Pathogenic monoclonal antibody against desmoglein 3 augments desmoglein 3 and p38 MAPK phosphorylation in human squamous carcinoma cell line

Pemphigus vulgaris is an autoimmune blistering disease characterized by cell–cell detachment of epidermal cells. Autoantibody against desmoglein (Dsg) 3, a transmembrane glycoprotein that mediates the association of desmosomes, plays a major role in blistering in pemphigus vulgaris (PV). The mechanisms of autoantibody-induced acantholysis have not been clarified. We previously reported that PV-IgG induces phosphorylation of Dsg3, decreases Dsg3 on the cell surface and forms Dsg3-depleted desmosomes in cultured keratinocytes, and that cell treatment with a potent pathogenic monoclonal antibody against Dsg3 (AK23 mAb) decreases the amount of Dsg3 in cultured keratinocytes. Although the precise mechanisms remain unclear, we have proposed the involvement of intracellular signal transduction resulting from the binding of autoantibodies to Dsg3. In this study, we examined whether AK23 mAb augments phosphorylation of Dsg3 and p38 mitogen-activating protein kinase (MAPK) in a human squamous cell line, DJM-1 cells. AK23 mAb increased serine phosphorylation of Dsg3 and augmented activation levels of p38 MAPK. These results indicate that antibodies bind to Dsg3, but not other antigens, in the IgG fraction and can induce activation of signal transduction.     

Novel nuclear shuttle peptide to increase transfection efficiency in esophageal mucosal cells

The major barrier to successful transfection appears to be passage of the DNA plasmid from the cytoplasm into the cell nucleus. The M9 nuclear localization peptide, a fragment of the naturally occurring heterogeneous nuclear ribonucleoprotein A1, which serves to shuttle messenger RNA across the nuclear membrane, has been proposed as a tool for enhancing transfection efficiency. We tested three different reporter plasmids to assess the ability of M9 to improve transfection efficiency in esophageal mucosal cells. The effect of M9 on the intracellular movement of plasmid was also assessed using fluorescent microscopy to trace rhodamine-labeled plasmid. The M9 nuclear shuttle peptide consistently increased the transfection efficiency. When transfection was carried out with specific plasmids, β-galactosidase enzyme activity, keratinocyte growth factor-1 growth factor levels, and the number of transfected cells expressing growth factor peptides were progressively increased with increasing M9 to plasmid ratios. Fluorescent microscopy demonstrated that the M9 shuttle allowed rhodamine-tagged plasmid to gain access to the nucleus, while it was located exclusively in the cytoplasm without the peptide. The M9 shuttle peptide increases transfection efficiency in esophageal mucosal cells, and therefore may have a useful role in gene therapy applications involving the esophagus. Presented at the Forty-Second Annual Meeting of The Society for Surgery of the Alimentary Tract, Atlanta, Ga., May 20–23, 2001 (poster presentation).     

异甘草酸镁对人表皮角质形成细胞炎症因子的产生和外周血白细胞趋化功能的影响

目的：研究异甘草酸镁对角质形成细胞产生促炎症细胞因子、黏附分子和对白细胞趋化功能的影响,以获得异甘草酸镁治疗炎症性皮肤病的药理学基础.方法：采用2 000 U/mL rhIFN-γ诱导人表皮角质形成细胞Ha-CaT细胞,用ELISA法检测不同浓度的异甘草酸镁对HaCaT细胞IL-8、IL-6和ICAM-1产生的影响;通过琼脂糖平板法在显微镜下观察药物对10 nmol/L甲酰甲二磺酰-亮氨酰-苯丙氨酸（fMLP）诱引的人白细胞移动距离的影响.结果：异甘草酸镁能剂量依赖性地抑制rhIFN-γ刺激引起的IL-8、IL-6和ICAM-1的产生,其IC50值分别为1.21、7.23和4.17μg/mL;对fMLP诱引的人外周血白细胞趋化功能有明显影响,其抑制作用呈剂量依赖性,IC50值为8.58 μg/mL.结论：异甘草酸镁可通过抑制人表皮角质形成细胞IL-8、IL-6、ICAM-1产生和白细胞趋化达到治疗炎症相关性皮肤病的作用.     

Caffeoylserotonin Protects Human Keratinocyte HaCaT Cells against H2O2‐Induced Oxidative Stress and Apoptosis through Upregulation of HO‐1 Expression via Activation of the PI3K/Akt/Nrf2 Pathway

Caffeoylserotonin (CaS) has strong radical scavenging activity as well as antioxidant activities, protecting cells from lipid peroxidation, intracellular reactive oxygen species generation, DNA damage, and cell death. The molecular mechanism by which CaS protects against oxidative stress is not well understood. Here, we analyzed the cytoprotective activity of CaS in hydrogen peroxide (H₂O₂)‐treated keratinocyte HaCaT cells. H₂O₂ induced apoptosis in the cells through activation of pro‐apoptotic p21, Bax, and caspase‐3. Pretreatment with CaS inhibited apoptotic gene expression and activated the anti‐apoptotic gene, Bcl‐xL. Although CaS did not directly affect heme oxygenase‐1 (HO‐1) expression, pretreatment with CaS augmented HO‐1 expression through an increase in NF‐E2‐related factor (Nrf2) stability and stimulation of Nrf2 translocation to the nucleus upon H₂O₂ exposure. H₂O₂ also induced the phosphorylation and subsequent activation of ERK, p38 MAPK, and Akt. Analysis using specific inhibitors of p38 MAPK and Akt demonstrated that only Akt activation was involved in HO‐1 and Nrf2 expressions. In addition, PI3K and PKC inhibitors suppressed HO‐1/Nrf2 expression and Akt phosphorylation. These results demonstrate that CaS protects against oxidative stress‐induced keratinocyte cell death in part through the activation of Nrf2‐mediated HO‐1 induction via the PI3K/Akt and/or PKC pathways, but not MAPK signaling. Copyright © 2013 John Wiley & Sons, Ltd.     

Some Effects of Hypothermia and Hypoxia on the Sensitivity of HeLa Cells to X-rays

Summary

In vivo studies on the alterations in radio-sensitivity produced in hypothermic mammals do not enable a distinction to be drawn between the effects of hypothermia per se and the concomitant hypoxia.

The present experiments on the radio-sensitivity of mammalian (HeLa) cells in vitro have permitted observations to be made on the separate effects of hypoxia and hypothermia.

It is concluded that, whereas hypoxia produces a decrease in radio-sensitivity, hypothermia produces no detectable change.     

Assessment of the hamster cheek pouch as a model for radiation-induced oral mucositis,and evaluation of the protective effects of keratinocyte growth factor using this model

Abstract

Purpose: Oral mucositis induced by radiotherapy impacts quality of life. Previous studies have reported on the use of the hamster as a model for radiation-induced oral mucositis; however, details regarding factors such as radiation dose response, effects on myeloperoxidase (MPO) activity and related histopathological changes remain unclear. In the present study using the hamster, we evaluated the dose dependency of radiation-induced oral mucositis and the effects of keratinocyte growth factor (palifermin).

Methods: Oral mucositis was induced in the cheek pouch by X-irradiation using single doses in the range 20–50 Gy. To evaluate the protective effect of palifermin, administration was carried out (5 mg/kg) on days 1, 2 and 3 or on days 9, 10 and 11 after single irradiation at a dose of 40 Gy.

Results: The oral mucositis score, MPO activity and histopathological findings of inflammation increased in a dose dependent manner. Palifermin treatment stimulated the proliferation of mucosal epithelial cells. Additionally, palifermin when administered on days 1, 2 and 3 after irradiation (40 Gy) reduced the severity of oral mucositis.

Conclusions: The hamster was found to be a suitable model for radiation-induced oral mucositis, with excellent results regarding the evaluation of radiation dose response and drug reactivity.     

Lymphocytes in patients with psoriasis promote proliferation of keratinocytes

Objective: To analyze the effect of lymphocytes on proliferation of keratinocytes in patients with psoriasis. Methods: Lymphocytes in lesion and peripheral blood were isolated and amplified, then cultured together with normal keratinocytes. By MTT method, the living cells were quantified in the mixed culture. Results: Compared with normal controls, lymphocytes from lesion and peripheral blood of psoriasis both promote the proliferation of keratinocytes (P<0. 01 and P<0. 05 respectively). The concentrations of IL-2 and IFN-7 in the mixture of lesion lymphocytes and keratinocytes were significantly higher than that of controls. Tripterygium glycosides inhibited this promotion. Conclusion: Lymphocytes in patients with psoriasis (mainly Th1 cell) play an important role in proliferation of keratinocytes. This psoriasis cell model is useful for studies on signal transduction in psoriasis.     

The squamous cell carcinoma antigens as relevant biomarkers of atopic dermatitis

BACKGROUND: Although it is thought that both Th1- and Th2-type inflammations are involved in the pathogenesis of atopic dermatitis (AD), it is controversial which immune response is more involved in regulating the clinical severity of AD. We recently found that the squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 are novel biomarkers of bronchial asthma, downstream of IL-4 and IL-13. OBJECTIVE: We examined whether SCCA1 and SCCA2 could also serve as biomarkers of AD, reflecting its Th2-type immune responses, and whether the expression level of SCCA was correlated with clinical severity of AD. METHOD: We compared the expression of SCCA1 and SCCA2 at the mRNA and protein levels in both involved and uninvolved skin of AD patients and in normal control skin. We next analysed induction of SCCA by IL-4 or IL-13 in keratinocytes. Finally, we compared the serum level of SCCA with laboratory parameters reflecting Th2-type inflammation and clinical severity in AD patients. RESULTS: SCCA1 and SCCA2 were highly expressed in involved skin of AD patients, compared with their uninvolved skin, at both mRNA and protein levels. SCCA protein was dominantly expressed in suprabasal keratinocytes in the epidermis of AD patients. Either IL-4 or IL-13, but not IFN-gamma or TNF, induced production of SCCA in keratinocytes. These result suggest that SCCA is induced in AD skin, probably due to direct actions of IL-4 and/or IL-13 on keratinocytes. Serum levels of SCCA were well correlated with eosinophil numbers and serum lactate dehydrogenase levels, and weakly with serum IgE levels, in AD patients. Furthermore, serum levels of SCCA were strongly correlated with clinical severity. CONCLUSIONS: Th2-type inflammation dominantly regulates the clinical severity of AD, and SCCA is a relevant biomarker of AD, reflecting both Th2-type inflammation and clinical severity.     

不同培养条件及时相对黑素细胞生物学特性的影响

目的观察不同培养条件及时相对体外培养的正常人黑素细胞生物学特性的影响。方法分离正常成人包皮黑素细胞,分别在添加有碱性成纤维细胞生长因子(bFGF)和霍乱毒素(CT)的RPMI1640培养基及角质形成细胞条件培养基中培养。培养的细胞用L-Dopa染色及S-100蛋白染色鉴定。观察其形态、增殖特性。结果培养细胞经生物学鉴定为黑素细胞。原代培养及条件培养基传代培养中黑素细胞有多个突起,而bFGF/CT培养基中传代培养时黑素细胞多呈梭形。两种培养条件下黑素细胞增殖特性无明显区别。结论两种方法均可稳定获得黑素细胞的体外选择性纯培养,角质形成细胞可通过细胞间接触及分泌细胞外因子影响黑素细胞的树突形成。     

Trefoil factor family 3 expression in the oral cavity

This study examined the expression, in oral keratinocytes and in the major and minor salivary glands, of Trefoil factor family 3 (TFF3) peptide. Trefoil factor family 3 messenger RNA (mRNA) and peptide were detected in cultures of normal oral keratinocytes by quantitative real‐time polymerase chain reaction (PCR) and western blotting, respectively. Trefoil factor family 3 was found, by immunohistochemical analyses, to be expressed in the basal layers of the oral mucosal epithelium. In salivary glands, immunohistochemical staining showed that TFF3 peptide expression was strongest in the mucous acini of the submandibular and the small salivary glands. Serous cells in the same glands showed weak staining. In the parotid gland, many serous acini showed weak positive staining, while other areas did not. In all glands examined, the intercalated, striated, and collecting ducts were moderately TFF3‐positive. Double immunostaining confirmed that mucous (MUC5B positive) cells were moderately or strongly positive for TFF3 and that some serous (MUC7 positive) cells showed restricted TFF3 expression, mostly in a granular pattern. The prevalence of the TFF3 peptide in the salivary secretions of healthy volunteers was detected by western blotting of saliva from minor salivary glands (four of five) and the parotid gland (one of five) and of mixed submandibular/sublingual saliva (five of five). In conclusion, the submandibular and small salivary glands appear to be the major producers of oral TFF3, but duct cells of all glands and keratinocytes of the oral mucosa may also contribute as sources of TFF3 in the oral cavity.