Dendritic cells under allergic condition enhance the activation of pruritogen-responsive neurons via inducing itch receptors in a co-culture study.

BACKGROUND: Itch sensitization has been reported in patients with chronic allergic skin diseases and observed in a mouse model of allergic contact dermatitis (ACD). There is evidence suggesting that neuroimmune interactions may contribute to itch sensitization, as an increase in dendritic cells (DCs) within ganglia has been observed during allergic conditions. However, how DCs interact with sensory neurons in ganglia during allergic conditions is still not known. This study aims to investigate the role of DCs in dorsal root ganglion (DRG) under ACD conditions, specifically focusing on itch sensitization within the DRG. The tolylene-2,4-diisocyanate (TDI) mouse model for ACD and the co-culture model of DCs and DRG neurons was employed in this study. RESULTS: We successfully induced ACD by TDI, as evidenced by the development of edema, elevated total serum IgE levels, and an observed itch reaction in TDI-sensitized mice. Calcium imaging and RT-qPCR analysis revealed that TDI-sensitized mice exhibited signs of peripheral sensitization, including a higher percentage of neurons responding to pruritogens and increased activation and expression of itch receptors in excised DRG of TDI-sensitized mice. Immunofluorescence and flow cytometric analysis displayed an increase of MHCII+ cells, which serves as a marker for DCs, within DRG during ACD. The co-culture study revealed that when DRG neurons were cultured with DCs, there was an increase in the number of neurons responsive to pruritogens and activation of itch receptors such as TRPA1, TRPV1, H1R, and TRPV4. In addition, the immunofluorescence and RT-qPCR study confirmed an upregulation of TRPV4. CONCLUSIONS: Our findings indicate that there is an increase of MHCII+ cells and itch peripheral sensitization in DRG under TDI-induced ACD condition. It has been found that MHCII+ cells in DRG might contribute to the itch peripheral sensitization by activating itch receptors, as shown through co-culture studies between DRG neurons and DCs. Further studies are required to identify the specific mediator(s) responsible for peripheral sensitization induced by activated DCs.

Early inflammatory events of mastitis-a pilot study with the isolated perfused bovine udder.

BACKGROUND: Bovine mastitis is an important health and cost factor in the milk industry. To elucidate whether isolated perfused bovine udders can be used to study early inflammatory events of mastitis, 1 mg of lipopolysaccharide (LPS) was instilled into quarters of 10 isolated perfused bovine udders. Three hours and 6 h after LPS instillation, tissue samples were taken from the gland cistern and base of the udder, subsequently stored in RNAlater and processed for the determination of inflammation-dependent gene regulation by real-time RT-qPCR. Gene expression analysis was performed using delta-delta Ct method. To translate mRNA results to protein, IL-1ß and IL-6 were determined in tissue homogenate by ELISA. RESULTS: The instillation of 1 mg LPS lead to an increased expression of pro-inflammatory cytokines and chemokines like TNF-α, CCL20, CXCL8 as well as of IL-1 ß, IL-6 and IL-10, lingual antimicrobial peptide (LAP) and S100A9. However, the degree of elevation differed slightly between gland cistern and udder base and markedly between 3 and 6 h after instillation, with a distinct increase in mediator expression after 6 h. IL-1ß protein increased in a time-dependent manner, whereas IL-6 was unchanged within 6 h of LPS instillation. CONCLUSION: Compared to in vivo studies with instillation of LPS into udders of living cows, a similar inflammation-dependent gene regulation profile can be mimicked in the isolated perfused bovine udder, indicating a supplementation of animal experiments.

Precision-cut bovine udder slices (PCBUS) as an in-vitro-model of an early phase of infection of bovine mastitis.

BACKGROUND: The aim of this study was to establish precision-cut bovine udder slices (PCBUS) as an in-vitro-model to investigate pathophysiological processes in the early phase of mastitis in order to have the possibility to investigate new therapeutic approaches for the treatment of such udder inflammation in later studies. Furthermore, this model should contribute to substitute in-vivo-experiments. Bovine mastitis is one of the most common and costly infectious diseases in the dairy industry, which is largely associated with the use of antimicrobial agents. Given this problem of antimicrobial resistance, it is essential to step up research into bacterial infectious diseases. Thus, the transfer of the in-vitro-model of precision-cut tissue slices to the bovine udder enables broad research into new therapeutic approaches in this area and can also be used to address issues in basic research or the characterisation of complex pathophysiological processes. RESULTS: A stimulation with LPS, PGN or the combination of both substances (LPS:PGN) demonstrates the ability of the PCBUS to react with a significant secretion of IL-1ß, TNF-α and PGE2. CONCLUSION: The slices represent an instrument for investigating pharmacological interactions with udder tissue, which can be useful for studies on pharmacological questions and the understanding of complex pathophysiological processes of infection and inflammation.

LEKTI domain 6 displays anti-inflammatory action in vitro and in a murine atopic dermatitis model.

BACKGROUND: Lympho-epithelial Kazal-type-related inhibitor (LEKTI) is a serine protease inhibitor consisting of multiple domains. A loss of function mutation is described in Netherton patients that show severe symptoms of atopic lesions and itch. OBJECTIVES: LEKTI domain 6 (LD6) has shown strong serine protease-inhibitory action in in vitro assays and thus it was tested in vitro and in vivo for potential anti-inflammatory action in models of atopic skin disease. METHODS: Human skin equivalents were treated with LD6 and an inflammatory reaction was challenged by kallikrein-related endopeptidase 5 (KLK5). Furthermore, LD6 was tested on dorsal root ganglia cells stimulated with KLK5, SLIGRL and histamine by calcium imaging. The effect of topically administered LD6 (0.4-0.8%) in lipoderm was compared to a topical formulation of betamethasone-diproprionate (0.1%) in a therapeutic setting on atopic dermatitis-like lesions in NC/Nga mice sensitized to house dust mite antigen. Endpoints were clinical scoring of the mice as well as determination of scratching behaviour. RESULTS: KLK5 induced an upregulation of CXCL-8, CCL20 and IL-6 in skin equivalents. This upregulation was reduced by pre-incubation with LD6. KLK5 as well as histamine induced calcium influx in a population of neurons. LD6 significantly reduced the calcium response to both stimuli. When administered onto lesional skin of NC/Nga mice, both LD6 and betamethasone-dipropionate significantly reduced the inflammatory reaction. The effect on itch behaviour was less pronounced. CONCLUSION: Topical administration of LD6 might be a new therapeutic option for treatment of lesional atopic skin.

FeMn with Phases of a Degradable Ag Alloy for Residue-Free and Adapted Bioresorbability.

The development of bioresorbable materials for temporary implantation enables progress in medical technology. Iron (Fe)-based degradable materials are biocompatible and exhibit good mechanical properties, but their degradation rate is low. Aside from alloying with Manganese (Mn), the creation of phases with high electrochemical potential such as silver (Ag) phases to cause the anodic dissolution of FeMn is promising. However, to enable residue-free dissolution, the Ag needs to be modified. This concern is addressed, as FeMn modified with a degradable Ag-Calcium-Lanthanum (AgCaLa) alloy is investigated. The electrochemical properties and the degradation behavior are determined via a static immersion test. The local differences in electrochemical potential increase the degradation rate (low pH values), and the formation of gaps around the Ag phases (neutral pH values) demonstrates the benefit of the strategy. Nevertheless, the formation of corrosion-inhibiting layers avoids an increased degradation rate under a neutral pH value. The complete bioresorption of the material is possible since the phases of the degradable AgCaLa alloy dissolve after the FeMn matrix. Cell viability tests reveal biocompatibility, and the antibacterial activity of the degradation supernatant is observed. Thus, FeMn modified with degradable AgCaLa phases is promising as a bioresorbable material if corrosion-inhibiting layers can be diminished.