Institutional experience with a rotational total skin electron irradiation (RTSEI) technique—A three decade review (1981–2012)

Total skin electron irradiation (TSEI) for patients with cutaneous lymphomas is technically challenging, and numerous approaches have been developed to overcome the many field matching problems associated with such a large and complex treatment volume. Since 1981 we have delivered TSEI using a rotational total skin electron irradiation (RTSEI) technique in conjunction with patch, treat and boost fields in order to provide complete skin and dose coverage. Initially we used a 6 MeV electron beam at an extended source-skin distance (SSD) on a modified linear accelerator. More recently we began using a high dose rate electron mode on a commercially available linear accelerator. The RTSEI technique allows the delivery of a seamless surface dose to the majority of the patient''s skin surface in a single treatment. In this review paper we present our three-decade experience with the technical development, dosimetry, treatment delivery and clinical outcomes of our RTSEI technique.     

Studying fatty aldehyde metabolism in living cells with pyrene-labeled compounds

The lack of fatty aldehyde dehydrogenase function in Sjögren Larsson Syndrome (SLS) patient cells not only impairs the conversion of fatty aldehydes into their corresponding fatty acid but also has an effect on connected pathways. Alteration of the lipid profile in these cells is thought to be responsible for severe symptoms such as ichtyosis, mental retardation, and spasticity. Here we present a novel approach to examine fatty aldehyde metabolism in a time-dependent manner by measuring pyrene-labeled fatty aldehyde, fatty alcohol, fatty acid, and alkylglycerol in the culture medium of living cells using HPLC separation and fluorescence detection. Our results show that in fibroblasts from SLS patients, fatty aldehyde is not accumulating but is converted readily into fatty alcohol. In control cells, in contrast, exclusively the corresponding fatty acid is formed. SLS patient cells did not display a hypersensitivity toward hexadecanal or hexadecanol, but 3-fold lower concentrations of the fatty alcohol than the corresponding fatty aldehyde were needed to induce toxicity in SLS patient and in control cells.     

Isolation,characterization and molecular cloning of new antimicrobial peptides belonging to the brevinin-1 and temporin families from the skin of Hylarana latouchii (Anura: Ranidae)

Around 40 species of Hylarana amphibians are distributed in tropical and subtropical Asia, and Chinese broad-folded frog, Hylarana latouchii (Boulenger, 1899) is one of them. In this study, six different cDNAs encoding four novel antimicrobial peptide precursors were cloned by screening the cDNA library of the Chinese broad-folded frog skin. The protein sequence analysis demonstrated that two deduced peptides belong to the brevinin-1 family, and the other two belong to temporin family of amphibian antimicrobial peptides. Thus, they were named as brevinin-1LT1 (FMGSALRIAAKVLPAALCQIFKKC), brevinin-1LT2 (FFGSVLKVAAKVLPAALCQIFKKC), temporin-LT1 (FLPGLIAGIAKML–NH₂) and temporin-LT2 (FLPIALKALGSIFPKIL–NH₂), respectively. Furthermore, brevinin-1LT1 and temporin-LT1 were purified by HPLC from the skin secretion of H. latouchii. In this work, all the peptides kill microbes by membrane-disturbing mechanisms, and this procedure was visualized via scanning electron microscopy (SEM).     

Improvement of Opal Multiplex Immunofluorescence Workflow for Human Tissue Sections

The Opal multiplex technique is an established methodology for the detection of multiple biomarkers in one section. The protocol encompasses iterative single stainings and heating-mediated removal of the primary and secondary antibodies after each staining round, leaving untouched the Opal fluorophores which are deposited onto the antigen of interest. According to our experience, repetitive heating of skin sections often results in tissue damage, indicating an urgent need for milder alternatives to strip immunoglobulins. In this study, we demonstrate that considerable heating-related damage was found not only in skin but also in tissues of different origin, mostly characterized by low cell density. Importantly, the morphology remained fully intact when sections were repetitively exposed to β-mercaptoethanol-containing stripping buffer instead of multiple heating cycles. However, target epitopes appeared sensitive at a differential degree to multiple treatments with stripping buffer, as shown by loss in staining intensity, but in all cases, the staining intensity could be restored by increment of the primary antibody concentrations. Application of β-mercaptoethanol-containing stripping buffer instead of heating for antibody removal markedly improved the quality of the Opal multiplex technique, as a substantial higher number of differently colored cells could be visualized within a well-conserved morphological context:     

A型肉毒毒素联合眼袋整形术对眼周皮肤松弛患者的疗效观察

目的:探讨A型肉毒毒素联合眼袋整形术对眼周皮肤松弛患者的治疗效果和对患者满意度的影响,为临床提供参考。方法:选取2015年6月至2016年6月间因眼周皮肤松弛来我院治疗的患者60例。患者根据自身意愿选择治疗方式,按其选择的治疗方式分组为对照组(n=28)和治疗组(n=32)。对照组采用单纯眼袋整形术进行治疗,治疗组采用A型肉毒毒素联合眼袋整形术治疗。治疗后对两组患者均随访6个月。观察记录患者手术后鱼尾纹的改善效果、眼袋减轻的情况和眼周皮肤光泽的改善效果。观察患者并发症的发生情况。随访结束时采用自制的满意度调查问卷对患者满意度进行调查。结果:经过治疗,治疗组鱼尾纹改善率为100.00%,明显高于对照组的60.71%;治疗组眼袋改善率为96.88%,明显高于对照组的32.14%;治疗组皮肤光泽改善率为87.50%,明显高于对照组的53.57%,差异均具有统计学意义(均P0.05)。治疗后,两组患者切口红肿、少量分泌物、小血肿的发生率比较无明显差异(P0.05),治疗组术眼红肿与外眦部切口瘀痕发生率明显少于对照组(P0.05)。治疗组总满意率为93.75%(30/32),明显高于对照组的67.86%(19/28),差异具有统计学意义(P0.05)。结论:A型肉毒毒素与眼袋整形术联合应用在眼周皮肤松弛的治疗上有较好的疗效和安全性,能明显降低患者术眼红肿与外眦部切口瘀痕的发生,并提高患者满意率,值得在临床上推广应用。     

Prostaglandin-E2 is produced by adult human epidermal melanocytes in response to UVB in a melanogenesis-independent manner

Excessive ultraviolet radiation (UVR) exposure induces erythema, mediated in part by prostaglandin-E₂ (PGE₂). While keratinocytes are a major PGE₂ source, epidermal melanocytes (EM) also express PGE₂-production machinery. It is unclear whether EM-produced PGE₂ contributes to UVR-induced skin inflammation, and whether this is correlated with melanogenesis. Epidermal melanocytes were cultured from skin phototype-1 and -4 donors, followed by assessment of PGE₂ production and melanogenesis. Epidermal melanocytes expressed cytoplasmic phospholipase-A₂, cyclooxygenase-1, cytoplasmic prostaglandin-E synthase and microsomal prostaglandin-E synthase-1, -2. Epidermal melanocytes produced PGE₂ under basal conditions, which increased further after arachidonic acid stimulation. Epidermal melanocytes expressed cyclooxygenase-2 (COX-2) mRNA and a selective COX-2 inhibitor (NS-398) reduced PGE₂ production. Ultraviolet B-induced PGE₂ production was positively correlated with skin phototype-1, despite variability between individual EM donors. By contrast, there was no correlation between PGE₂ production by EM and their melanogenic status. Thus, EM may contribute to UVR-induced erythema, with role of donor skin phototype more important than their melanogenic status.     

Capillary blood flow imaging within human finger cuticle using optical microangiography

We report non‐invasive 3D imaging of capillary blood flow within human finger cuticle by the use of Doppler optical microangiography (DOMAG) and ultra‐high sensitive optical microangiography (UHS‐OMAG) techniques. Wide velocity range DOMAG method is applied to provide red blood cell (RBC) axial velocity mapping in capillary loops with ranges of ±0.9 mm/s and ±0.3 mm/s. Additionally, UHS‐OMAG technique is engineered to acquire high resolution image of capillary morphology. The presented results are promising to facilitate clinical trials of treatment and diagnosis of various diseases such as diabetes, Raynaud's phenomenon, and connective tissue diseases by quantifying cutaneous blood flow changes within human finger cuticle. (© 2013 WILEY‐VCH Verlag GmbH &Co. KGaA, Weinheim)     

Antimicrobial function of the GAPDH-related antimicrobial peptide in the skin of skipjack tuna,Katsuwonus pelamis

A 3.4 kDa of antimicrobial peptide was purified from an acidified skin extract of skipjack tuna, Katsuwonus pelamis, by preparative acid-urea–polyacrylamide gel electrophoresis and C₁₈ reversed-phase HPLC. A comparison of the N-terminal amino acid sequence of the purified peptide with that of other known polypeptides revealed high sequence homology with the YFGAP (Yellowfin tuna Glyceraldehyde-3-phosphate dehydrogenase-related Antimicrobial Peptide); thus, this peptide was identified as the skipjack tuna GAPDH-related antimicrobial peptide (SJGAP). SJGAP showed potent antimicrobial activity against Gram-positive bacteria, such as Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus, and Streptococcus iniae (minimal effective concentrations [MECs], 1.2–17.0 μg/mL), Gram-negative bacteria, such as Aeromonas hydrophila, Escherichia coli D31, and Vibrio parahaemolyticus (MECs, 3.1–12.0 μg/mL), and against Candida albicans (MEC, 16.0 μg/mL) without significant hemolytic activity. Antimicrobial activity of this peptide is heat-stable but salt-sensitive. According to the secondary structural prediction and the homology modeling, this peptide consists of three secondary structural motifs, including one α-helix and two parallel β-strands, and forms an amphipathic structure. This peptide showed neither membrane permeabilization ability nor killing ability, but did display a small degree of leakage ability. These results suggest that SJGAP acts through a bacteriostatic process rather than bactericidal one. SJGAP is another GAPDH-related antimicrobial peptide isolated from skipjack tuna and likely plays an important role for GAPDH in the innate immune defense of tuna fish.     

Novel Immunohistochemical Techniques Using Discrete Signal Amplification Systems for Human Cutaneous Peripheral Nerve Fiber Imaging

Confocal imaging uses immunohistochemical binding of specific antibodies to visualize tissues, but technical obstacles limit more widespread use of this technique in the imaging of peripheral nerve tissue. These obstacles include same-species antibody cross-reactivity and weak fluorescent signals of individual and co-localized antigens. The aims of this study were to develop new immunohistochemical techniques for imaging of peripheral nerve fibers. Three-millimeter punch skin biopsies of healthy individuals were fixed, frozen, and cut into 50-µm sections. Tissues were stained with a variety of antibody combinations with two signal amplification systems, streptavidin-biotin-fluorochrome (sABC) and tyramide-horseradish peroxidase-fluorochrome (TSA), used simultaneously to augment immunohistochemical signals. The combination of the TSA and sABC amplification systems provided the first successful co-localization of sympathetic adrenergic and sympathetic cholinergic nerve fibers in cutaneous human sweat glands and vasomotor and pilomotor systems. Primary antibodies from the same species were amplified individually without cross-reactivity or elevated background interference. The confocal fluorescent signal-to-noise ratio increased, and image clarity improved. These modifications to signal amplification systems have the potential for widespread use in the study of human neural tissues.