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Glutathione transferases (GSTs; EC 2.5.1.18) form a group of multifunctional enzymes that are involved in phase II of the cellular detoxification mechanism and are associated with increased susceptibility to cancer development and resistance to anticancer drugs. The present study aims to evaluate the ligandability of the human GSTM1-1 isoenzyme (hGSTM1-1) using a broad range of structurally diverse pesticides as probes. The results revealed that hGSTM1-1, compared to other classes of GSTs, displays limited ligandability and ligand-binding promiscuity, as revealed by kinetic inhibition studies. Among all tested pesticides, the carbamate insecticide pirimicarb was identified as the strongest inhibitor towards hGSTM1-1. Kinetic inhibition analysis showed that pirimicarb behaved as a mixed-type inhibitor toward glutathione (GSH) and 1-chloro-2,4-dinitrobenzene (CDNB). To shine a light on the restricted hGSTM1-1 ligand-binding promiscuity, the ligand-free crystal structure of hGSTM1-1 was determined by X-ray crystallography at 1.59 Å-resolution. Comparative analysis of ligand-free structure with the available ligand-bound structures allowed for the study of the enzyme’s plasticity and the induced-fit mechanism operated by hGSTM1-1. The results revealed important structural features of the H-site that contribute to xenobiotic-ligand binding and specificity. It was concluded that hGSTM1-1 interacts preferentially with one-ring aromatic compounds that bind at a discrete site which partially overlaps with the xenobiotic substrate binding site (H-site). The results of the study form a basis for the rational design of new drugs targeting hGSTM1-1.

Atopic dermatitis (AD) is a common inflammatory skin disease, and its pathogenesis is closely associated with microbial homeostasis in the gut, namely the gut-skin axis. Particularly, recent metagenomics studies revealed that the abundance of two major bacterial species in the gut, Faecalibacterium prausnitzii and Akkermansia muciniphila , may play a critical role in the pathogenesis of AD, but the effect of these species in AD has not yet been elucidated. To evaluate the potential beneficial effect of F. prausnitzii or A. muciniphila in AD, we conducted an animal model study where F. prausnitzii EB-FPDK11 or A. muciniphila EB-AMDK19, isolated from humans, was orally administered to 2,5-dinitrochlorobenzene (DNCB)-induced AD models using NC/Nga mice at a daily dose of 10 8 CFUs/mouse for six weeks. As a result, the administration of each strain of F. prausnitzii and A. muciniphila improved AD-related markers, such as dermatitis score, scratching behavior, and serum immunoglobulin E level. Also, the F. prausnitzii and A. muciniphila treatments decreased the level of thymic stromal lymphopoietin (TSLP), triggering the production of T helper (Th) 2 cytokines, and improved the imbalance between the Th1 and Th2 immune responses induced by DNCB. Meanwhile, the oral administration of the bacteria enhanced the production of filaggrin in the skin and ZO-1 in the gut barrier, leading to the recovery of functions. Taken together, our findings suggest that F. prausnitzii EB-FPDK11 and A. muciniphila EB-AMDK19 have a therapeutic potential in AD, which should be verified in humans.

We have implemented an improved, cost-effective, and highly reproducible protocol for a simple and rapid differentiation of the human leukemia monocytic cell line THP-1 into surrogates for immature dendritic cells (iDCs) or mature dendritic cells (mDCs). The successful differentiation of THP-1 cells into iDCs was determined by high numbers of cells expressing the DC activation markers CD54 (88%) and CD86 (61%), and the absence of the maturation marker CD83. The THP-1-derived mDCs are characterized by high numbers of cells expressing CD54 (99%), CD86 (73%), and the phagocytosis marker CD11b (49%) and, in contrast to THP-1-derived iDCs, CD83 (35%) and the migration marker CXCR4 (70%). Treatment of iDCs with sensitizers, such as NiSO4 and DNCB, led to high expression of CD54 (97%/98%; GMFI, 3.0/3.2-fold induction) and CD86 (64%/96%; GMFI, 4.3/3.2-fold induction) compared to undifferentiated sensitizer-treated THP-1 (CD54, 98%/98%; CD86, 55%/96%). Thus, our iDCs are highly suitable for toxicological studies identifying potential sensitizing or inflammatory compounds. Furthermore, the expression of CD11b, CD83, and CXCR4 on our iDC and mDC surrogates could allow studies investigating the molecular mechanisms of dendritic cell maturation, phagocytosis, migration, and their use as therapeutic targets in various disorders, such as sensitization, inflammation, and cancer.

Glutathione S -transferase (GSTs) are members of multifunction enzymes in organisms and mostly known for their roles in insecticide resistance by conjugation. Spodoptera litura (Fabricius) is a voracious agricultural pest widely distributed in the world with high resistance to various insecticides. The function of GSTs in the delta group of S. litura is still lacking. Significantly up-regulation of SlGSTd1 was reported in four pyrethroids-resistant populations and a chlorpyrifos-selected population. To further explore its role in pyrethroids and organophosphates resistance, the metabolism and peroxidase activity of SlGSTD1 were studied by heterologous expression, RNAi, and disk diffusion assay. The results showed that K m and V max for 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity of SlGSTD1were 1.68 ± 0.11 mmol L −1 and 76.0 ± 2.7 nmol mg −1  min −1 , respectively. Cyhalothrin, beta-cypermethrin, and chlorpyrifos had an obvious inhibitory effect on SlGSTD1 activity, especially for fenvalerate, when using CDNB as substrate. Fenvalerate and cyhalothrin can be metabolized by SlGSTD1 in E. coli and in vitro. Also, silencing of SlGSTd1 significantly increased the toxicity of fenvalerate and cyhalothrin, but had no significant effect on the mortality of larvae treated by beta-cypermethrin or chlorpyrifos. SlGSTD1 possesses peroxidase activity using cumene hydroperoxide as a stress inducer. The comprehensive results indicate that SlGSTD1 is involved in fenvalerate and cyhalothrin resistance of S. litura by detoxication and antioxidant capacity.

Atopic dermatitis (AD) is a common inflammatory skin disease characterized by a complex, heterogeneous pathogenesis including skin barrier dysfunction, immunology, and pruritus. Although epidermal growth factor (EGF) is essential for epithelial homeostasis and wound healing, the effect of EGF on AD remains to be explored. To develop a new therapy for AD, the anti-AD potential of EGF was investigated by inducing AD-like skin lesions in NC/Nga mice using 2,4-dinitrochlorobenzene (DNCB). EGF was administrated to NC/Nga mice to evaluate its therapeutic effect on DNCB-induced AD. EGF treatment improved dermatitis score, ear thickness, epidermal hyperplasia, serum total immunoglobulin E level, and transepidermal water loss in NC/Nga mice with DNCB-induced AD. In addition, levels of skin barrier-related proteins such as filaggrin, involucrin, loricrin, occludin, and zonula occludens-1 (ZO-1) were increased by EGF treatment. These beneficial effects of EGF on AD may be mediated by EGF regulation of Th1/Th2-mediated cytokines, mast cell hyperplasia, and protease activated receptor-2 (PAR-2) and thymic stromal lymphopoietin (TSLP), which are triggers of AD. Taken together, our findings suggest that EGF may potentially protect against AD lesional skin via regulation of skin barrier function and immune response.

The mouse model of 2,4-dinitrochlorbenzene (DNCB)-induced human-like atopic dermatitis (hlAD) has been widely used to test novel treatment strategies and compounds. However, the study designs and methods are highly diverse, presenting different hlAD disease patterns that occur after sensitization and repeated challenge with DNCB on dorsal skin. In addition, there is a lack of information about the progression of the disease during the experiment and the achieved pheno- and endotypes, especially at the timepoint when therapeutic treatment is initiated. We here examine hlAD in a DNCB-induced BALB/cJRj model at different timepoints: (i) before starting treatment with dexamethasone, representing a standard drug control (day 12) and (ii) at the end of the experiment (day 22). Both timepoints display typical AD-associated characteristics: skin thickening, spongiosis, hyper- and parakeratosis, altered cytokine and gene expression, increased lipid mediator formation, barrier protein and antimicrobial peptide abnormalities, as well as lymphoid organ hypertrophy. Increased mast cell infiltration into the skin and elevated immunoglobulin E plasma concentrations indicate a type I allergy response. The DNCB-treated skin showed an extrinsic moderate sub-acute hlAD lesion at day 12 and an extrinsic mild sub-acute to chronic pheno- and endotype at day 22 with a dominating Th2 response. A dependency of the filaggrin formation and expression in correlation to the disease severity in the DNCB-treated skin was found. In conclusion, our study reveals a detailed classification of a hlAD at two timepoints with different inflammatory skin conditions and pheno- and endotypes, thereby providing a better understanding of the DNCB-induced hlAD model in BALB/cJRj mice.

Atopic dermatitis is a chronic complex inflammatory skin disorder that requires sustainable treatment methods due to the limited efficacy of conventional therapies. Sargassum serratifolium , an algal species with diverse bioactive substances, is investigated in this study for its potential benefits as a therapeutic agent for atopic dermatitis. RNA sequencing of LPS-stimulated macrophages treated with ethanolic extract of Sargassum serratifolium (ESS) revealed its ability to inhibit a broad range of inflammation-related signaling, which was proven in RAW 264.7 and HaCaT cells. In DNCB-induced BALB/c or HR-1 mice, ESS treatment improved symptoms of atopic dermatitis within the skin, along with histological improvements such as reduced epidermal thickness and infiltration of mast cells. ESS showed a tendency to improve serum IgE levels and inflammation-related cytokine changes, while also improving the mRNA expression levels of Chi3l3, Ccr1, and Fcεr1a genes in the skin. Additionally, ESS compounds (sargachromanol (SCM), sargaquinoic acid (SQA), and sargahydroquinoic acid (SHQA)) mitigated inflammatory responses in LPS-treated RAW264.7 macrophages. In summary, ESS has an anti-inflammatory effect and improves atopic dermatitis, ESS may be applied as a therapeutics for atopic dermatitis.

We have integrated dermal dendritic cell surrogates originally generated from the cell line THP-1 as central mediators of the immune reaction in a human full-thickness skin model. Accordingly, sensitizer treatment of THP-1-derived CD14 - , CD11c + immature dendritic cells (iDCs) resulted in the phosphorylation of p38 MAPK in the presence of 1-chloro-2,4-dinitrobenzene (DNCB) (2.6-fold) as well as in degradation of the inhibitor protein kappa B alpha (IκBα) upon incubation with NiSO 4 (1.6-fold). Furthermore, NiSO 4 led to an increase in mRNA levels of IL-6 (2.4-fold), TNF-α (2-fold) and of IL-8 (15-fold). These results were confirmed on the protein level, with even stronger effects on cytokine release in the presence of NiSO 4 : Cytokine secretion was significantly increased for IL-8 (147-fold), IL-6 (11.8-fold) and IL-1β (28.8-fold). Notably, DNCB treatment revealed an increase for IL-8 (28.6-fold) and IL-1β (5.6-fold). Importantly, NiSO 4 treatment of isolated iDCs as well as of iDCs integrated as dermal dendritic cell surrogates into our full-thickness skin model (SM) induced the upregulation of the adhesion molecule clusters of differentiation (CD)54 (iDCs: 1.2-fold; SM: 1.3-fold) and the co-stimulatory molecule and DC maturation marker CD86 (iDCs ~1.4-fold; SM:~1.5-fold) surface marker expression. Noteworthy, the expression of CD54 and CD86 could be suppressed by dexamethasone treatment on isolated iDCs (CD54: 1.3-fold; CD86: 2.1-fold) as well as on the tissue-integrated iDCs (CD54: 1.4-fold; CD86: 1.6-fold). In conclusion, we were able to integrate THP-1-derived iDCs as functional dermal dendritic cell surrogates allowing the qualitative identification of potential sensitizers on the one hand, and drug candidates that potentially suppress sensitization on the other hand in a 3D human skin model corresponding to the 3R principles (“replace”, “reduce” and “refine”).

In the past decades studies investigating the dendritic cell (DC) activation have been conducted almost exclusively in animal models. However, due to species-specific differences in the DC subsets, there is an urgent need for alternative in vitro models allowing the investigation of Langerhans cell (LC) and dermal dendritic cell (DDC) activation in human tissue. We have engineered a full-thickness (FT) human skin tissue equivalent with incorporated LC surrogates derived from the human myeloid leukemia-derived cell line Mutz-3, and DDC surrogates generated from the human leukemia monocytic cell line THP-1. Topical treatment of the skin models encompassing Mutz-LCs only with nickel sulfate (NiSO 4 ) or 1-chloro-2,4-dinitrobenzene (DNCB) for 24 h resulted in significant higher numbers of CD1a positive cells in the dermal compartment, suggesting a sensitizer-induced migration of LCs. Remarkably, exposure of the skin models encompassing both, LC and DDC surrogates, revealed an early sensitizer-induced response reflected by increased numbers of CD1a positive cells in the epidermis and dermis after 8 h of treatment. Our human skin tissue equivalent encompassing incorporated LC and DDC surrogates allows the investigation of DC activation, subsequent sensitizer identification and drug discovery according to the principles of 3R.

A topic dermatitis (AD) is a common chronic and recurrent skin disorder. The protective effects of sodium butyrate (NaB), a metabolite of short-chain fatty acid breakdown by the gut microbiota, have been widely reported in numerous inflammatory diseases. However, the effect of NaB treatment alone on AD has not been reported. In the current study, AD was induced in BALB/c mice with 2,4-dinitrochlorobenzene (DNCB) for 28 days with NaB (200 mg/kg) treatment by gavage. NaB attenuated AD-induced skin bleeding, scarring, dryness, abrasions and erosions. In addition, NaB inhibited inflammatory cells infiltration and attenuated the expression of inflammatory cytokines and chemokines. Mechanistically, NaB reduced histone deacetylase 3 (HDAC3) expression and NF-κB p65 nuclear translocation by increasing the lysine acetylation levels of STAT1 and NF-κB p65 in AD. Taken together, our study suggests that NaB inhibits inflammatory mediators and ameliorates AD by inhibiting HDAC3 expression, thereby upregulating STAT1 and NF-κB p65 lysine acetylation levels and reducing NF-κB p65 nuclear translocation. Therefore, this study provides a new theoretical basis for NaB in the treatment of AD.