Epidermal differentiation: the role of proteases and their inhibitors

Dermatological diseases range from minor cosmetic problems to life-threatening conditions, as seen in some severe disorders of keratinization and cornification. These disorders are commonly due to abnormal epidermal differentiation processes, which result in disturbed barrier function of human skin. Elucidation of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis is therefore of great importance for the understanding and therapy of these disorders. Much of the barrier function of human epidermis against the environment is provided by the cornified cell envelope (CE), which is assembled by transglutaminase (TGase)-mediated cross-linking of several structural proteins and lipids during the terminal stages of normal keratinocyte differentiation. The major constituents of the stratum corneum and the current knowledge on the formation of the stratum corneum will be briefly reviewed here. The discovery of mutations that underlie several human diseases caused by genetic defects in the protein or lipid components of the CE, and recent analyses of mouse mutants with defects in the structural components of the CE, catalyzing enzymes, and lipid processing, have highlighted their essential function in establishing the epidermal barrier. In addition, recent findings have provided evidence that a disturbed protease-antiprotease balance could cause faulty differentiation processes in the epidermis and hair follicle. The importance of regulated proteolysis in epithelia is well demonstrated by the recent identification of the SPINK5 serine proteinase inhibitor as the defective gene in Netherton syndrome, cathepsin C mutations in Papillon-Lefevre syndrome, cathepsin L deficiency infurless mice, targeted ablation of the serine protease Matriptase/MTSP1, targeted ablation of the aspartate protease cathepsin D, and the phenotype of targeted epidermal overexpression of stratum corneum chymotryptic enzyme in mice. Notably, our recent findings on the role of cystatin M/E and legumain as a functional dyad in skin and hair follicle cornification, a paradigm example of the regulatory functions exerted by epidermal proteases, will be discussed.     

Thermoregulation during mild exercise at different circadian times

Eight healthy subjects exercised at 90 watts on a cycle ergometer on four occasions, at times close to the minimum, maximum rate of rise, maximum, and maximum rate of fall of their resting core temperature. The duration of exercise was determined by the time taken for the core (rectal) temperature to reach an equilibrium value. Forearm skin blood flow and temperature were measured regularly during the exercise, as were heart rate and ratings of perceived exertion. Sweat loss was calculated by weighing the subjects nude before and after the exercise. The rise of heart rate was not significantly different at the four times of exercise, though the rating of perceived exertion was greatest at 05:00 h. Resting core temperatures showed a significant circadian rhythm at rest (the timing of which confirmed that exercise was being performed at the required times), but the amplitude of this rhythm was decreased significantly by the exercise. The initial rate of rise of core temperature, and the total rise from the resting to the equilibrium value, were both inversely proportional to resting temperature. The time-course of the rise was accurately described by a negative-exponential model, but this model gave no evidence that the kinetics of the equilibration process depended upon the time of day. The thermoregulatory responses to the rise in core temperature—the amount of total sweat loss and rises in forearm skin blood flow and temperature—differed according to the time of exercise. In general, the responses were significantly greater at 17:00 h compared with 05:00 h, and at 23:00 h compared with 11:00 h. The results accord with predictions made on the basis of previous work by us in which core temperature rhythms have been separated into components due to the endogenous body clock and due to the direct effects of spontaneous activity. The results are discussed in terms of the ecological implications of the differing capabilities of humans to deal with heat loads produced by spontaneous activity or mild exercise at different phases of the circadian rhythm of resting core temperature.     

Evidence of hair loss after subacute exposure to 2-chloroethyl ethyl sulfide, a mustard analog, and beneficial effects of N-acetyl cysteine

Mustard gas has been used as a vesicant chemical warfare agent. However, a suitable biomarker for monitoring mustard gas exposure is not known. We observed that the hairs of the guinea pigs exposed intratracheally to subacute doses of 2-chloroethyl ethyl sulfide (CEES), a mustard analog, came out very easily though there was no sign of skin lesions or skin damage. Also the hairs looked rough and dry and lost the shiny glaze. There was no recovery from this hair loss, though the animals never became hairless, following CEES exposure. Hairs were observed in this study both visually and with light microscopy. Treatment with N-acetylcysteine (NAC) prior to CEES exposure could prevent the hair loss completely. Hence, sudden hair loss might be a good biomarker for subacute exposure of mustard gas to subjects at risks when the victims might have no other visible symptom of toxicity.     

Differential rAAV2 transduction efficiencies and insulin secretion profiles in pure and co-culture models of human enteroendocrine L-cells and enterocytes

BACKGROUND: Cell-based therapies for treating insulin-dependent diabetes (IDD) can provide a more physiologic regulation of blood glucose levels in a less invasive fashion than insulin injections. Previously, we developed an engineered human enteroendocrine L-cell model for regulated insulin release via recombinant adeno-associated virus serotype 2, or rAAV2, transduction. The aim of this study was to evaluate the efficiency and selectivity of rAAV2-mediated insulin gene delivery to enteroendocrine L-cells in co-culture with a prevailing number of enterocytes, which are the predominant cell type in intestinal epithelium. METHODS: We tested rAAV2 transduction in pure and co-culture models of human cell lines of enterocytes (Caco-2 and T84 cell lines) and enteroendocrine L-cells (NCI-H716 cell line). Non-viral, chemical-mediated transfection was used as a control. Transduced and transfected co-cultures were subjected to insulin secretion studies. RESULTS: In pure cultures, rAAV2 exhibited a low transduction efficiency towards both Caco-2 and T84 enterocytes, as opposed to a strong reporter expression in permissive NCI-H716 L-cells. In co-cultures of NCI-H716 L-cells and Caco-2 or T84 enterocytes, rAAV2 exhibited differential transduction efficiency with a strong preference towards NCI-H716 L-cells. The rAAV2-transduced co-culture achieved regulated insulin release against stimulation, whereas the chemically transfected co-culture failed to respond. CONCLUSIONS: This study demonstrated that rAAV2-mediated insulin gene transfer can differentiate human intestinal cell types in vitro, in particular enterocyte and enteroendocrine L-cell lines. We consider the AAV2 vector a useful tool in developing enteroendocrine L-cell-specific insulin gene delivery for IDD treatment, in terms of AAV2 avoiding enterocytes and targeting selectively L-cells.     

Performance enhancement in rally car drivers via heat acclimation and race simulation

To investigate the combined use of an interactive racecar simulator and heat acclimation on psychomotor (driving) performance, eight rally drivers underwent 4 days of repeated heat (50 degrees C) exposure (1 h x day(-1)) during which they performed a simulated rally drive (3x12-min stages each separated by a 2-min break), after first cycling for 15 min at 125 W to induce some degree of fatigue and heat storage prior to beginning the rally. During the rally stages, a generic set of pace notes were read to the subject by a co-driver. In each simulation, sweat loss, heart rate, core (rectal) and skin temperatures were recorded and driving and psychomotor performance were assessed by recording stage times and time to complete a psychomotor test. Levels of physiological and perceived thermal strain were also recorded. Significant decreases in rally stage times (88 s; P<0.005), psychomotor test time (18 s; P<0.01), final core (0.25 degrees C; P<0.001) and skin (0.44 degrees C; P<0.005) temperatures, heart rate (16 beats x min(-1); P<0.05) and physiological (15 W x m(-2); P<0.005) and perceived thermal (3.7 units; P<0.01) strain were evident by the end of the final simulation, and a significant (P<0.05) increase in sweat sensitivity (+0.33 g x h(-1) x degrees C(-1)) was also recorded. These results suggest that both heat acclimation and race simulation can improve the psychomotor performance of rally drivers, although the relative contribution of each factor was not determined here. However, in a practical setting, these factors would not be used in isolation. After performing the acclimation and simulation protocol prior to an actual rally, drivers subjectively reported improvements in tolerating a high thermal load and in their ability to control the rally vehicle.     

Analysis of a two-stage case-control study with cluster sampling of controls: application to nonmelanoma skin cancer

We present a pseudolikelihood approach for analyzing a two-stage population-based case-control study with cluster sampling of controls. These methods were developed to analyze data from a study of nonmelanoma skin cancer (NMSC). This study was designed to evaluate the role of ultraviolet radiation (UVB) on NMSC risk while adjusting for age group, which is known for all subjects, and for other individual-level factors, such as susceptibility to sunburn, which are known only for participants in the case-control study. The methods presented yield estimates of relative and absolute risk, with standard errors, while accounting naturally for the two-stage sampling of the cohort and cluster sampling of controls.     

Accelerated tissue aging and increased oxidative stress in broiler chickens fed allopurinol

Uric acid has been hypothesized as being one of the more important antioxidants in limiting the accumulation of glycosylated endproducts in birds. Study 1 was designed to quantitatively manipulate the plasma concentrations of uric acid using hemin and allopurinol while study 2 determined their effects on skin pentosidine, the shear force value of Pectoralismajor muscle, plasma glucose, body weight and chemiluminescence monitored oxidative stress in broiler chickens. Hemin was hypothesized to raise uric acid concentrations thereby lowering oxidative stress whereas allopurinol was hypothesized to lower uric acid concentrations and raise measures of oxidative stress. In study 1 feeding allopurinol (10 mg/kg body weight) to 8-week-old broiler chicks (n=50) for 10 days decreased plasma uric acid by 57%. However, hemin (10 mg/kg body weight) increased uric acid concentrations 20%. In study 2, 12-week-old broiler chicks (n=90) were randomly assigned to either an ad libitum (AL) diet or a diet restricted (DR) group. Each group was further divided into three treatments (control, allopurinol or hemin fed). Unexpectedly, hemin did not significantly effect uric acid concentrations but increased (P<0.05) measures of chemiluminescence dependent oxidative stress in both the DR and AL birds probably due to the ability of iron to generate oxygen radicals. Allopurinol lowered concentrations of uric acid and increased (P<0.05) the oxidative stress in the AL birds at week 22, reduced (P<0.05) body weight in both the AL and DR fed birds at 16 and 22 weeks of age, and markedly increased (P<0.001) shear force values of the pectoralismajor muscle. Skin pentosidine levels increased (P<0.05) in AL birds fed allopurinol or hemin fed birds, but not in the diet restricted birds at 22 weeks. The significance of these studies is that concentrations of plasma uric acid can be related to measures of oxidative stress, which can be linked to tissue aging.     

MiR-203 及其调控机制的研究进展

MicroRNA(miRNA)是一种长18-25个碱基的单链非编码RNA,其广泛参与机体众多病理、生理过程。MiR-203是一种上皮组织特异性表达的miRNA。研究发现miR-203在皮肤发育、皮肤相关疾病、上皮组织肿瘤等病理、生理过程中发挥重要调控作用。一些疾病的研究还发现了miR-203与表观遗传学机制的相互作用,而miR-203调控机制的研究集中在P63、SOCS-3及相关的信号通路等方面。     

SD大鼠与巴马小型猪的皮肤组织学比较

目的研究及观察SD大鼠和巴马小型猪皮肤的正常比较组织学。方法取SD大鼠和巴马小型猪不同部位的皮肤进行石蜡切片、HE染色,光学显微镜观察。结果两种动物的皮肤组织学结构在以下方面存在着显著差异：1.SD大鼠的毛囊成簇分布,平均3～9成群,而巴马小型猪的毛囊较稀少;2.SD大鼠表皮较薄,没有透明层,基底细胞缺乏异质性,真皮与表皮连接面平坦,没有皮钉;而在巴马小型猪皮肤表皮和真皮连接区,有上下交错的表皮皮钉和真皮乳头;3.SD大鼠的真皮结构相对松散,真皮血管系统不发达,而巴马小型猪皮肤的真皮网织层和乳头层交界的地方,水平分布着很多的浅表小静脉和小动脉丛,这种血管分布的方式与人类皮肤中的血管分布极为类似;4.SD大鼠的汗腺只局限于足垫的皮肤,汗腺上皮只有一种细胞类型,腺细胞呈立方形或矮柱状,胞核圆形,导管短而弯曲,由两层上皮细胞组成。而巴马小型猪皮肤的汗腺是顶泌汗腺,分布于真皮和脂肪相接的真皮深层,分泌部为粗管,管腔大,盘曲成团。腺细胞呈立方形或扁平,胞核圆形或长梭形。腺细胞与基膜之间也有肌上皮细胞。导管较细而直,开口于毛囊上段。