Gene therapy: pursuing restoration of dermal adhesion in recessive dystrophic epidermolysis bullosa

The replacement of a defective gene with a fully functional copy is the goal of the most basic gene therapy. Recessive dystrophic epidermolysis bullosa (RDEB) is characterised by a lack of adhesion of the epidermis to the dermis. It is an ideal target for gene therapy as all variants of hereditary RDEB are caused by mutations in a single gene, COL7A1, coding for type VII collagen, a key component of anchoring fibrils that secure attachment of the epidermis to the dermis. RDEB is one of the most severe variants in the epidermolysis bullosa (EB) group of heritable skin diseases. Epidermolysis bullosa is defined by chronic fragility and blistering of the skin and mucous membranes due to mutations in the genes responsible for production of the basement membrane proteins. This condition has a high personal, medical and socio‐economic impact. People with RDEB require a broad spectrum of medications and specialised care. Due to this being a systemic condition, most research focus is in the area of gene therapy. Recently, preclinical works have begun to show promise. They focus on the virally mediated ex vivo correction of autologous epithelium. These corrected cells are then to be expanded and grafted onto the patient following the lead of the first successful gene therapy in dermatology being a grafting of corrected tissue for junctional EB treatment. Current progress, outstanding challenges and future directions in translating these approaches in clinics are reviewed in this article.     

Human tissue kallikrein expression in the stratum corneum and serum of atopic dermatitis patients

Human tissue kallikreins are a family of 15 trypsin- or chymotrypsin-like secreted serine proteases (KLK1-KLK15). Many KLKs have been identified in normal stratum corneum (SC) and sweat, and are candidate desquamation-related proteases. We report quantification by enzyme-linked immunosorbent assay (ELISA) of KLK5, KLK6, KLK7, KLK8, KLK10, KLK11, KLK13 and KLK14 in the SC and serum of atopic dermatitis (AD) patients by ELISA, and examine their variation with clinical phenotype, correlation with blood levels of eosinophils, lactate dehydrogenase (LDH) and immunoglobulin E. The overall SC serine protease activities were also measured. In the SC of AD, all KLKs, except KLK11, were significantly elevated. The elevation of chymotrypsin-like KLK7 was predominant, compared with trypsin-like KLKs. The SC overall plasmin- and furin-like activities were significantly elevated, while trypsin- and chymotrypsin-like activities did not differ significantly. In the serum of AD patients, KLK8 was significantly elevated and KLK5 and KLK11 were significantly decreased. However, their serum levels were not modified by corticosteroid topical agents. The alterations of KLK levels in the SC of AD were more pronounced than those in the serum. KLK7 in the serum was significantly correlated with eosinophil counts in the blood of AD patients, while KLK5, KLK8 and KLK11 were significantly correlated with LDH in the serum. In conclusion, we report abnormal kallikrein levels in the SC and the serum of AD patients. KLKs might be involved in skin manifestation and/or focal/systemic inflammatory reactions in AD. Our data may contribute to a better understanding of the pathogenesis of AD.     

A majority of proliferating T cells in cutaneous malignant T cell lymphomas may lack the high affinity IL-2 receptor (CD25)

IL-2 is a major growth factor for all T-cell subsets acting via a specific membrane receptor. To investigate its role in the pathogenesis of cutaneous T-cell lymphomas (CTCLs), we analysed the expression of high-affinity IL-2 receptors (IL-2Rs) on proliferating cells in these disorders. We showed by immunohistochemical double staining that most cycling cells do not express high-affinity IL-2Rs. Four T-cell lines were established from patients with CTCLs. Two lines required both IL-2 and IL-4 for growth, one line required IL-2 and one line IL-4 alone. The last of these lacked expression of the IL-2R -chain. Thus, IL-2 may not be the only, or the most important, growth stimulus in CTCLs and reactive skin infiltrates. T helper cells, which dominated the infiltrate, might represent TH2 cells.     

Transgene expression in human epidermal keratinocytes: cell cycle arrest of productively transfected cells

We have analysed the consequences of liposome mediated gene transfer into human primary epidermal keratinocytes and compared non-Epstein-Barr Virus (EBV) and EBV based expression vectors that carry the genes encoding human Growth Hormone (hGH) or Enhanced Green Fluorescent Protein (EGFP). Different kinetics between the non-EBV and EBV based vectors were revealed upon subcultivation of hGH transfected keratinocytes. The keratinocytes transfected with non-EBV based vector showed a rapid reduction in hGH production. Although the EBV based vector resulted in more stable expression, this was also reduced over time. Chromatin inactivation by deacetylation was investigated by treatment with sodium butyrate and found not to be the reason for the decreasing expression. Keratinocytes divided into subpopulations enriched for either stem cells or transit amplifying cells, based on beta1-integrin expression and function, do not differ significantly with respect to susceptibility to productive transfection. However, when the keratinocytes were transfected with the EGFP gene and sorted live by FACS into EGFP negative and positive populations, only the negative cells were capable of forming significant numbers of colonies. This is consistent with the observation that the ability to incorporate BrdU was dramatically reduced in the EGFP expressing population within 24-48 h post transfection indicating an almost complete cell cycle arrest. p53 levels were unaffected by the procedures, and the keratinocyte cell line HaCat, mutated in both p53 alleles, also shows a marked reduction in clonogenic potency upon transfection. There was a slight increase of TUNEL positive apoptotic nuclei in the positive population at early time points. However, the apoptotic index was still very low. When we measured the frequency of involucrin expressing cells, we found an increase in the productively transfected population over time indicating an initiation of terminal differentiation. In contrast to the transfected cultures, keratinocytes that were transduced using a retroviral vector showed no decrease in colony forming efficiency. In conclusion we find that transgene expressing cells from transfected cultures of epidermal keratinocytes undergo cell cycle arrest and initiate terminal differentiation by mechanisms which are independent of p53 levels.     

Isolating a pure population of epidermal stem cells for use in tissue engineering

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.     

银屑病患者骨髓CD34+细胞Notch受体及其靶基因表达的研究

目的 探讨银屑病患者骨髓CD34+细胞表面两种Notch受体的表达及其下游靶基因Hes-1的表达。方法 分离12例银屑病患者及10例正常对照骨髓CD34+细胞,以β-肌动蛋白为内参照,采用Western印迹法半定量检测Notch受体1、2及其下游靶基因Hes-1的蛋白表达水平。结果 银屑病患者CD34+造血细胞Notch1表达水平（0.9488 ± 0.3221）明显高于正常人对照（0.6693 ± 0.1465）;Hes-1蛋白在银屑病患者表达（0.8579 ± 0.3729）亦高于正常人对照（0.5728 ± 0.1787）,两者差异均有统计学意义（P < 0.05）,而Notch2蛋白的表达在患者组（0.7568 ± 0.3461）和正常人对照组（0.8312 ± 0.2887）之间差异无统计学意义（P > 0.05）。结论 Notch1受体及其下游基因Hes-1的高表达可能参与银屑病患者骨髓造血细胞低增殖活性的发生。     

Interleukin-17- and protease-activated receptor 2-mediated production of CXCL1 and CXCL8 modulated by cyclosporine A, vitamin D3 and glucocorticoids in human keratinocytes

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor which mediates a variety of functions in the skin including cutaneous inflammation. SLIGKV-NH(2) , an agonist peptide for PAR2, enhanced the interleukin (IL)-17-induced production of two CXC chemokines, CXCL1 (GRO-α) and CXCL8 (IL-8), in normal human epidermal keratinocytes (NHEK) in a concentration-dependent manner. The enhanced production of those chemokines was suppressed by a PAR2-specific siRNA. The SLIGKV-NH(2) -induced production of both CXCL1 and CXCL8 was markedly reduced by cyclosporine A. The enhanced production of CXCL1 was suppressed by 1α, 24R-dihydroxyvitamin D(3) , an active form of vitamin D(3) , and weakly by glucocorticoids, dexamethasone and clobetasol propionate, whereas production of CXCL8 was not altered by any of those receptor agonists. In psoriatic skin, the thickened upper spinous layer of the epidermis was positive for PAR2 protein and the expression of the IL17A mRNA was increased. These results suggest that the IL-17-induced pro-inflammatory reaction is enhanced by the activation of PAR2 in keratinocytes, and that the effect of PAR2 is differentially modulated by cyclosporine A, the active form of vitamin D(3) and glucocorticoids.     

复方泽漆冲剂对血热型寻常型银屑病患者血清中MMP-2、MMP-9和IL-18的影响

目的观察复方泽漆冲剂对血热型寻常型银屑病(PV)患者血清中MMP-2、MMP-9和IL-18的变化,探讨复方泽漆冲剂对血热型PV患者血清MMP-2、MMP-9和L-18浓度的影响;方法经纳入的血热型患者分治疗组(28例)采用复方泽漆冲剂,对照组(28例)采用自拟方,4周后采用ELISA法对56例PV患者治疗前后血清中MMP-2、MMP-9和IL-18水平进行检测;结果治疗组及对照组患者血清MMP-2、MMP-9和IL-18治疗后浓度均显著低于治疗前水平P0.05;结论复方泽漆冲剂能降低血热型PV患者血清中MMP-2、MMP-9和IL-18的水平,观察MMP-2、MMP-9和IL-18浓度变化对PV的治疗和指导用药有重要的临床价值。     

银屑病患者血清抗EB病毒抗体的检测

目的：探讨EB病毒感染与银屑病的关系。方法：采用酶免疫法检测53例银屑病患者血清EB病毒抗壳抗原／IgG、IgM抗体，抗早期抗原－D／IgG抗体。抗核心抗原－1／IgG抗体。结果：银屑病患者血清抗ER病毒早期抗原－D／IgG抗体阳性率明显高于正常对照组（P＜0．001）。结论：银屑病患者体内EB病毒可能处于激活状态。     

Detection of herpesvirus DNA in peripheral blood mononuclear cells and skin lesions of patients with pemphigus by polymerase chain reaction

Pemphigus is an autoimmune disease where both endogenous (genetic) and exogenous (environmental) factors play a part. Viral infections, in particular herpesvirus infections, have been identified as a possible triggering factor for pemphigus. In this study, using the polymerase chain reaction, we studied peripheral blood mononuclear cells (PBMC) and skin biopsies from patients with pemphigus, and in some of these were able to demonstrate the presence of DNA sequences of herpes simplex virus 1/2 (50% in PBMC and 71% in skin biopsies), Epstein-Barr virus (15% in PBMC and 5% in skin biopsies) and human herpesvirus 6 (20% in PBMC only). However, the inability to detect herpesvirus DNA consistently in these cases suggests that viral infection may only be an occasional factor triggering the outbreak or exacerbation of the disease. The possible role of interferons and interleukins in the pathogenesis of virus-induced pemphigus is discussed.     

Mutation analysis of the ATP2A2 gene in Taiwanese patients with Darier's disease

BACKGROUND: Darier's disease (DD) is an autosomal dominant skin disorder characterized by abnormal keratinization and acantholysis. Pathogenic mutations in the ATP2A2 gene encoding SERCA2, a calcium pump of the sarco/endoplasmic reticulum, have recently been identified. OBJECTIVES: To identify mutations of the ATP2A2 gene in Taiwanese patients with DD. METHODS: Mutation analysis of genomic DNA was performed on five families with DD and two sporadic cases. All 21 exons and the flanking intron boundaries were amplified and followed by direct sequencing. Restriction fragment analysis or direct sequencing in each family and in normal controls further verified the mutations. RESULTS: Mutations in the functional domains of the ATP2A2 gene were identified and verified in all seven pedigrees. They consisted of four mis-sense mutations (R131Q, P680L, G703S, G807R), one altered splice-site mutation (2980 + 5insA) and one frameshift deletion mutation (1457-1458delAG). Of these, R131Q, which was reported twice previously, was detected in two unrelated families. The remaining five were novel mutations. CONCLUSIONS: Six pathogenic mutations in the ATP2A2 gene were identified in seven Taiwanese DD pedigrees. The results confirmed that most mutations in the ATP2A2 gene are private and of the mis-sense type.     

Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus

Please cite this paper as: Efficient and selective tumor cell lysis and induction of apoptosis in melanoma cells by a conditional replication‐competent CD95L adenovirus. Experimental Dermatology 2010; 19 : e56–e66. Abstract: The high mortality of melanoma demands the development of new strategies, and gene therapy may be considered provided improvements in efficacy and selectivity. Overexpression of the death ligand CD95L/FasL has been shown in previous studies as highly effective for apoptosis induction in melanoma cells. For efficient and selective targeting of melanoma, a conditional replication‐competent adenoviral vector was constructed (Ad5‐FFE‐02), which drives CD95L expression by a tetracycline‐inducible promoter. For restricting its replication to melanoma cells, the adenoviral E1A gene is controlled by a tyrosinase‐derived promoter. Furthermore, adenoviral E1B was deleted and a mutated E1A was used to preferentially support replication in tumor cells. Proving its high selectivity and efficiency, strong expression of E1A and doxycycline‐dependent induction of CD95L were characteristic for tyrosinase‐positive melanoma cells after Ad5‐FFE‐02 transduction, whereas absent in non‐melanoma cell lines. Importantly, Ad5‐FFE‐02‐mediated cell lysis was restricted to melanoma cells, and induction of apoptosis was found only in tyrosinase and CD95 expressing cells. Finally, the combination of adenoviral replication and CD95L‐mediated apoptosis resulted in an enhanced repression of melanoma cell growth. This new adenoviral vector may provide a basis for an efficient targeting of melanoma.     

早幼粒细胞白血病基因在寻常性银屑病患者外周血淋巴细胞中表达的研究

目的 检测寻常性银屑病患者外周血淋巴细胞中早幼粒细胞白血病（PML）基因的表达,以探索PML基因与银屑病之间的相互关系。方法 收集年龄18 ~ 65岁、未经系统治疗的寻常性银屑病患者50例,健康人对照45例。分离外周血淋巴细胞,使用RNAprep pure 血液总RNA提取试剂盒提取淋巴细胞RNA,用实时荧光定量逆转录聚合酶链反应检测外周血淋巴细胞PML基因mRNA的表达;流式细胞仪检测30例银屑病患者和25例健康人对照外周血淋巴细胞PML蛋白荧光强度值。分别用独立样本t检验对检测结果进行统计分析。结果 50例银屑病患者和45例健康人对照外周血淋巴细胞PML mRNA相对定量（RQ）值分别为14.98 ± 3.64和5.50 ± 1.10,银屑病患者组显著高于健康人对照组（两组比较,t = 16.79,P ＜ 0.05）。30例银屑病患者和25例健康人对照外周血淋巴细胞PML蛋白荧光强度值分别为3.13 ± 0.27和2.43 ± 0.21,银屑病患者组亦显著高于健康人对照组（两组比较,t = 6.93,P ＜ 0.05）。结论 PML基因mRNA及蛋白在银屑病患者外周血淋巴细胞中高表达,提示PML基因可能在银屑病的发病中起一定作用。     

Imaging of pH and pO2 gives insight in molecular processes of irradiated cells

One of the major challenges in radiation therapy is the interference with tissue repair processes due to hypoxic characteristics and pH dysregulation. In this study, we present dual imaging of pH and oxygenation in vitro based on luminescent biocompatible sensor foils that allow studying the effects of irradiation on different cell types in culture. Different sensitivities of fibroblast and oral squamous carcinoma cells were observed by complementing oxygen and pH differences with proliferation assays. This study highlights especially the distinct role of oxygen after irradiation and the difference in proliferation processes of irradiated normal dermal cells in contrast to irradiated tumor cells.     

水痘—带状疱疹病毒与皮损中各类细胞关系的电镜观察

为了探讨（水痘－带状疱疹病毒）（ＶＺＶ）与皮损中各类细胞之间的宿主关系和亲和性，用透射电镜观察了水痘、带状疱疹患者皮损，结果发现ＶＺＶ在皮肤的宿主细胞范围广泛，包括表皮细胞、成纤维细胞、血管内皮与周皮细胞、许旺细胞以及巨噬细胞和肥大细胞，其中对表皮细胞、成纤维细胞的亲和性最强，与中性粒细胞和淋巴细胞无宿主关系。中性粒细胞在皮肤局部显示出较强的吞噬、消化ＶＺＶ现象，而巨噬细胞在防御和播散（ＶＺＶ）中均起作用     

白塞病皮损中淋巴细胞凋亡和 Fas、 bcl-2的表达

为探讨白塞病Fas、bcl-2和细胞凋亡在皮损中的表达及其在发病机理中的作用，对19例白塞病（活动期12例，非活动期7例），用TUNEL原位检测皮损淋巴细胞凋亡；用免疫组化法观察Fas、bcl-2阳性表达。结果表明活动期白塞病 巴细胞凋亡及Fas有弱的表达，bcl-2有较强表达，。非活动期Fas及凋亡表达增强；bcl-2仅有弱的表达，两组Fas、bcl-2及凋亡指数（AI）的表达有显著差异（P＝0．001，P＝0．002，P＝0．009）。两组淋巴细胞的AI与Fas表达呈正相关（r=0.772,P=0.000);与bcl-2表达呈负相关（r=-0.899,P=0.000).Fas介导的淋巴细胞凋亡的缺陷可能是白塞病发病的的重要原因，bcl-2有抑制凋亡的作用。     

银屑病患者骨髓高增殖潜能集落形成细胞P16基因mRNA表达的研究

目的：研究银屑病患者骨髓高增殖潜能集落形成细胞（HPP-CFC）的集落形成能力及P16基因mRNA表达,探讨银屑病患者骨髓造血干细胞体外增殖活性及原因。方法：密度梯度离心法分离银屑病患者及正常对照骨髓单个核细胞,培养于含人干细胞因子（SCF）、人粒-巨噬细胞系集落剌激因子（GM-CSF）、白介素（IL）-3、IL-6细胞因子组合的甲基纤维素半固体培养基,培养14d时计数HPP-CFC集落,然后收集集落。提取纯化集落细胞的总RNA,应用反转录（RT）-PCR方法检测HPP-CFC集落细胞的P16基因mRNA的表达。结果：①在甲基纤维素半固体培养基中,银屑病患者骨髓HPP-CFC集落数显著低于正常对照组,且集落形态较小;②银屑病患者骨髓HPP-CFC集落细胞的P16基因mRNA表达高于正常对照组,差异有统计学意义。结论：银屑病患者骨髓HPP-CFC集落形成能力降低;银屑病患者骨髓HPP-CFC的P16基因mRNA表达阳性率增高,推测P16基因可能参与了银屑病患者骨髓造血干细胞体外增殖活性异常的发生。     

miR-23a-3p､miR-155､miR-124a表达变化与艾滋病患者高效抗逆转录病毒治疗效果的相关性分析

目的探究miR-23 a-3 p?miR-155?miR-124a表达变化与艾滋病患者高效抗逆转录病毒治疗(HAART)效果的相关性。方法选取2019年1月至2020年1月武汉市金银潭医院收治的152例艾滋病患者,按照随机数字表法分为胸腺五肽组和HAART组,各76例。胸腺五肽组给予胸腺五肽治疗,HAART组给予HAART。对比两组患者治疗有效率及不良反应发生情况,分析miR-23 a-3 p?miR-155?miR-124a表达量与治疗效果的相关性。结果治疗后两组患者CD4⁺计数?CD8⁺计数上升,人类免疫缺陷病毒(HIV)载量下降,且与胸腺五肽组相比,HAART组CD4⁺计数?CD8⁺计数较高,HIV病毒载量较低,差异具有统计学意义(P<0.05);治疗后两组患者miR-23 a-3 p?miR-155?miR-124a表达下降,且与胸腺五肽组相比,HAART组患者miR-23 a-3 p?miR-155?miR-124a表达较低,差异具有统计学意义(P<0.05);CD4⁺T淋巴细胞计数与miR-23 a-3 p?miR-155?miR-124a表达均呈负相关(r=-0.336?-0.525?-0.298,P=0.003?0.001?0.010)。结论艾滋病患者采用HAART,有较好的临床效果,且miR-23 a-3 p?miR-155?miR-124a表达会随着治疗效果的不同而发生变化。     

Long‐term effects of omalizumab on peripheral blood cells and C‐reactive protein levels in patients with chronic spontaneous urticaria

Omalizumab's mechanism of action is not well‐understood yet despite its strong therapeutic efficacy in chronic spontaneous urticaria (CSU). To determine the overall effect of omalizumab on peripheral blood cell counts and serum C‐reactive protein levels (sCRP) during a 1‐year follow‐up in patients with CSU. Data of 74 patients (male/female: 20/54) were reviewed from medical charts. Leucocyte counts, percentages of peripheral blood cells(lymphocyte, monocyte, neutrophil [PPBN], eosinophil, basophil [PPBB]) and sCRP were recorded at baseline, 3rd, 6th, 12th months of omalizumab treatment. Although a dramatic increase in the mean PPBB (±SD) was observed at the 3rd month, PPBB (%) gradually decreased after the 3rd month (PPBB: 0.38 ± 0.21 [baseline] vs. 0.59 ± 0.3 [3rd month], p = .002). However, 12th month PPBB remained higher than baseline (PPBB:0.38 ± 0.21 [baseline] vs. 0.46 ± 0.27 [12th month], p = .03). A dramatic decrease in the mean PPBN (%) was noticed within the first 3 months (PPBN:62.85 ± 8.97 [baseline] vs. 58.37 ± 9.07 [3rd month], p = .04), and 12th month PPBN remained lower than baseline values (PPBN: 62.85 ± 8.97 [baseline] vs. 60.31 ± 8.02 [12th month], p = .045).Mean sCRP (mg/dL) decreased rapidly within the first 3 months (sCRP: 1.09 ± 1.53 [baseline] vs. 0.56 ± 0.45 [3rd month], p = .17) and 12th month sCRP still remained lower than baseline levels (sCRP: 1.09 ± 1.53 [baseline] vs. 0.83 ± 1.06 [12th month], p = .01). Omalizumab substantially increases PPBB,and reduces PPBN accompanied by a reduction in sCRP especially in the first 3 months; however, these effects may continue in the long‐term. The alterations in peripheral blood cell ratios and sCRP may contribute to the therapeutic effect of omalizumab in CSU.