[Le Blon, D.; Hoornaert, C.; Daans, J.; De Vocht, N.; Reekmans, K.; Berneman, Z.; Ponsaerts, P.] Univ Antwerp, Lab Expt Hematol, B-2020 Antwerp, Belgium. [Le Blon, D.; Hoornaert, C.; Daans, J.; De Vocht, N.; Reekmans, K.; Hens, N.; Goossens, H.; Berneman, Z.; Ponsaerts, P.] Univ Antwerp, Vaccine & Infect Dis Inst, B-2020 Antwerp, Belgium. [Guglielmetti, C.; Verhoye, M.; Van der Linden, A.] Univ Antwerp, Bioimaging Lab, B-2020 Antwerp, Belgium. [Dooley, D.; Lemmens, E.; Hendrix, S.] Hasselt Univ, Biomed Res Inst, Dept Morphol, Diepenbeek, Belgium. [Santermans, E.; Hens, N.] Hasselt Univ, Ctr Stat, I Biostat, Hasselt, Belgium. [Hens, N.] Univ Antwerp, Chermid, B-2020 Antwerp, Belgium.

Mast cells (MCs) are found abundantly in the central nervous system and play a complex role in neuroinflammatory diseases such as multiple sclerosis and stroke. In the present study, we show that MC-deficient KitW-sh/W-sh mice display significantly increased astrogliosis and T cell infiltration as well as significantly reduced functional recovery after spinal cord injury compared to wildtype mice. In addition, MC-deficient mice show significantly increased levels of MCP-1, TNF-α, IL-10 and IL-13 protein levels in the spinal cord. Mice deficient in mouse mast cell protease 4 (mMCP4), an MC-specific chymase, also showed increased MCP-1, IL-6 and IL-13 protein levels in spinal cord samples and a decreased functional outcome after spinal cord injury. A degradation assay using supernatant from MCs derived from either mMCP4−/− mice or controls revealed that mMCP4 cleaves MCP-1, IL-6, and IL-13 suggesting a protective role for MC proteases in neuroinflammation. These data show for the first time that MCs may be protective after spinal cord injury and that they may reduce CNS damage by degrading inflammation-associated cytokines via the MC-specific chymase mMCP4.

A disintegrin and metalloprotease 17 (ADAM17) is a sheddase with important substrates including tumor necrosis factor-α (TNF-α) and its receptors, the p75 neurotrophin receptor (p75NTR), and members of the epidermal growth factor family. The rationale of this study was to inhibit ADAM17-induced shedding of soluble TNF-α in order to reduce detrimental inflammation after spinal cord injury (SCI). However, using the specific ADAM17 blocker BMS-561392 in neuronal and glial cell cultures, we show that proper functioning of ADAM17 is vital for oligodendrocyte and microglia survival in a p44 MAPK-dependent manner. In contrast, genetic ablation of ADAM17 specifically increases microglial death. Surprisingly, although blocking ADAM17 in vivo does not substantially change the ratio between membrane-bound and soluble TNF-α, it increases expression of the pro-apoptotic marker Bax and microglial apoptosis while impairing functional recovery after SCI. These data suggest that ADAM17 is a key survival factor for microglial cells after SCI.

We have previously shown that the angiotensin AT2-receptor (AT2R) is upregulated in neuronal injury and when stimulated with angiotensin II promotes reinnervation and remyelination. The present study aimed to test AT2R-stimulation by the novel selective, non-peptide AT2R-agonist Compound 21 (C21) as a potential therapeutic approach for the treatment of experimental spinal cord injury in mice. Complementary experiments in primary neurons and organotypic cultures served to identify underlying neuroprotective mechanisms. Spinal cord injury was induced by controlled extradural contusion (10 mN for 10 sec) at T9 in anaesthetised female Balb/C mice. 0.5 μl fluorescent dextrane was injected into the left motor cortex for ex vivo tracking of motor neurons. Animals were treated with C21 (0.3 mg/kg/day i.p.) or vehicle for 4 weeks. Locomotor deficits were evaluated daily by Basso Mouse Scale and foot print analysis. The impact of C21 (1 μM) on reinnervation was evaluated in a co-culture system consisting of green-fluorescent-protein-(GFP)-positive entorhinal cortices and hippocampal target tissue. Neuronal differentiation and apoptosis were investigated in primary murine, neuronal cells. After spinal cord injury, C21 significantly attenuated locomotor deficits when compared to vehicle treatment (1.6 points on Basso Scale). Functional recovery was accompanied by a significantly increased number of motor neurons crossing the lesional area. In vitro, C21 significantly induced axonal ingrowth from entorhinal cortices into the hippocampal target tissue (+50%) as well as neurite outgrowth (+25%) from primary neurons. C21-induced neurite outgrowth was absent in neurons derived from AT2R-deficient mice. Treatment with C21 further significantly induced expression of anti-apoptotic Bcl-2 (+75.7%), of the neurotrophin BDNF (+53.7%), the neurotrophin receptor TrkB (+57.4%) and the marker for neurite sprouting, GAP43 (+103%). Specific stimulation of the AT2 receptor with the non-peptide AT2R-agonist C21 ameliorated locomotor function after experimental spinal cord injury in mice through the promotion of axonal outgrowth resulting in reinnervation and through anti-inflammatory, anti-apoptotic and neuroprotective mechanisms. Thus AT2R-stimulation may be considered a novel therapeutic approach for disease states requiring neuroprotection and neuroregeneration.

Integrins are transmembrane adhesion proteins that convey critical topobiological information and exert crucial signalling functions. In skin and hair follicle biology, β1 integrins and their ligands are of particular interest. It is not yet known whether β1 integrins play any role in the regulation of human hair growth and the expression pattern of β1 integrin in the human pilosebaceous unit remains ill-defined. Here, we show that pilosebaceous immunoreactivity for β1 integrin is most prominent in the outermost layer of the outer root sheath and the surrounding connective tissue sheath of human scalp hair follicles in situ and in vitro. Sites of β1 integrin immunoreactivity co-express fibronectin and tenascin-C. Contrary to previous reports, β1 integrin immunoreactivity in situ was not significantly upregulated in the human bulge region. Functionally, two β1 integrin-activating antibodies (12G10, TS2/16) and ligand-mimicking RGD peptides promoted the growth of microdissected, organ-cultured human scalp hair follicles in vitro and inhibited spontaneous hair follicle regression. This supports the concept that β1 integrin-mediated signalling is also important in human hair growth control. The physiologically relevant organ culture assay employed here is a potential research tool for exploring whether targeted stimulation of β1 integrin-mediated signalling is a suitable candidate for human hair loss management.

Background/purpose: This work was performed to optimize extraction conditions for D‐Squame® tape skin samples for use in the skin multiple analyte profile (SkinMAP) method, a Linco Research Corporation bead‐based assay for skin analytes. The experiments were designed to help identify sources of variability during extraction that may be amenable to further control.Methods: Two experimental designs were used to study factors influencing the extraction of skin samples from D‐Squame® tapes. Visually healthy skin samples were obtained from both female and male adult volar forearms. Factors studied in two experiments included: four surfactant (SDS) levels (0.02–0.2%), two buffer types [Citrate‐phosphate buffered saline at pH 5.5, phosphate‐buffered saline (PBS) at pH 7.4], two buffer volumes (1.0, 1.5 mL), two propylene glycol (PG) levels (0.1%, 1.0%), two extraction temperatures (7–10°C, 22–30+°C), two extraction times (30, 60 min), and location in sonication bath (two vectors). The response biomarkers were cortisol, fibronectin, human serum albumin, involucrin, keratin‐6 and keratins 1, 10. Skin sampling sites were also evaluated as sources of variation.Results: There was no single set of extraction conditions in our experiments that maximized recovery of all the biomarkers. SDS level had the most consistently significant (P<0.05) and directional effects on biomarker recoveries. In general, higher SDS resulted in higher recovery of all biomarkers. There was less consistency and fewer significant results for the other extraction factors.Conclusions: These data enable us to better manage SkinMAP studies and interpret their results. The use of 1.5 mL PBS containing 0.2% SDS and 0.5% PG with 30 min sonication at low (near 4°C) temperature is optimal for the quantitation of a range of SkinMAP analytes. In order to protect researchers from obtaining inflated false positive rates, it is crucial to design such studies and analyze the data using appropriate statistical methodology, especially for those studies involving only a small number of subjects.

Neurotrophins regulate cutaneous innervation, act as growth and motility factors on structural skin cells such as keratinocytes and fibroblasts, modulate cutaneous immune function and even serve as stress mediators in skin biology. The multilayered neurotrophin interaction with skin biology through high affinity specific tyrosinekinase receptors and the Janus-faced p75 receptor, which depending on ligand and co-receptor expression can serve as a low-affinity pan-neurotrophin receptor or a high affinity proneurotrophin receptor, guaranties this neuroendocrine peptide family a central position in the control of skin homeostasis in health and disease. It is a challenging task for future research efforts to integrate our knowledge on differential neurotrophin expression patterns and signaling pathways into complex concepts of neuroendocrine tissue remodeling and pathogenetic processes. In addition, we need to improve our understanding of the role of neurotrophin processing enzymes, associated co-receptors and intracellular adaptor molecules in specific cutaneous cell populations to design precise interaction tools for research and treatment. Such tools will allow us to utilize this ancient growth factor family in the management of neurotrophin responsive pathogenetic pathways and cutaneous diseases such as neurogenic inflammation, peripheral nerve degeneration, wound healing, atopic dermatitis or psoriasis.

The skin develops probably the densest and most complex innervation of all mammalian organs, consisting of sensory and autonomic nerves loaded with a plethora of neuropeptides and neurotransmitters. Skin innervation, as well as the expression patterns of neurotrophins and their receptors, is subject to dramatic changes during not only morphogenesis but also adult tissue remodeling under physiological or inflammatory conditions. Bilateral neuroimmune interactions are the basis of adaptive responses to tissue remodeling (such as hair cycling), psycho-emotional stress or skin inflammation. Dermatitis and hair loss may be exacerbated by stress-induced neurogenic inflammation. In addition, selected inflammatory skin diseases are associated with increased innervation. Finally, inflammatory cytokines influence the cutaneous expression of neurotrophins, as well as neurotrophin-induced neurite outgrowth following axotomy. Here, we review key studies on bilateral neuroimmune interactions in the skin under both healthy and disease conditions to provide a basis for future research on the role of inflammation in peripheral nerve regeneration.

In the healthy mammalian CNS, mast cells (MCs) are thought to be located mostly in the thalamus. In this study, we have systematically assessed the presence of MCs in the hippocampal formation (HF) and in the thalamus of normal male and female B10.PL mice. Giemsa+ and Toluidine Blue+ MCs were detected by histomorphometric analyses at perivascular and intraparenchymal sites of both the hippocampus and the entorhinal cortex. We found a mean number of 4.4 MCs in the HF of female and 3.3 MCs in male B10.PL mice. In contrast to the HF, no MCs were present in the thalamus of these mice. Notably, all HF-MCs showed immunoreactivity for Kit, the receptor for the MC growth and maturation factor SCF, as assessed by FITC–avidin/Kit double labelling. We demonstrate that the majority of brain MCs is found in the hippocampus and entorhinal cortex of B10.PL mice, though the total number of MCs is small compared to other mouse strains or rats. The presence of most brain MCs in the HF of B10.PL mice suggests a potential role of MCs in hippocampal physiology and pathology.

Nerve growth factor (NGF) promotes proliferation via its high affinity receptor (TrkA). Its precursor proNGF promotes apoptosis via the pan-neurotrophin-receptor p75. Recently, we have identified NGF and p75 as important hair growth terminators. However, if proNGF is involved or if NGF can also promote hair growth via TrkA is unclear. By RT-PCR we found that NGF/proNGF mRNA levels peak during early anagen in murine back skin, whereas NGF/proNGF protein levels peak during catagen, indicating high turnover in early anagen and protein accumulation in catagen. By immunohistochemistry, NGF and TrkA are found in the proliferating compartments of the epidermis and hair follicle throughout the cycle. In contrast, strong proNGF is found in the highly differentiated inner root sheath and adjacent to the p75+ regressing epithelial strand in catagen. Commercial 7S NGF, which contains both NGF and proNGF, promotes anagen development in organ-cultured early anagen mouse skin, whereas it promotes catagen development in late anagen skin. Together, our findings suggest an anagen-promoting or anagen-supporting role for NGF/TrkA, and a catagen-promoting role for proNGF/p75 interactions. This has important implications for the future design of specific neurotrophin receptor ligands as novel pharmaceuticals in the modification of tissue remodeling processes such as hair growth or wound healing.

After traumatic brain lesion, microglial cells are rapidly activated, migrate toward the sites of injury, and cause secondary damage that accounts for most of the loss of brain function. In the present study, we have characterized a new macrophage/microglia activation factor (MA-F). Using the monocytic cell line U937, we were able to demonstrate that MAF is upregulated after TPA-induced differentiation into macrophages. We have generated a specific antibody against MAY In BV-2 microglial cells, MAF is partially co-localized with 1134, a classical microglial marker. In addition, we have analyzed the in vivo expression patterns of MAF after entorhinal cortex lesion. We were able to show a substantial upregulation of MAF on selected CD11b(+) and IB4(+) macrophages/microglial cells in the deafferented hippocampus and in the perilesional region, while no MAF expression was detectable on the contralateral side. Confocal microscopy revealed a lysosome-like expression pattern in BV-2 cells, as well as in ECL-associated macrophages/microglial cells in vivo. Furthermore, we were able to demonstrate that U937 cells with downregulated MAF converted slower and to a significantly reduced extent to the macrophageal phenotype after TPA treatment. In addition, MAF downregulation in BV-2 microglial cells substantially reduced the phagocytotic uptake of dextran beads. Our data indicate that MAF is expressed in selected macrophages/microglial cells around the lesion and in the degenerating hippocampus after ECL. Furthermore, MAF expression in monocytic cells seems to play a functional role in the differentiation to a phagocytosing phenotype and may be, at least partially, required for phagocytotic activity, specifically in lesioned tissue after brain trauma. (c) 2005 Wiley-Liss, Inc.

After chemical, biological, or physical damage, growing (i.e. anagen) hair follicles develop abnormalities that are collectively called hair follicle dystrophy. Comparatively lower follicular damage induces the "dystrophic anagen" response pathway (=prolonged, dystrophic anagen, followed by severely retarded follicular recovery). More severe follicular damage induces the dystrophic catagen pathway (=immediate anagen termination, followed by a dystrophic, abnormally shortened telogen and maximally fast follicular recovery). In order to recognize these distinct damage response strategies of the hair follicle in a clinical or histopathological context, we have used the well-established C57BL/6J mouse model of cyclophosphamide-induced alopecia to define pragmatic classification criteria for hair follicle dystrophy (e.g., structure and pigmentation of the hair shaft, location, and volume of ectopic melanin granules, distension of follicular canal, number of TdT-mediated dUTP nick end labeling positive keratinocytes in the hair bulb; neural cell-adhesion molecule immunoreactivity and alkaline phosphatase activity as markers for the level of damage to the follicular papilla). These classification criteria for hair follicle dystrophy are useful not only in chemotherapy-induced alopecia models, but also in the screening of drug-treated or mutant mice in a highly standardized, accurate, sensitive, reproducible, easily applicable, and quantifiable manner.

BACKGROUND:The hair follicle continually undergoes dynamic remodelling in a cyclical manner involving tightly coordinated patterns of cell proliferation, differentiation and apoptosis. The oncoprotein c-Myc is a key regulator of these events in epidermal keratinocytes, but its importance in the hair growth cycle has not previously been determined.OBJECTIVES:To determine the role of c-Myc in the hair growth cycle.METHODS:We characterized the hair follicle phenotype of transgenic mice that permit expression of a switchable form of c-Myc (c-Myc-ER) in the suprabasal epithelial layers of the epidermis and hair follicle.RESULTS:c-Myc activation increased epithelial cell proliferation in the outer root sheath and distal hair follicle, without any substantial alteration in levels of apoptosis. Moreover, chronic c-Myc activation resulted in marked desynchronization of the murine hair growth cycle, uncoupling of hair cycle-related skin thickness and enlargement of the sebaceous gland.CONCLUSIONS:These data implicate c-Myc in the control of hair growth cycling and hair cycle-related epidermal and sebaceous gland homeostasis. We suggest that c-Myc may be activating follicular stem cells either directly or indirectly and that this has important implications for control of the 'hair cycle clock', hair growth and epidermal maintenance.