Optical clearing in collagen- and proteoglycan-rich osteochondral tissues.

OBJECTIVE: Recent developments in optical clearing and microscopy technology have enabled the imaging of intact tissues at the millimeter scale to characterize cells via fluorescence labeling. While these techniques have facilitated the three-dimensional (3D) cellular characterization within brain and heart, study of dense connective tissues of the musculoskeletal system have been largely unexplored. Here, we quantify how optical clearing impacted the cell and tissue morphology of collagen-, proteoglycan-, and mineral-rich cartilage and bone from the articulating knee joint. METHODS: Water-based fructose solutions were used for optical clearing of bovine osteochondral tissues, followed by imaging with transmission and confocal microscopy. To confirm preservation of tissue structure during the clearing process, samples were mechanically tested in unconfined compression and visualized by cryo-SEM. RESULTS: Optical clearing enhanced light transmission through cartilage, but not subchondral bone regions. Fluorescent staining and immunolabeling was preserved through sample preparations, enabling imaging to cartilage depths five times deeper than previously reported, limited only by the working distance of the microscope objective. Chondrocyte volume remained unchanged in response to, and upon the reversal, of clearing. Equilibrium modulus increased in cleared samples, and was attributed to exchange of interstitial fluid with the more viscous fructose solution, but returned to control levels upon unclearing. In addition, cryo-SEM-based analysis of cartilage showed no ultrastructural changes. CONCLUSION: We anticipate large-scale microscopy of diverse connective tissues will enable the study of intact, 3D interfaces (e.g., osteochondral) and cellular connectivity as a function of development, disease, and regeneration, which have been previously hindered by specimen opacity.

TRAIL contributes to the apoptotic effect of 13-cis retinoic acid in human sebaceous gland cells.

BACKGROUND: The full mechanism of action of isotretinoin [13-cis retinoic acid (13-cis RA)] in treating acne is unknown. 13-cis RA induces key genes in sebocytes that are involved in apoptosis, including Tumor necrosis factor Related Apoptosis Inducing Ligand (TRAIL). OBJECTIVES: In this study, we investigated the role of 13-cis RA-induced TRAIL within SEB-1 sebocytes. METHODS: Using 13-cis RA and recombinant human TRAIL (rhTRAIL) protein, we assessed induction of TRAIL and apoptosis in SEB-1 sebocytes, normal keratinocytes and patient skin biopsies. RESULTS: Treatment with rhTRAIL protein increased TUNEL-positive staining in SEB-1 sebocytes. TRAIL siRNA significantly decreased the percentage of TUNEL-positive SEB-1 sebocytes in response to 13-cis RA treatment. Furthermore, TRAIL expression increased in the skin of patients with acne after 1 week of isotretinoin therapy compared with baseline. TRAIL expression localized within sebaceous glands. Unlike sebocytes, TRAIL protein expression was not increased in normal human epidermal keratinocytes in response to 13-cis RA, nor did rhTRAIL induce apoptosis in keratinocytes, suggesting that TRAIL is key in the sebocyte-specific apoptotic effects of 13-cis RA. CONCLUSIONS: Taken together, our data suggest that TRAIL, like the neutrophil gelatinase-associated lipocalin, is involved in mediating 13-cis RA apoptosis of sebocytes.

Isotretinoin increases skin-surface levels of neutrophil gelatinase-associated lipocalin in patients treated for severe acne.

BACKGROUND: A clear-cut need exists for safe and effective alternatives to the use of isotretinoin in severe acne. Lack of data regarding the specifics of isotretinoin's mechanism of action has hampered progress in this area. Recently, the protein neutrophil gelatinase-associated lipocalin (NGAL) has been identified as a mediator of the apoptotic effect of isotretinoin on sebocytes. OBJECTIVES: To establish further the clinical relevance of NGAL and to elucidate the factors that induce NGAL expression in sebocytes. METHODS: Methods were developed to isolate and quantify skin-surface levels of NGAL from normal subjects and patients with acne undergoing treatment with isotretinoin. RESULTS: Patients with acne were found to have higher skin levels of NGAL compared with normal subjects. Studies in SEB-1 sebocytes indicate that NGAL expression is increased in response to Propionibacterium acnes and interleukin (IL)-1ß. In patients, isotretinoin increases NGAL levels by 2·4-fold on the skin surface and this increase precedes decreases in sebum and P. acnes counts. CONCLUSIONS: These data support the hypothesis that NGAL is an important mediator of the early effects of isotretinoin on the sebaceous glands and provide insights into the mechanisms that regulate NGAL expression in the skin.

Oxidative activity of the type 2 isozyme of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) predominates in human sebaceous glands.

Sebum production is regulated by the opposing effects of androgens and estrogens. The intracrine activity of steroid metabolizing enzymes is important in regulating sebum production because these enzymes can convert weak steroids from the serum into potent androgens and estrogens within the sebaceous gland (SG). 17Beta-hydroxysteroid dehydrogenase (17beta-HSD) interconverts weak and potent sex steroids via redox reactions. In this regard, it may function as a gatekeeping enzyme regulating the hormonal milieu of the SG. Six isozymes of 17beta-HSD have been identified that differ in their substrate preference and their preference to produce weak or potent sex steroids via oxidation or reduction, respectively. The goals of this study are: (i) to identify which isozyme (s) of 17beta-HSD is active in SG; (ii) to determine if its activity differs in facial skin compared with nonacne-prone skin that may account for the regional differences in sebum production; (iii) to compare the activity of 17beta-HSD in intact glands and in SG homogenates; and (iv) to determine if 13-cis retinoic acid inhibits 17beta-HSD activity. Human SG were assayed for 17beta-HSD activity using estrogens, androgens, and progestins as substrates. Oxidative activity of the type 2 isozyme predominated in all samples tested. Although transcripts for the types 1, 2, 3, and 4 isozymes were detected using reverse transcriptase-polymerase chain reaction, only mRNA for the predominant type 2 isozyme and the type 4 isozyme were detected in northern analysis. Greater reductive activity of 17beta-HSD was noted in SG from facial areas compared with nonacne-prone areas, suggesting an increased net production of potent androgens in facial areas. Oxidation was more predominant over reduction in intact SG compared with homogenized SG, thus supporting the hypothesis that 17beta-HSD protects against the effects of potent androgens in vivo. Activity of the type 2 17beta-HSD was not inhibited by 13-cis retinoic acid. In conclusion, SG possess the cellular machinery needed to transcribe the genes for the type 1-4 isozymes of 17beta-HSD. At the protein level, however, oxidative activity of the type 2 isozyme predominates, suggesting that 17beta-HSD isozyme activity may be translationally regulated.

The melanocortin 5 receptor is expressed in human sebaceous glands and rat preputial cells.

Melanocortins regulate pigmentation, adrenal hormone secretion, immune functions, lipid metabolism, and feeding behaviors in rodents. These peptides include adrenocorticotrophic hormone, melanocyte stimulating hormone, beta-lipotrophin, and the endorphins. Lipid metabolism in sebaceous glands and preputial glands of rodents is regulated by alpha-melanocyte stimulating hormone, the major agonist for melanocortin receptors. Five melanocortin receptor subtypes have been identified that differ in their tissue localization and affinities for melanocortin ligands. Targeted disruption of the melanocortin 5 receptor in transgenic mice results in widespread dysfunction of exocrine glands, including a marked decrease in sebum production. A role for melanocortins in the modulation of human sebum production has not been established. The goal of this study is to determine which melanocortin receptors are expressed in human sebaceous glands. Messenger RNA was isolated from human sebaceous glands and the reverse transcriptase polymerase chain reaction was performed using primers specific for each of the melanocortin receptor subtypes. Transcripts were detected for the melanocortin 5 receptor. A polyclonal chicken antihuman antibody to the melanocortin 5 receptor localized to sebaceous glands, eccrine glands, hair follicles, and epidermis in human skin, rat skin, cultured human sebocytes, and rat preputial cells. Presence of the melanocortin 5 receptor protein in human sebaceous glands and rat preputial glands was further verified by Western blotting. These data support further investigation of the role of melanocortins in the regulation of human sebum production and support the use of the rat preputial system as an experimental model in sebaceous gland physiology.

Activity of the type 1 5 alpha-reductase exhibits regional differences in isolated sebaceous glands and whole skin.

The presence of 5 alpha-reductase (5 alpha-R) in skin may indicate that the androgen regulation of sebaceous glands and sebum production requires the local conversion of testosterone to dihydrotestosterone. The goals of this study were to identify which isozyme of 5 alpha-R (type 1 or type 2) is expressed in sebaceous glands from facial areas, scalp, and non-acne-prone areas; to determine if 5 alpha-R activity is concentrated in sebaceous glands; to assess whether there are regional differences in this enzyme's activity; and to test the effects of azasteroid inhibitors and 13-cis retinoic acid on 5 alpha-R in these tissues. Sebaceous glands were microdissected from facial skin, scalp, and non-acne-prone skin (arm, breast, abdomen, leg), and the activity of 5 alpha-R was determined. A total of 49 samples from 23 male and 21 female subjects without acne (age range, 16 to 81 years, 56 +/- 20 years [mean +/- SD]) was analyzed. The biochemical properties of the enzyme in each of the samples tested are consistent with those of the type 1 5 alpha-R. Minimal to no type 2 5 alpha-R was detected. The level of 5 alpha-R activity was significantly higher in the sebaceous glands compared to whole skin in facial skin (p = 0.047), scalp (p = 0.039), and non-acne-prone skin (p = 0.04). Enzyme activity in sebaceous glands from facial skin and scalp was significantly higher than in a comparable amount of sebaceous gland material obtained from non-acne-prone areas (32 +/- 6 [mean +/- SEM]), 35 +/- 7 (mean +/- SEM) versus 6.0 +/- 3.0 (mean +/- SEM) pmol/min/mg protein, p = 0.014 and 0.007, respectively). Finasteride and 13-cis retinoic acid were poor inhibitors of the enzyme with 50% inhibitory concentration values greater than 500 nM. These data demonstrate that in the skin from older patients without acne the type 1 isozyme of 5 alpha-R predominates, its activity is concentrated in sebaceous glands and is significantly higher in sebaceous glands from the face and scalp compared to non-acne-prone areas, and the action of 13-cis retinoic acid in the control of acne is not at the level of 5 alpha-R. Furthermore, we suggest that specific inhibition of the type 1 5 alpha-R may offer a viable approach to the management of sebum production and, hence, acne.

Ad lib caffeine consumption, symptoms of caffeinism, and academic performance.

The authors explored the relationship between ad lib caffeine consumption in college students and the incidence of caffeinism, characterized by heightened anxiety, depression, and various psychophysiological reactions. Students were randomly selected from four groups (abstainers from caffeine and low, moderate, and high consumers). A survey battery assessed the effects of caffeine, incidence of psychophysiological disorders, state-trait anxiety, and depression. The moderate and high consumer groups combined reported significantly higher trait anxiety and depression scores when compared with abstainers. The high consumer group also reported significantly higher levels of symptoms of caffeinism, higher frequency of psychophysiological disorders, and lower academic performance.

Multilamellar bodies as potential scattering particles in human age-related nuclear cataracts.

PURPOSE: To characterize within human age-related nuclear cataracts rare spherical objects covered by multiple membranes, termed multilamellar bodies (MLBs). METHODS: Adult human normal, transparent lenses were obtained from eye bank donors and age-related nuclear cataracts were obtained immediately after extracapsular extraction. Each sample was Vibratome sectioned fresh into 200 microm thick sections that were fixed and embedded for light or electron microscopy. Confocal images were recorded from sections stained with the lipid soluble dye, DiI. RESULTS: Light micrograph montages of the equatorial plane containing the fetal and embryonic nuclei were examined. Rare, but distinct, circular 1-3 microm diameter objects were observed consistently in the cataracts. These objects did not appear to be components of the complex intercellular interfaces. Serial sections indicated that the objects were spherical, or contained a spherical component. For about 20,000 fiber cell cross-sections in each lens, the frequency of MLBs was 10 times higher in cataracts than in the normal lens nuclei. Although extensive searching with the electron microscope was necessary, the size, circular profile and multiple layers of thin (5 nm) membranes easily identified the MLBs. Interiors of the MLBs displayed variable textures. Confocal images indicated that the coverings were enriched in lipid compared to the adjacent plasma membranes. The calculated density of the MLBs in the cataractous nuclei was about 3800/mm3, which represents a volume fraction of 0.00003. CONCLUSIONS: Because the MLBs are large compared to the wavelength of light, display interiors with variable staining textures and have lipid-rich coverings, they appear to be ideal candidates for large scattering particles that may contribute to the forward light scattering in nuclear cataracts.

The interactive effect of personality, time of day, and caffeine: a test of the arousal model.

The personality dimension of introversion/extraversion is one of the few personality dimensions that can be reliably identified from study to study and investigator to investigator. The importance of this demension within personality theory is due both to the stability of the trait and the influential theory of H. J. Eysenck. The basic assumption in Eysenck's theory of introversion/extraversion is that the personality differences between introverts and extraverts reflect some basic difference in the resting level of cortical arousal or activation. Assuming that there is a curvilinear relationship (an inverted U) between levels of stress and performance leads to a test of this arousal theory. That is, moderate increases in stress should hinder the performance of introverts who are presumably already highly aroused. However, the same moderate increase in stress might help the performance of the presumably underaroused extraverts. Revelle, Amaral, and Turriff reported that the administration of moderate doses of caffeine hindered the performance of introverts and helped the performance of extraverts on a cognitive task similar to the verbal test of the Graduate Record Examination. Assuming that caffeine increases arousal, this interaction between introversion/extraversion and drug condition supports Eysenck's theory. This interaction was explored in a series of experiments designed to replicate, extend, and test the generality of the original finding. The interaction between personality and drug condition was replicated and extended to additional cognitive performance tasks. However, these interactions were affected by time of day and stage of practice, and the subscales of introversion/extraversion, impulsivity, and sociability, were differentially affected. In the morning of the first day, low impulsives were hindered and high impulsives helped by caffeine. This pattern reversed in the evening of the first day, and it reversed again in the evening of Day 2. We concluded that the results from the first day of testing require a revision of Eysenck's theory. Instead of a stable difference in arousal between low and high impulsives, it appeared that these groups differed in the phase of their diurnal arousal rhythms. The result is that low impulsives are more aroused in the morning and less aroused in the evening than are the high impulsives. A variety of peripheral or strategic explanations (differences in caffeine consumption, guessing strategies, distraction, etc.) for the observed performance increments and decrements were proposed and tentatively rejected. It seems probable that some fundamental change in the efficiency with which information is processes is responsible for these performance changes.

Individual differences in diurnal rhythms and multiple activation states: a reply to M. W. Eysenck and Folkard.

Three issues raised by M. W. Eysenck and Folkard are discussed. These include (a) just what individual difference variable is mediating the time of day and caffeine effects; (b) what the difference is in the diurnal rhythms of low and high impulsives; and (c) whether it is necessary to postulate multiple activation states. Suggestions for future research are then given.

Androgen metabolism in sebaceous glands from subjects with and without acne.

OBJECTIVE: To determine if there are differences in the activity of 17beta-hydroxysteroid dehydrogenase and 5alpha-reductase (responsible for the production of testosterone and dihydrotestosterone, respectively) in sebaceous glands obtained from men and women with and without acne. DESIGN: Single-center examination of androgen levels and sebaceous gland enzyme activity in a cohort of volunteers. SETTING: Academic referral center. PATIENTS: Thirty-four subjects, consisting of 8 women with acne, 10 women without acne, 8 men with acne, and 8 men without acne. INTERVENTIONS: Single visit for blood sampling and 2 biopsies of forehead skin. MAIN OUTCOME MEASURES: Serum levels of androgens were determined and compared with the activity of 5alpha-reductase and 17beta-hydroxysteroid dehydrogenase in sebaceous glands microdissected from skin samples. RESULTS: No significant differences in the activity of 5alpha-reductase or 17beta-hydroxysteroid dehydrogenase in sebaceous glands according to the presence of acne were noted in either men or women. The activity of 5alpha-reductase and 17beta-hydroxysteroid dehydrogenase was significantly greater in sebaceous glands from men (n = 16) than women (n = 17). The oxidative activity of 17beta-hydroxysteroid dehydrogenase was 2-fold higher in men than women. Serum levels of dehydroepiandrosterone sulfate, androstenedione, testosterone, and dihydrotestosterone were significantly higher in women with acne than in women without acne. No differences in serum androgen levels were noted in men on the basis of the presence of acne. CONCLUSIONS: Higher serum androgen levels are associated with the presence of acne in women. A role for locally produced androgens in this process, however, cannot be excluded.

Immunolocalization of 5alpha-reductase isozymes in acne lesions and normal skin.

BACKGROUND: Dihydrotestosterone mediates androgen-dependent diseases, such as acne, hirsutism, and androgenetic alopecia. This hormone is produced from testosterone by the 5alpha-reductase enzyme. There are 2 isozymes of 5alpha-reductase (types 1 and 2) that differ in their localization within the body and even within the skin. Activity of the type 1 isozyme predominates in sebaceous glands, where it may be involved in regulation of sebum production. Since specific inhibition of 5alpha-reductase type 1 may represent a novel therapeutic approach to acne, it is important to define the localization of these isozymes in normal sebaceous follicles and acne lesions. OBSERVATIONS: Skin biopsy specimens were obtained from the backs of 11 subjects: 8 with acne and 3 without acne. Sections of normal follicles, open comedones, closed comedones, and inflammatory lesions were incubated with antibodies to types 1 and 2 5alpha-reductase. In all samples, the type 1 antibody localized specifically to sebaceous glands, and the type 2 antibody localized to the companion layer of the hair follicle (the innermost layer of the outer root sheath) and granular layer of the epidermis. Localization of the type 2 isozyme was also noted within the walls of open and closed comedones and in endothelial cells from sections of inflammatory lesions. CONCLUSIONS: The immunolocalization of 5alpha-reductase isozymes in normal sebaceous follicles and acne follicles is similar to the pattern described in terminal hair follicles and corresponds with the findings of biochemical studies that have demonstrated predominance of type 1 activity in sebaceous glands. The function of type 2 5alpha-reductase in comedones or endothelial cells in inflammatory lesions is unknown.

Eysenck's arousal theory of introversion-extraversion: a converging measures investigation.

Convergent electroencephalographic, performance, and psychometric measures of arousal were used to test predictions based on H. J. Eysenck's (1967) theory. Twelve introverts and 12 extraverts had their brainstem auditory evoked responses (BAERs) monitored under 3 levels of caffeine administration (0.0, 1.5, and 3.0 mg/kg) and 3 levels of task demand (resting, simple response time, and complex response time). Results revealed a decrease in the latency of Wave V of the BAER as a function of personality, with introverts evidencing significantly shorter latency of Wave V as compared with extraverts. Faster conduction time between Waves I-III and I-V were also found in introverts. Caffeine at either the 1.5 or 3.0 mg/kg level was associated with decreased latency of Wave V, compared with the placebo condition. No personality differences in subjective arousal or reaction time performance were found across any of the conditions.

Epidural abscesses of the spine: case comparisons.

Epidural abscesses of the spine generally arise from staphylococcal infections that by hematologic spread or direct invasion affect the integrity of the vertebral column or meningeal coverings of the spinal cord. A case comparison approach highlights goal-oriented nursing interventions which assist the patient in the adaptation to and recovery from this unique central nervous system infection.

Compliance, low literacy, and locus of control.

Compliance remains a critical, complex issue in health care today. As outlined, many interrelated factors affect levels of compliance and noncompliance. Assessing literacy and locus of control may assist the health care professional in identifying persons less likely to comply with medical treatments. Although compliance remains difficult to predict, these components should be considered carefully in patient-professional relationships and patient education.

Specialty nursing council: a peer support group for nurses in independent roles.

As nurses move into more nontraditional, expanded roles, they sometimes lack a social and professional support group within the work setting. Feeling isolated and lacking support, several clinical nurse specialists (CNSs) at a large county institution began the Specialty Nursing Council. The Specialty Nursing Council provides a means of networking and support for nurses in specialty roles in a three institution health care campus. Monthly meetings are conducted to bring members together and for continuing education programs. The keys to success in forming this type of council are: 1) a dedicated group that plans, organizes, and distributes informational material; 2) common goals and objectives; 3) direct benefits to members.