wound during scarring. In addition, several clinical observations indicate that damage to the peripheral nervous system influences wound healing, resulting in delayed wound healing or chronic wounds, underlining the role of innervation and neuromediators for normal cutaneous tissue repair development. Promising tissue engineering strategies, including the use of biomaterials, skin substitutes, and stem cells, could provide novel alternative treatments in wound healing and help in improving patient's sensory recovery

Major Role for TRPV1 and InsP3R in PAR2-Elicited Inflammatory Mediator Production in Differentiated Human Keratinocytes

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Major Role for TRPV1 and InsP3R in PAR2-Elicited Inflammatory Mediator Production in Differentiated Human Keratinocytes

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