A small molecule mitigates hearing loss in a mouse model of Usher syndrome III.

Usher syndrome type III (USH3), characterized by progressive deafness, variable balance disorder and blindness, is caused by destabilizing mutations in the gene encoding the clarin-1 (CLRN1) protein. Here we report a new strategy to mitigate hearing loss associated with a common USH3 mutation CLRN1(N48K) that involves cell-based high-throughput screening of small molecules capable of stabilizing CLRN1(N48K), followed by a secondary screening to eliminate general proteasome inhibitors, and finally an iterative process to optimize structure-activity relationships. This resulted in the identification of BioFocus 844 (BF844). To test the efficacy of BF844, we developed a mouse model that mimicked the progressive hearing loss associated with USH3. BF844 effectively attenuated progressive hearing loss and prevented deafness in this model. Because the CLRN1(N48K) mutation causes both hearing and vision loss, BF844 could in principle prevent both sensory deficiencies in patients with USH3. Moreover, the strategy described here could help identify drugs for other protein-destabilizing monogenic disorders.

CCL5 and CCL20 mediate immigration of Langerhans cells into the epidermis of full thickness human skin equivalents.

Epidermal Langerhans cells (LC) play a key role in initiation and regulation of immune responses. Whereas LC migration out of the epidermis upon environmental assault is extensively studied, the mechanisms involved in the (re)population of the epidermis with LC are poorly understood. Here, we investigated the immigration of LC derived from the human MUTZ-3 cell line (MUTZ-LC) into the epidermis of a full thickness skin equivalent, comprising a fully differentiated epidermis on a fibroblast-populated dermis. MUTZ-LC were used to determine which epidermis-derived chemokines play a role in mediating LC trans-dermal migration into the epidermis. We found evidence for a role of keratinocyte-derived CCL5 and CCL20 in the chemo-attraction of MUTZ-LC. Neutralizing antibodies against CCL5 and CCL20 blocked LC migration towards keratinocytes. Secretion of these two chemokines was associated with incorporation of MUTZ-LC into the epidermis of full thickness skin equivalents. In conclusion, our findings suggest that epidermis derived CCL5 and CCL20 are pivotal mediators in recruitment of LC into the epidermis.

Technical advance: Langerhans cells derived from a human cell line in a full-thickness skin equivalent undergo allergen-induced maturation and migration.

In this report, the construction of a functional, immunocompetent, full-thickness skin equivalent (SE) is described, consisting of an epidermal compartment containing keratinocytes, melanocytes, and human LCs derived from the MUTZ-3 cell line (MUTZ-LC) and a fibroblast-populated dermal compartment. The CD1a(+)Langerin(+)HLA-DR(+) MUTZ-LCs populate the entire epidermis at a similar density to that found in native skin. Exposure of the SE to subtoxic concentrations of the allergens NiSO(4) and resorcinol resulted in LC migration out of the epidermis toward the fibroblast-populated dermal compartment. A significant dose-dependent up-regulation of the DC maturation-related CCR7 and IL-1ß transcripts and of CD83 at the protein level upon epidermal exposure to both allergens was observed, indicative of maturation and migration of the epidermally incorporated LC. We have thus successfully developed a reproducible and functional full-thickness SE model containing epidermal MUTZ-LC. This model offers an alternative to animal testing for identifying potential chemical sensitizers and for skin-based vaccination strategies and provides a unique research tool to study human LC biology in situ under controlled in vitro conditions.

Irritant-induced migration of Langerhans cells coincides with an IL-10-dependent switch to a macrophage-like phenotype.

Langerhans cells (LCs) migrate after topical exposure of the skin to irritants, despite the supposed independence of irritant contact dermatitis from adaptive immunity. Whereas allergen-activated LCs are known to migrate to the draining lymph nodes (LNs), the fate of migrated LCs upon topical irritant exposure is unknown. Here, we identified a phenotypic switch of LCs after their migration into the dermis upon irritant exposure. With the aid of ex vivo intact human skin and epidermal sheets, we show that dermal fibroblasts are necessary for an IL-10-dependent postmigrational phenotypic switch of LCs into macrophage-like cells. Exposure of ex vivo skin to a panel of seven irritants resulted in a decrease in the number of CD1a(+) cells and an increase in CD14(+)/CD68(+) cells in the dermis. Neutralizing antibodies against IL-10 totally inhibited the phenotypic LC-to-macrophage transition, but did not influence the migration of CD1a(+) cells. Exposure of epidermal sheets to irritants resulted in a fibroblast-dependent LC-to-CD14(+)/CD68(+) switch coinciding with migration, which could be totally inhibited by neutralizing antibodies against either IL-10 or CCL2/CCL5 (two chemokines responsible for epidermal-to-dermal migration). We have thus identified an IL-10-dependent phenotypic switch of LCs into macrophage-like cells upon irritant exposure and emigration from the epidermis.

Epidermis-to-dermis migration of immature Langerhans cells upon topical irritant exposure is dependent on CCL2 and CCL5.

Skin irritation is generally not considered to be an immunological event; however, alterations in the density of Langerhans cells (LC) in the epidermis do occur, which is indicative of LC migration. In this study, we investigated the migration of LC out of the epidermis after skin exposure to contact irritants and identified the chemokines involved. With the aid of ex vivo-intact human skin and epidermal sheets we show that dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of ex vivo-intact human skin to a panel of seven irritants (SDS, salicylic acid, phenol, isopropanol, DMSO, TritonX, or benzalkonium chloride) resulted in decreased numbers of CD1a(+) cells in the epidermis and the accumulation of CD1a(+) cells in the dermis. In contrast to allergen exposure, neutralizing antibodies to either CXCL12 or CCL19/CCL21 did not inhibit LC migration out of the epidermis. Exposure of epidermal sheets to the prototypical irritant SDS resulted in a TNF-alpha-dependent LC migration towards dermal fibroblasts. This was a result of CCL2/MCP-1 and CCL5/RANTES chemokine secretion by fibroblasts: injection of CCL2- and CCL5-neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. We have thus identified a novel role for TNF-alpha-inducible dermis-derived CCL2 and CCL5 in initiating migration of irritant-exposed human LC out of the epidermis.

Comparison of a novel CXCL12/CCL5 dependent migration assay with CXCL8 secretion and CD86 expression for distinguishing sensitizers from non-sensitizers using MUTZ-3 Langerhans cells.

As the induction of contact hypersensitivity is the result of a series of cellular processes, including maturation and migration of epidermal dendritic cells (Langerhans cells (LC)), a battery of assays based on these in vivo events might provide a robust in vitro predictability model for distinguishing sensitizers from non-sensitizers. Therefore, assays with read-out for changes in CD86 expression and CXCL8 secretion were compared with a novel functional assay based on the in vitro migratory behaviour of LC. In all three assays LC derived from the human myeloid-leukaemia-cell-line MUTZ-3 (MUTZ-LC) were used. Exposure of MUTZ-LC to a panel of five sensitizers and three non-sensitizers resulted in increased CD86 expression in only 3/5 sensitizers, but also in 1/3 non-sensitizers. In contrast, CXCL8 secretion was uniformly increased after exposure to all sensitizers, but not after exposure to non-sensitizers. In a transwell migration assay, preferential migration of sensitizer-exposed MUTZ-LC towards CXCL12 was observed (5/5 sensitizers), whereas non-sensitizer-exposed MUTZ-LC only migrated towards CCL5 (3/3 non-sensitizers). In conclusion, the novel MUTZ-LC migration assay and analysis of CXCL8 secretion proved to be more successful than analysis of CD86 in predicting sensitizers from non-sensitizers and therefore warrant further investigation in the field of in vitro assay development.

CXCL12 is essential for migration of activated Langerhans cells from epidermis to dermis.

The initial step in Langerhans cell (LC) migration from the epidermis to the lymph node involves migration of maturing LC into the dermis. Here, we investigated the migration of LC out of the epidermis after exposure of the skin to contact allergens. Ex vivo intact human skin, epidermal sheets, and LC derived from the MUTZ-3 cell line (MUTZ-LC) were used to determine whether dermal fibroblasts play a role in mediating LC migration towards the dermis. Exposure of epidermal sheets or MUTZ-LC to allergens (nickel sulphate, 2,4-dinitrochlorobenzene, and cinnamaldehyde) or a cytokine maturation cocktail resulted in LC migration towards dermal fibroblasts. This was due to upregulation of CXCR4 on maturing LC and secretion of CXCL12/stromal derived factor-1 chemokine by fibroblasts. Neutralizing antibodies to either CXCL12 or CXCR4 completely blocked migration. Injection of CXCL12 neutralizing antibodies into intact human skin totally inhibited LC migration into the dermis. In contrast, neutralizing antibodies to CCL19/CCL21 did not inhibit migration into the dermis. We describe a novel and essential role of dermis-derived CXCL12 in initiating migration of maturing human LC to the dermis thus permitting their further journey to the draining lymph nodes.

Expression and immunogenicity of the Plasmodium falciparum circumsporozoite protein: the role of GPI signal sequence.

Previous studies have shown that the immunogenicity of rodent malaria parasite-derived circumsporozoite protein (CS) can be improved by deleting the glycosyl-phosphatidyl-inositol (GPI) signal sequence. To study whether GPI signal sequence deletion would also improve immunogenicity of CS derived from the major plasmodium species causing mortality in humans (P. falciparum), we tested different variants of the P. falciparum CS protein in the context of a live vector-based vaccine carrier (rAd35). We demonstrate that deletion of the GPI signal sequence from CS did not result in altered expression or secretion. In contrast, cellular localization was clearly altered, which perhaps helps to explain the significant improvement of anti-CS antibody and T-cell responses observed in mice using deletion variants in the context of the rAd35 carrier. Our results show that rational design of antigens is warranted for further development of malaria vaccines.

Human CD46-transgenic mice in studies involving replication-incompetent adenoviral type 35 vectors.

Wild-type strains of mice do not express CD46, a high-affinity receptor for human group B adenoviruses including type 35. Therefore, studies performed to date in mice using replication-incompetent Ad35 (rAd35) vaccine carriers may underestimate potency or result in altered vector distribution. Here, it is reported that CD46 transgenic mice (MYII-strain) express CD46 in all major organs and that it functions as a receptor for rAd35 vectors. Similar to monkeys and humans, MYII mice highly express CD46 in their lungs and kidneys and demonstrate low expression in muscle. Upon intravenous administration, rAd35 vector genomes as well as expression are detected in lungs of MYII mice, in contrast to wild-type littermates. Expression was predominantly detected in lung epithelial cells. Upon intramuscular administration, the initial level of luciferase expression is higher in MYII mice as compared with wild-type littermates, in spite of the fact that CD46 expression is low in muscle of MYII mice. The higher level of expression in muscle of MYII mice results in prolonged gene expression as assessed by CCD camera imaging for luciferase activity. Finally, a significant dose-sparing effect in MYII mice as compared with wild-type littermates on anti-SIVgag CD8+ T-cell induction following intramuscular vaccination with an rA35.SIVgag vaccine was observed. This dose-sparing effect was also observed when reinfusing dendritic cells derived from MYII mice after exposure to rAd35.SIVgag vaccine as compared with rAd35.SIVgag exposed dendritic cells from wild-type littermates. It was concluded that MYII mice represent an interesting preclinical model to evaluate potency and safety of rAd35 vectors.

Histone deacetylase inhibitor BL1521 induces a G1-phase arrest in neuroblastoma cells through altered expression of cell cycle proteins.

Histone deacetylase inhibitors (HDACi) have been discovered as potential drugs for cancer treatment. The effect of BL1521, a novel HDACi, on the cell cycle distribution and the induction of apoptosis was investigated in a panel of MYCN single copy and MYCN amplified neuroblastoma cell lines. BL1521 arrested neuroblastoma cells in the G1 phase and induced up to 30% apoptosis. Downregulation of CDK4, upregulation of p21(WAF1/CIP1) and an increase of hypophosphorylated retinoblastoma protein were observed, indicating a possible mechanism for the cell-cycle arrest. BL1521 also induced downregulation of p27, which may underlie the observed induction of apoptosis.