2D-IR spectroscopy of carbohydrates: Characterization of thiocyanate-labeled ß-glucose in CHCl3 and H2O.

Carbohydrates constitute one of the four key classes of biomacromolecules but have not been studied by 2D-IR spectroscopy so far. Similarly as for proteins, a lack of native vibrational reporter groups, combined with their huge structural diversity, leads to spectrally congested infrared spectra already for single carbohydrates. Biophysical studies are further impeded by the strong overlap between water modes and carbohydrate modes. Here, we demonstrate the application of the known vibrational reporter group thiocyanate (SCN) as a label in glucose. In this first study, we are able to perform IR and 2D-IR spectroscopy of ß-glucose with SCN at the C2 position in chloroform. Upon improved synthesis and the removal of all protecting groups, we successfully performed 2D-IR spectroscopy of ß-glucose in H2O. All experimental results are compared to those of methyl-thiocyanate as a reference sample. Overall, we show that the concept of using site-specific vibrational reporter groups can be transferred to carbohydrates. Thus, biophysical studies with 2D-IR spectroscopy can now expand to glycoscience.

Enhanced expression of the antimicrobial peptide LL-37 in lesional skin of adults with atopic eczema.

BACKGROUND: Atopic eczema (AE) is a common multifactorial chronic skin disease associated with a defective skin barrier and increased susceptibility to skin infections. The human cathelicidin LL-37 plays a role in the host defence of skin. Studies have demonstrated deficient expression of LL-37 in skin of AE patients. OBJECTIVES: The aim of this study was to investigate the expression of LL-37 in lesional skin compared with nonlesional skin in patients with different severity of AE, patients with other eczema and healthy subjects. METHODS: Twenty patients with AE, four patients with other eczema and 10 healthy subjects were included. Severity of AE was graded using SCORing of atopic dermatitis (SCORAD). Skin biopsies were taken from lesional and nonlesional skin from all patients and from skin of healthy controls. The levels of LL-37 mRNA were analysed by quantitative reverse transcriptase-polymerase chain reaction. Evaluation of dermal and epidermal protein expression of LL-37 and the degree of inflammation was performed by immunohistochemical stainings. RESULTS: Patients with AE and patients with other eczema had significantly (P < 0.05) higher levels of LL-37 in lesional skin than in nonlesional skin. The expression of LL-37 was not statistically associated to severity of AE valued by SCORAD. Nonlesional skin from patients did not differ from skin of healthy subjects in terms of LL-37 expression. In the presence of epidermal injury or vesicles the LL-37 peptide was always detected. CONCLUSIONS: Patients with AE exhibit enhanced expression of LL-37 in lesional skin compared with nonlesional, suggesting a role of LL-37 in AE that might be associated with the process of re-epithelialization.

Effect of mycophenolate mofetil on rat kidney grafts with prolonged cold preservation.

The impact of mycophenolate mofetil (MMF) on initial renal transplant function is not well characterized. We tested how MMF may modulate graft function and survival in a syngeneic rat kidney transplantation model after prolonged cold preservation. Donor kidneys were preserved in University of Wisconsin for either 24 or 39 h prior to transplantation into nephrectomized rats. Recipients received MMF (20 mg/kg/day) or vehicle. Mycophenolic acid (MPA) blood concentrations were measured by high-performance liquid chromatography. The inflammatory response, tubular epithelial proliferation, and histologic damage 3 days post-transplantation were assessed microscopically. In the 24 h cold storage (c.s.) group serum-creatinine was measured. In the 39 h c.s. group 1-week recipient survival was determined. After 24 h of c.s., recipient survival was 100%. The number of T-cell infiltrates was low and not influenced by MMF, whereas renal ED1+ cell infiltration was significantly suppressed by MMF. Tubular cell proliferation was enhanced by MMF. Serum-creatinine levels and renal histology were comparable between MMF and vehicle-treated animals. In the 39 h c.s. group, recipient survival was 20% in MMF-treated vs 90% in vehicle-treated animals (P=0.001). MMF effectively suppressed inflammatory cell infiltration and inhibited tubular cell proliferation. MMF-induced structural damage was most striking in the renal papilla. In rat kidney grafts with moderate preservation injury (24 h c.s.), MMF, given at an immunosuppressive dose, showed predominantly antiinflammatory effects without compromising graft function. In grafts with severe preservation injury (39 h c.s.), MMF caused irreversible structural damage and inhibited tubular cell regeneration resulting in renal failure.

Elemental analysis mirrors epidermal differentiation.

Using a scanning nuclear microprobe, the distribution of elements and trace elements of skin cross sections of normal skin, non-lesional psoriatic skin and in dry atopic skin have been mapped. In non-lesional psoriatic skin and in dry atopic skin the epidermal Ca-gradient is higher than that of normal skin. In addition, abnormally high Fe and Zn levels were recorded in the stratum granulosum and corneum regions in the pathological skin. It is suggested that these findings correlate to an increased cell turnover in the basal cell layer of the psoriatic and atopic skins. The ratio of Ca/Zn in stratum corneum of paralesional psoriatic skin is approximately 8:1 compared to 12: 1 in normal skin and 15: 1 in atopic skin. This suggests that the differentiation process in paralesional psoriatic skin may actually be an example of disturbed programmed cell death.

[Despite better knowledge of pathogenesis and treatment: atopic eczema--a disease with increasing incidence in Sweden]. / Trots bättre kunskaper om patogenes och behandling: atopiskt eksem--en sjukdom som ökar i Sverige.

The past twenty years have witnessed an increasing incidence of atopic dermatitis in Western Europe. The article consists in a discussion of the pathogenesis, clinical signs and treatment of this common skin disease. Both an IgE-mediated reaction on epidermal Langerhans cells, and a physiological/biochemical defect of the skin barrier structure may be crucial factors of the multifactorial pathogenesis. Local treatment with corticosteroids and moisturisers remains the basic approach, though the development of new more specific treatments is under way. Although much remains to be learned about atopic dermatitis, today all patients can be offered effective treatment resulting in improved quality of life.

Aspects on the physiology of human skin: studies using particle probe analysis.

The cellular part of the skin, the epidermis, is a very thin structure, approximately 120 microns thick, a fact which has hindered the exploration of the physiology of the epidermis in normal and pathological conditions. An additional complication is the fact that the epidermis contains layers of cells at different stages of differentiation. Therefore, conventional physiological capillary probes cannot, with any satisfactory precision, be located within a specified cell of a specified layer of the skin in vivo. Hence, alternative ways for the exploration of skin physiology have been sought for. In the past, analysis of the elemental content of skin was done was done as bulk measurements, and surprisingly wide ranges of elemental content were recorded. The width of these ranges was most certainly due to the sampling methods used rather than the sensitivity of the chosen method of analysis. Also, these older measurements did not discriminate between the different strata, and therefore the information provided little if any substance for a functional analysis of processes involved in normal and pathological differentiation of the epidermis. Particle probes, however, have been able to overcome such methodological problems. Over a period of 15 years we have studied normal human skin, normal-looking, paralesional skin from psoriatics, and skin from persons suffering from atopic dermatitis using PIXE analysis. In recent years, trace elements have been shown to work as secondary messengers or regulatory substances. As an example, calcium (Ca2+) has proven to be a very important signalling substance in a great variety of cellular systems. Studies with the transmission electron microscope (TEM) as well as histochemical methods have allowed an understanding of the role of Ca2+ in the differentiation process of the epidermis. Ca2+ has also been shown to play an important role in apoptosis (programmed cell death), which is currently a hot subject for the obvious reason that the final differentiation step between the stratum granulosum level and the stratum corneum represents a particular aspect of programmed cell death. The importance of the balance between calcium and zinc in apoptosis has been clearly demonstrated in a number of cellular systems, but we have still to clarify the validity of topical treatment with Zn ointments in different skin conditions. Substantial iron (Fe) losses via psoriatic lesions were demonstrated more than two decades ago, and these data were given new meaning when we found that a more discrete loss occurs in clinically normal-looking psoriatic skin. Obviously, such findings stress the importance of understanding the relation between the elemental content and normal and abnormal physiology. The ultimate goal of particle probe studies is to provide an understanding of the formation of a mature stratum corneum with a functional barrier reflected in physiological/biochemical mechanisms behind the properties of changed skin in patients afflicted with skin disorders of genetic or constitutional origin. This paper aims to give an overview of the state of the art in skin physiology made possible through the use of particle probes.

Pixe analysis of pathological skin with special reference to psoriasis and atopic dry skin.

The nuclear microprobe is used for element analysis of human skin cross sections, providing new insight into the physiology of normal and pathological conditions. Special interest is focused on trace elements as they work as secondary messengers or regulatory substances. The distribution of ions in normal tissues serves as reference for pathological changes. Calcium (Ca2+) is assumed to take an important role in the differentiation process of the epidermis. This paper presents new data on elemental and trace elemental distributions in skin. Samples are prepared from skin biopsies obtained from patients with skin disorder and from individuals with no records of skin disorder serving as controls. Using the nuclear microprobe, both elemental maps and quantitative depth profiles are obtained. Previous findings of abnormal Fe distribution in psoriatic skin are confirmed, and new observations of altered Zn and Ca profiles in atopic skin are reported. The relation to possible physiological/biochemical mechanisms and apoptosis ("programmed cell death") is discussed. The study is a part in a larger survey aiming at an understanding of the formation of a mature stratum corneum with a functional barrier, and its changed properties in cases of skin disorder.

Human skin physiology studied by particle probe microanalysis.

Particle probe methods (electron probe and proton probe X-ray microanalysis) have been applied to investigate the distribution of elements and water over the different layers of the epidermis. For major elements, electron probe X-ray microanalysis (XRMA) provides the advantage of superior spatial resolution, but for trace element analysis the more sensitive proton probe (particle induced X-ray emission, PIXE) analysis has to be used. On a dry weight basis, the concentration of S is rather constant across the epidermis, whereas the concentrations of P, K, Cl and Na show gradients with high levels in stratum germinativum (basale) and stratum spinosum but low levels in the stratum granulosum and stratum corneum. Essentially, Fe and Zn are confined to the basal region in normal skin. The concentration of Ca, however, increased steadily from the basal region to the stratum corneum. The probe technique allows quantitative analysis of stratum-specific changes in elemental content in a variety of pathological conditions, e.g., changes induced by nickel, detergents and other chemicals, or in psoriatic skin. Of particular interest are findings of increased Fe and Zn in non-involved psoriatic skin. Since the different layers of the skin have different elemental concentrations and react differently under pathological conditions, the probe techniques are far superior to bulk chemical analysis in elucidating physiological and pathological processes in the skin.

Friction, capacitance and transepidermal water loss (TEWL) in dry atopic and normal skin.

The biophysical properties of non-eczematous skin at three locations in atopics and non-atopics were characterized using non-invasive physical methods. Skin friction was measured with a newly developed sliding friction instrument, the degree of hydration with a capacitance meter (Corneometer CM 820), and the transepidermal water loss (TEWL) was determined using an Evaporimeter EP1. The areas examined (dorsum of the hand, volar forearm and lower back) showed lower values of friction and capacitance in the atopic patients than did corresponding sites in the normal controls. In most areas a significant correlation between friction and capacitance was found. The TEWL was increased in atopic skin, but TEWL seems to correlate neither to friction nor to capacitance.

Dry skin in atopic dermatitis.

Atopic dermatitis (AD) is a common, chronically recurring skin disorder. Dry skin is a common finding in patients with AD, apart from the dermatitis. Although there are obvious clinical signs of an impaired barrier function of the skin, few investigators have studied this aspect of AD. The stratum corneum, where the barrier is located, has been studied with different techniques in patients with AD, and the results are now presented. The water-binding capacity of dry atopic skin was found to be reduced when measured with an in vitro microbalance technique. TEWL (transepidermal water loss) measured with and Evaporimeter Ep1, was increased in dry skin and in clinically normal skin of atopics on predilection areas. Water content was decreased in dry atopic skin, when measured with the Corneometer CM 420. In a quantitative electron microscopic study, the lamellar bodies were found to have an increased relative volume in dry atopic skin. When using chromatographic analysis, preliminary data suggested reduced amounts of extractable stratum corneum lipids in patients with AD. In a clinical study, 80% of the patients with AD regarded their skin as being dry. Fifty percent were found to have areas of dry skin, on clinical examination. By scanning electron microscopy (SEM), the surface pattern of dry atopic skin was found to be coarse and irregular. When using profilometry, quantitative differences in roughness parameters were found in dry atopic vis-à-vis to normal skin.(ABSTRACT TRUNCATED AT 250 WORDS)

'Dry' skin in atopic dermatitis. I. A clinical study.

A common finding in patients with atopic dermatitis is the occurrence of 'dry' skin on non-eczematous regions. 'Dry' skin is here defined as a clinical condition meaning a rough, finely scaling non-inflamed skin surface. The frequency and extension of 'dry' skin were examined in 50 patients with atopic dermatitis and were compared with those in 50 non-atopics. A discrepancy was found in both groups between the subjective opinion of the presence of 'dry' skin and the objectively noted 'dry' skin. Among the atopics, 48% were found to have 'dry' skin compared with 14% among the controls (p less than 0.01). The most frequent location of 'dry' skin in both groups was the back. Intolerance to wool was found to be significantly high (p less than 0.01) in the atopic group, although it was also quite common in non-atopics. In order to correlate the clinical observation to skin morphology, a replica-technique was used to visualize the surface of 'dry' skin in the scanning electron microscope.

'Dry' skin in atopic dermatitis. II. A surface profilometry study.

In patients with atopic dermatitis, the skin is often 'dry' on non-eczematous areas, and feels rough to the touch. In the scanning electron microscope (SEM) this roughness corresponds to a change in the skin surface, from regular major and minor furrows into a coarse and irregular pattern. In the present study the topography of normal skin and 'dry' atopic skin was quantitatively recorded using a profilometry method. A computer-based three-dimensional reconstruction of the skin surface was made from replicas, and common roughness parameters were calculated. It was found that in the 'dry' atopic skin roughness parameters Ra, Rq, and Rmax, were significantly increased, whereas the number of peaks, Rn, was decreased. Parameters describing the shape (skewness, kurtosis, and delta a) of the profile were not significantly different. These topographical data clearly support the visual impression of the surface pattern of 'dry' atopic skin found in SEM.