Image1_Case report: Difference in outcomes between two cases of Hailey-Hailey disease treated with apremilast.tif

Hailey-Hailey disease (HHD) is a rare autosomal dominant acantholytic dermatosis clinically characterized by recurrent erythematous plaques and erosions mainly on the intertriginous regions. Although HHD seriously affects quality of life, conventional treatments often fail to provide long-term relief for most patients. The effectiveness of apremilast, a phosphodiesterase-4 inhibitor, against severe HHD was first reported in 2018, and after further testing, this agent is currently expected to be established as an efficacious and safe therapeutic option. Here we report two cases of HHD treated with apremilast which showed opposite outcomes. Although the case with extremely severe symptoms showed remarkable and long-lasting improvement with apremilast used after acute treatment with oral corticosteroid, the other case, with milder symptoms treated only with apremilast, showed no improvement. Our transcriptome analysis using skin samples collected prior to apremilast administration revealed the involvement of the NF-κB signaling pathway, which is related to the responses to bacteria and other organisms. However, this pathway was more strongly activated in case 2 than in case 1, suggesting that the steroid treatment preceding apremilast may have been effective and supportive in the apremilast-responding case. One of the two cases highlights the potential of apremilast as a treatment option for HHD, but the other underlines the difficulties in managing HHD and the complexity of the disease background. The accumulation of cases and larger clinical studies are expected to precisely evaluate the safety and efficacy of apremilast, and the potential for therapies in combination with conventional treatments.</p

Image2_Case report: Difference in outcomes between two cases of Hailey-Hailey disease treated with apremilast.tif

Hailey-Hailey disease (HHD) is a rare autosomal dominant acantholytic dermatosis clinically characterized by recurrent erythematous plaques and erosions mainly on the intertriginous regions. Although HHD seriously affects quality of life, conventional treatments often fail to provide long-term relief for most patients. The effectiveness of apremilast, a phosphodiesterase-4 inhibitor, against severe HHD was first reported in 2018, and after further testing, this agent is currently expected to be established as an efficacious and safe therapeutic option. Here we report two cases of HHD treated with apremilast which showed opposite outcomes. Although the case with extremely severe symptoms showed remarkable and long-lasting improvement with apremilast used after acute treatment with oral corticosteroid, the other case, with milder symptoms treated only with apremilast, showed no improvement. Our transcriptome analysis using skin samples collected prior to apremilast administration revealed the involvement of the NF-κB signaling pathway, which is related to the responses to bacteria and other organisms. However, this pathway was more strongly activated in case 2 than in case 1, suggesting that the steroid treatment preceding apremilast may have been effective and supportive in the apremilast-responding case. One of the two cases highlights the potential of apremilast as a treatment option for HHD, but the other underlines the difficulties in managing HHD and the complexity of the disease background. The accumulation of cases and larger clinical studies are expected to precisely evaluate the safety and efficacy of apremilast, and the potential for therapies in combination with conventional treatments.</p

Relationship between cytotoxic activities of gas phase extracts of cigarette smoke and cigarette brand.

<p>The gas phase extracts of cigarette smoke (designated cCSE) at the original tar concentration of 10 mg/ml were prepared from cigarettes of various brands by continuous smoking protocol as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#pone-0107856-g001" target="_blank">Fig. 1</a>. The cCSEs were subjected to MTS reduction assay for evaluation of their cytotoxic activities, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#pone-0107856-g003" target="_blank">Fig. 3</a>. MTS reduction activity in the absence of the gas phase extract was represented as 100%. Values represent means ± SE of three experiments, each in triplicate. P, Peace (JT, Japan; 28 mg tar, 2.3 mg nicotine), HL, Hi-Lite (JT, Japan; 17 mg tar, 1.4 mg nicotine), SS, Seven Stars (JT, Japan; 14 mg tar, 1.2 mg nicotine), M, Mevius (JT, Japan; 10 mg tar, 0.8 mg nicotine), MSL, Mevius Super Light (JT, Japan; 6 mg tar, 0.5 mg nicotine), Ma, Marlboro (Phillip Morris, USA; 12 mg tar, 1.0 mg nicotine), LS, Lucky Strike (British American Tobacco, UK; 11 mg tar, 0.9 mg nicotine), K9, Kent 9 mg (British American Tobacco, UK; 9 mg tar, 0.8 mg nicotine).</p

Schematic diagram of an apparatus for preparation of gas phase extracts of cigarette smoke.

<p>A standard method for preparation of the gas phase extract of cigarette smoke is as follows. Four cigarettes of Hi-Lite brand, unless otherwise specified, were sequentially combusted and the main-stream smoke was continuously sucked through a Cambridge filter at a constant flow rate of 1.050 l/min by an aspirator, to remove the tar phase. The remaining gas phase was bubbled through a glass ball filter (pore size: 20–30 µm) into phosphate buffered saline (PBS, 15 ml) in a graduated cylinder kept at 25°C. After combustion of cigarette, the filter was dried in air for 12 h at 25°C, and the dry weight of the tar phase trapped on the Cambridge filter was obtained by subtracting the weight of filter before use from that after use. The concentration of the gas phase extract was expressed as the virtual tar concentration (mg tar/ml PBS), assuming that the tar phase trapped on the Cambridge filter is dissolved in the PBS. Four cigarettes of Hi-Lite brand gave the dry tar weight of approximately 150 mg. Notably, cytotoxicity of the gas phase extracts depends not on cigarette brands but on the virtual tar concentration.</p

Sensitivities of various cultured cells to the gas phase extracts of cigarette smoke.

<p>The gas phase extracts of cigarette smoke (cCSE) at the virtual tar concentration of 10 mg/ml were prepared from Hi-Lite brand cigarettes by continuous smoking protocol, and they were subjected to MTS reduction assay using various cultured cells. MTS reduction activity was represented as a percentage of the control value in the absence of cCSE. Values represent means ± SE of three experiments, each in triplicate. (A) C6, rat glioma cells; HEK293T, human embryonic kidney cells; CHO, Chinese hamster ovary cells; HeLa, human cervical carcinoma cells; U937, human monocytes; RAW264.7, mouse macrophages; HUVEC, immortalized human umbilical vein endothelial cells; A7r5, rat aorta smooth muscle cells. (B) SBC-3, human lung small cell carcinoma; H1299, human lung squamous cell carcinoma; A549, human lung adenocarcinoma.</p

Cytotoxic activities of gas phase extracts of cigarette smoke and the number of cigarette.

<p>Concentration-response curves of the gas phase extracts of cigarette smoke prepared from varying numbers of cigarettes (Hi-Lite brand) (A) and of the phosphate buffered saline (PBS) in the second graduated cylinder (B) for inhibition of MTS reduction activity. (A) The gas phase extract of cigarette smoke (designated cCSE) was prepared from varying numbers (2–40) of cigarettes (Hi-Lite brand) based on continuous smoking protocol, while a new Cambridge filter was used every 4 cigarettes. Inset: Concentration-response curves of the cCSE prepared from 20 or 40 cigarettes with a change in scale of concentrations on x axis. (B) In the apparatus for preparation of cCSE, the second graduated cylinder with 15 ml of PBS was incorporated downstream of the first one, and cCSE was prepared from either 4 or 14 cigarettes (Hi-Lite brand). The cytotoxicity of PBS in the original and second graduated cylinders was evaluated using MTS reduction assay. MTS reduction activity in the absence of the gas phase extract was represented as 100%. Values represent means ± SE of three experiments, each in triplicate. 4-1 (14-1) and 4-2 (14-2) represent the cytotoxic activities of the PBS in the first (original) and second graduated cylinders prepared from 4 (14) cigarettes, respectively.</p

Pharmacological properties of cytotoxic activities of two types of gas phase extracts of cigarette smoke.

<p>The gas phase extracts of cigarette smoke at the virtual tar concentration of 10 mg/ml PBS were prepared from Hi-Lite brand cigarettes by either continuous (cCSE) or puff smoking protocol (pCSE), and they were subjected to MTS reduction assay (A) and LDH leakage assay (B). For determination of the effects of inhibitors of protein kinase C or NADPH oxidase, 5 µM BIS I or 1 µM DPI was added to the culture medium of C6 glioma cells, respectively, 30 min before the start of 4-h incubation with cCSE or pCSE. In panel A, MTS reduction activity was represented as a percentage of the control value in the absence of CSEs (PBS) within the vehicle-treated group. In panel B, LDH activity leaked into culture medium was represented as a percentage of total activity in the medium of cells lysed by 0.2% Triton X-100. Values represent means ± SE of three experiments, each in triplicate. **P<0.01 vs PBS-treated cells within either of three groups (Vehicle-, BIS I- and DPI-treated groups); ^##P<0.01 vs cCSE- or pCSE-treated cells within the vehicle-treated group.</p

The EC₅₀ and maximal values for inhibition of MTS reduction activity of the cCSEs prepared from varying numbers (2–40) of cigarettes (Hi-Lite brand).

a<p>The cCSEs at the original tar concentration of 10 mg/ml were subjected to MTS reduction assay in C6 glioma cells.</p><p>The concentration-response curves for inhibition of MTS reduction activity were constructed, and the EC₅₀ values and maximal inhibition were determined. The concentrations of cCSEs were represented by the virtual tar concentrations. MTS reduction activity in the absence of the cCSEs was represented as 100%. Values represent means ± SE of three experiments, each in triplicate.</p><p>**P<0.01 vs the value for 2 cigarettes.</p><p>The EC₅₀ and maximal values for inhibition of MTS reduction activity of the cCSEs prepared from varying numbers (2–40) of cigarettes (Hi-Lite brand).</p

Cytotoxic activities of gas phase extracts of cigarette smoke and smoking methods.

<p>The gas phase extracts of cigarette smoke were prepared from Hi-Lite brand cigarettes by either continuous smoking protocol (cCSE) as described in the legend for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#pone-0107856-g001" target="_blank">Fig. 1</a> or puff smoking (pCSE) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107856#s2" target="_blank">Materials and Methods</a>. The original gas phase extracts at the virtual tar concentration of 10 mg/ml PBS were prepared, and they were subjected to MTS reduction assay (A), LDH leakage assay (B), DNA fragmentation assay (C) and PI uptake assay (D, E) in cultured C6 glioma cells for evaluation of their cytotoxic activities. In MTS reduction assay, substrate reduction activity was represented as a percentage of the value in the absence of the gas phase extract; in LDH leakage assay, LDH activity leaked into culture medium was represented as a percentage of total activity in the medium of cells lysed by 0.2% Triton X-100; in PI uptake assay, the number of the cells positive for PI uptake was represented as a percentage of total number of cells identified by Hoechst 33342 for nuclear staining. Values in panels A, B and E represent means ± SE of three experiments, each in triplicate. The results in panels C and D are representative of three separate experiments.</p

Comparison of concentrations of carbonyl compounds in cCSE and pCSE.

a<p>The cCSE and pCSE at the original tar concentration of 10 mg/ml were prepared from Hi-Lite brand cigarettes by either continuous (cCSE) or puff smoking protocol (pCSE).</p><p>cCSE and pCSE were fractionated by HPLC and each fraction was analyzed for cytotoxic activities using PI uptake assay. The positive fractions were analyzed by GC/MS after derivatization with a carbonyl reagent PFBOA. Values represent means ± SD of three experiments.</p><p>Comparison of concentrations of carbonyl compounds in cCSE and pCSE.</p