Objective To establish atopic dermatitis(AD)-like models in BALB/c mice using three chemical inducers,calcipotriol(MC903),2,4-dinitrochlorobenzene(DNCB),and oxazolone(OXA),and to explore the occurrence of ferroptosis in the different models.Methods Healthy 7-week-old female BALB/c mice were divided randomly into eight groups(n=8 mice per group)based on the induction site(ear/dorsal skin)and inducer:ear/dorsal control groups,MC903 ear/dorsal model groups,DNCB ear/dorsal model groups,and OXA ear/dorsal model groups.Models were established by topical application of the respective agents at specified concentrations.Mice in the MC903 ear/dorsal groups underwent continuous induction for 14 d.Mice in the DNCB and OXA ear/dorsal groups were sensitized for 3 consecutive days,4 days after the sensitization was completedand then challenged 12 times on day 8 and every other day for up to day 30.Skin lesions were observed and skin thickness was measured.Plasma levels of reactive oxygen species(ROS),interleukin(IL)-4,interferon(IFN)-γ,and malondialdehyde(MDA)were detected,the skin was examined by histopathological staining and ultrastructural observation,and expression levels of ferroptosis-related proteins(glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1),long-chain-fatty-acid-CoA ligase 4(ACSL4),transferrin receptor 1(TfR1))were detected by Western Blot.Results Compared with each control mice,all model mice exhibited obvious redness,swelling,scratching,desquamation,and rough thickening of the skin,and skin thickness was significantly increased(P＜0.01).ROS,IFN-γ,IL-4,and MDA levels were elevated to varying extents(P＜0.05)and histopathological features,including epidermal hyperplasia,keratinocyte degeneration,dermal vascular congestion,and immune cell infiltration,were detected in model mice.Transmission electron microscopy also revealed mitochondrial membrane rupture,increased density,and cristae reduction.Expression levels of ferroptosis markers were dysregulated,including significantly decreased GPX4/FTH1(P＜0.05)and increased ACSL4/TfR1 expression(P＜0.05).Conclusions All three chemicals successfully induced AD-like phenotypes in BALB/c mice through site-specific applications.Ferroptosis is involved in the pathological process of AD,but heterogeneity exists among inducers and modeling sites.

Objective To establish atopic dermatitis(AD)-like models in BALB/c mice using three chemical inducers,calcipotriol(MC903),2,4-dinitrochlorobenzene(DNCB),and oxazolone(OXA),and to explore the occurrence of ferroptosis in the different models.Methods Healthy 7-week-old female BALB/c mice were divided randomly into eight groups(n=8 mice per group)based on the induction site(ear/dorsal skin)and inducer:ear/dorsal control groups,MC903 ear/dorsal model groups,DNCB ear/dorsal model groups,and OXA ear/dorsal model groups.Models were established by topical application of the respective agents at specified concentrations.Mice in the MC903 ear/dorsal groups underwent continuous induction for 14 d.Mice in the DNCB and OXA ear/dorsal groups were sensitized for 3 consecutive days,4 days after the sensitization was completedand then challenged 12 times on day 8 and every other day for up to day 30.Skin lesions were observed and skin thickness was measured.Plasma levels of reactive oxygen species(ROS),interleukin(IL)-4,interferon(IFN)-γ,and malondialdehyde(MDA)were detected,the skin was examined by histopathological staining and ultrastructural observation,and expression levels of ferroptosis-related proteins(glutathione peroxidase 4(GPX4),ferritin heavy chain 1(FTH1),long-chain-fatty-acid-CoA ligase 4(ACSL4),transferrin receptor 1(TfR1))were detected by Western Blot.Results Compared with each control mice,all model mice exhibited obvious redness,swelling,scratching,desquamation,and rough thickening of the skin,and skin thickness was significantly increased(P＜0.01).ROS,IFN-γ,IL-4,and MDA levels were elevated to varying extents(P＜0.05)and histopathological features,including epidermal hyperplasia,keratinocyte degeneration,dermal vascular congestion,and immune cell infiltration,were detected in model mice.Transmission electron microscopy also revealed mitochondrial membrane rupture,increased density,and cristae reduction.Expression levels of ferroptosis markers were dysregulated,including significantly decreased GPX4/FTH1(P＜0.05)and increased ACSL4/TfR1 expression(P＜0.05).Conclusions All three chemicals successfully induced AD-like phenotypes in BALB/c mice through site-specific applications.Ferroptosis is involved in the pathological process of AD,but heterogeneity exists among inducers and modeling sites.

Objective: To investigate the etiology composition of enterovirus (EV) in patients with severe hand, foot, and mouth disease (HFMD) in children. To assess the diagnostic value of cerebrospinal fluid (CSF) tests in severe HFMD, and to find the key laboratory tests for severe HFMD. Methods: A total of 288 hospitalized cases of children clinically diagnosed with severe HFMD in Hangzhou Children′s Hospital were included from March to July 2016. Throat swabs were collected and enterovirus nucleic acids were detected by fluorescence quantitative reverse transcription (RT)-PCR. Synchronous CSF and serum samples were collected for EV-A71 and CV-sackievirus A16 (CV-A16)-IgM antibody detection. CSF samples underwent routine and biochemical tests. Normally distributed continuous variables were compared using t test. Non-normal distribution continuous variables were compared using Mann-Whitney U test. Differences between categorical variables were compared with χ² test. Results: The total positive rate of enterovirus nucleic acid EV-A71/CV-A16/EV by fluorescence quantitative RT-PCR in the 288 cases of children clinically diagnosed with HFMD was 83.7% (241/288), including EV-A71 55.2% (159/288), CV-A16 4.9% (14/288) and the other enterovirus 23.6% (68/288). Among the other enterovirus group, there were 29.4% CV-A6 (20/68) , 16.2% CV-A4 (11/68) and CV-A10 10.3% (7/68). The total positive rate of combined serum and CSF detection of EV nucleic acid and EV-A71 and CV-A16 IgM antibody was 98.3% (283/288). EV nucleic acid positive rate was 83.7% (24/288). The positive rates were statistically different (χ² =37.289, P=0.000). The CSF nucleated cells count in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (Z=-4.472 and -9.991, respectively, both P<0.05). The CSF nucleated cells positive rate in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (χ²=43.857 and 133.078, respectively, both P<0.05). The CSF protein level in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (Z=-3.151 and -5.255, respectively, both P<0.05). The CSF protein positive (>400 mg/L) rate in EV-A71 positive subgroup was higher than those in CV-A16 positive subgroup and other enteroviruses subgroup (χ²=4.956 and 11.795, respectively, both P<0.05). The CSF nucleated cell counts and positive rates in EV-A71 IgM antibody-positive subgroup were both higher than those in antibody-negative subgroup (both P<0.05). The CSF protein level and elevated proportion in antibody-positive subgroup were both higher than those in antibody-negative subgroup (both P<0.05). The lactate dehydrogenase concentration in antibody-positive subgroup was significantly higher than those in antibody-negative subgroup (P<0.05). The EV-A71 IgM antibody in serum was significantly correlated with the antibody in CSF (r=0.600, P<0.05). Conclusions EV-A71 is still the most important pathogen of severe HFMD in Hangzhou in 2016. Other enterovirus such as CV-A6, CV-A4 increases compared to those in 2014 and 2015. The CSF routine and biochemical tests and the IgM antibody levels can serve as an important indicator for the diagnosis of children with severe HFMD.