Qualification of a precise and easy-to-handle sweat casting imprint method for the prediction and quantification of anti-perspirant efficacy.

A time- and cost-effective sweat casting method using the forearm as test site to assess the efficacy of several anti-perspirant formulations with a low number of test subjects has been evaluated and qualified. The imprint sweat casting method is based on a 2-component silcone-imprint technique to measure the efficacy of more than eight products in parallel with the same test subject. In studies using aluminum chlorohydrate (ACH) formulations as test anti-perspirants, a clear-cut correlation could be demonstrated between sweat gland activities measured by the imprint method and gravimetric measurement of sweat gland activities. Concentration-dependent inhibition of sweat gland activity could be observed with the imprint technique up to an ACH concentration of 15%, and all formulations containing 2% ACH or above resulted in statistically significant reduction of sweat gland activity (P < 0.001) when compared with untreated control areas. Furthermore, the SDs of individual studies using the imprint technique were in a range of +/-20% of sweat gland activity, which can be regarded rather low for in vivo measurements of a complex process like sweat secretion. A group-wise comparison between the measurements of anti-perspirant activity as determined by the imprint protocol and the Food and Drug Administration (FDA) Guideline compliant gravimetric hot-room protocol revealed that the test results for anti-perspirant activity obtained with the imprint protocol are similar to those obtained with the hot-room protocol. Moreover, the data generated with the imprint protocol have a high predictive value for the outcome of a later guideline-compliant hot-room test. As the imprint casting method tends to be a little more sensitive for formulations with low anti-perspirant activity, and seems to be associated with less interassay variability than the standard gravimetric hot-room test, the imprint casting method may select products which later fail to pass the standard gravimetric hot-room test. Meanwhile the imprint sweat casting has proven to be a robust method useful to support efficacy-oriented product development. Therefore, in later stages of utilization it might even evolve into an efficient claim substantiation tool.

Contact angle measurement - a reliable supportive method for screening water-resistance of ultraviolet-protecting products in vivo.

Substantivity of sunscreen formulations is affected by the wash-out rate of ultraviolet-absorber and -reflector compounds in water. Water-resistance of sunscreen formulations is currently determined according to a standardized European Cosmetic Toiletry and Perfumery Association (COLIPA) protocol, encompassing the determination of a minimal erythemal dose before and after a defined immersion step in water. It can be supposed that the higher the wettability of a treated skin area, the higher is the wash-out rate of sunscreen compounds. This present report addresses the validity of determining the wettability of treated skin alone as a measure for the water-resistance of sunscreen products. The report addresses the robustness, accuracy and congruence of a recently developed wettability test, based on the measurement of the contact angle (CA) of a sessile water drop on treated skin areas. Contact angle data of 66 sunscreen formulations are compared with the corresponding results of 81 water-resistance tests, using the sun protection factor (SPF)/immersion/SPF method. Sunscreen products tested by the CA method were applied to the skin of the volar forearm of test subjects at a defined dose and drying-time, using a standardized application and recording device. Contact angles between a sessile water drop and skin were recorded by a Charge-Coupled Device (CCD) camera and subjected to automatic contour analysis. Taking the SPF/immersion/SPF method as gold standard, accuracy parameters of the CA method were determined. By using an appropriate cut-off level of CAs, the CA method has a specificity and positive-predictive value of 100%, and turns out to be a reliable screening method to identify water-resistant formulations. Based on our findings, those formulations that give CAs above 30 degrees may be categorized water-proof without further testing by the COLIPA water-resistance method.

Barrier characteristics of different human skin types investigated with X-ray diffraction, lipid analysis, and electron microscopy imaging.

The stratum corneum requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. The lipids are organized in intercellular membranes exhibiting short- and long-periodicity lamellar phases. In recent years, the phase behavior of barrier lipid mixtures has been studied in vitro. The relationship of human stratum corneum lipid composition to membrane organization in vivo, however, has not been clearly established. Furthermore, the special function of the different ceramide species in the stratum corneum is largely unknown. We examined lipid organization and composition of stratum corneum sheets from different subtypes of healthy human skin (normal, dry, and aged skin). Lipid organization was investigated using X-ray diffraction and demonstrated that the 4.4 nm peak attributed to the long periodicity phase was frequently missing for skin with a low Cer(EOS)/Cer(total) ratio, indicating an important part for Cer(EOS), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to sphingosine (S). A deficiency in the 4. 4 nm peak was predominantly observed in young dry skin. In one case of aged skin, however, and less often in young normal skin this peak was also missing. Furthermore, the ceramide composition of samples without the 4.4 nm peak showed a deficiency of Cer(EOH), which contains 6-hydroxy-4-sphingenine (H), and an increase in Cer(NS) and Cer(AS), which contain nonhydroxy (N) or alpha-hydroxy fatty acids (A). In addition, a 3.4 nm peak attributed to crystalline cholesterol occurred in most cases of aged and dry skin, but was not observed in young normal skin. Our results do not indicate a definite pattern of correlation between lipid organization and types of human skin. They demonstrate, however, that Cer(EOS) and Cer(EOH) are key elements for the molecular organization of the long periodicity lamellar phase in the human stratum corneum.

Two ceramide subfractions detectable in Cer(AS) position by HPTLC in skin surface lipids of non-lesional skin of atopic eczema.

The non-involved skin of atopic eczema (NEAE) is characterized by severe dryness and an impaired barrier function of the stratum corneum as indicated by an increased transepidermal water loss. Previous studies have demonstrated that this barrier impairment coincides with marked alterations in the amount and composition of stratum corneum ceramides. The aim of this study was to identify specific alterations in NEAE that may be used in the diagnosis of the atopic eczema. Using a classical procedure for high performance thin layer chromatography we could confirm earlier results: apart from Cer(EOH), which contains omega-hydroxy fatty acid (O) ester-linked to linoleic acid (E) and amide-linked to 6-hydroxy-4-sphingenine (H), the quantities of all ceramide fractions were significantly decreased. Furthermore, Cer(EOH)/Certotal was significantly increased, whereas the percentage of Cer(EOS), which contains sphingosine (S), and Cer(NP), which contains non-hydroxy fatty acid (N) amide-linked to phytosphingosine (P), were significantly decreased. Using a modified procedure for high performance thin layer chromatography we could demonstrate the formation of a double peak in the position of Cer(AS), which contains alpha-hydroxy fatty acid (A), in lipids of NEAE. The subfractions of the double peak comprised 15% and 12% of Certotal. MALDITOF mass spectrometry suggested that the double peak was formed by a homologous series of mono-hydroxylated and mono-unsaturated ceramides of different chain length, e.g., Cer(AS) subfractions containing either (C16,18) or (C22,24,26) alpha-hydroxy fatty acids. In contrast, in normal skin a single peak in Cer(AS) position, which comprised 22% of Certotal, was mainly formed by the long chain subfraction. In some cases this single peak displayed a small shoulder at its right flank, but never showed a clear peak separation when developed with NEAE samples. Furthermore, even in senile xerosis, or in either non-involved skin of psoriasis or seborrhoic eczema, only a single peak occurred in Cer(AS) position. Accordingly, the double peak might be specific for NEAE and turn out to be a marker for atopic eczema.

The acute effects of smokeless tobacco on transport and barrier function of buccal mucosa.

The acute effects of smokeless tobacco (ST) on buccal mucosal transport and barrier function were studied by means of in vivo and in vitro techniques. In humans, in vivo exposure to 0.5 g ST transiently increased the transmural electrical potential difference (PD). However, despite continued exposure, PD returned to baseline within 20 min. The mechanisms for these changes were explored by use of dog buccal mucosa mounted in Ussing chambers. Luminal exposure to a Ringer-extract of ST (EOST) increased PD and short-circuit current (Isc) and decreased electrical resistance (R), with changes reversible upon removal of EOST from the bath. Further, radioisotopic fluxes showed that the increase in Isc in EOST-exposed tissues was accompanied by increased absorption of electrolytes (Na, Cl, and other ions), and the decrease in R was accompanied by increased permeability to mannitol. Light microscopy of tissues exposed to EOST showed no morphological changes after exposure to 0.5 g of ST, but after exposure to 1.5-2.5 g of ST, dilated intercellular spaces were identified. Contact of aqueous media with ST led to the release of electrolytes and other soluble compounds into solution. To determine the effect of electrolyte release on buccal function, we exposed mucosae luminally to a solution with ion composition and/or osmolality similar to EOST or to one with an EOST previously dialyzed against Ringer. Solutions with similar ion composition and/or osmolarity changed PD, Isc, and R in a manner similar to EOST, while dialyzed-EOST had no effect. In addition, luminal nicotine produced effects different from EOST, decreasing PD and Isc and increasing R.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative investigation of human stratum corneum ceramides.

The stratum corneum (SC) requires ceramides, cholesterol, and fatty acids to provide the cutaneous permeability barrier. SC lipids can be analyzed by normal-phase high-performance thin-layer chromatography (HPTLC). However, without further analysis, some uncertainty remains about the molecular composition of lipids represented by every TLC band of an unknown sample. We therefore analyzed each ceramide band further by subjecting the isolated lipids to a direct coupling of reversed-phase high-performance liquid chromatography and electrospray ionization-mass spectrometry (HPLC/ESI-MS, or LC/MS). LC/MS analysis and ESI-MS/MS negative ion and collision-induced dissociation experiments revealed that ceramide band 4 contained not only N-(omega-OH-acyl)acyl-6-OH-sphingosine, Cer(EOH), but also N-(alpha-OH-acyl)-sphingosine. Band 5 exclusively contained N-acyl-6-OH-sphingosine. Our results demonstrate the benefit of LC/MS analysis for selective identification of human SC ceramides. Moreover, the combination of HPTLC for pre-separation and LC/MS for identification of lipids is an even more powerful tool for detailed ceramide analysis.

Coenzyme Q10, a cutaneous antioxidant and energizer.

The processes of aging and photoaging are associated with an increase in cellular oxidation. This may be in part due to a decline in the levels of the endogenous cellular antioxidant coenzyme Q10 (ubiquinone, CoQ10). Therefore, we have investigated whether topical application of CoQ10 has the beneficial effect of preventing photoaging. We were able to demonstrate that CoQ10 penetrated into the viable layers of the epidermis and reduce the level of oxidation measured by weak photon emission. Furthermore, a reduction in wrinkle depth following CoQ10 application was also shown. CoQ10 was determined to be effective against UVA mediated oxidative stress in human keratinocytes in terms of thiol depletion, activation of specific phosphotyrosine kinases and prevention of oxidative DNA damage. CoQ10 was also able to significantly suppress the expression of collagenase in human dermal fibroblasts following UVA irradiation. These results indicate that CoQ10 has the efficacy to prevent many of the detrimental effects of photoaging.

Subcutaneous splenosis.

Splenosis is the ectopic implantation of splenic tissue, usually as the result of trauma. Intraperitoneal, intrathoracic, and retroperitoneal sites of implantation have been reported. The authors report a case of subcutaneous splenosis involving the abdominal wall that was imaged with CT and scintigraphy.

Cytoprotection by sucralfate: role of sulfate ions.

Sucralfate and its component, sucrose octasulfate, are both SO4(2-)-containing compounds shown to protect against acid-peptic injury in rabbit and/or cat esophagi. To determine if sulfate ions (SO4(2-) contributed to this protection, a series of in vitro and in vivo experiments were performed in acid-exposed rabbit esophagi. In the Ussing chamber SO4(2-)-containing solutions significantly reduced the acid-induced decline in electrical resistance (R) observed in controls. This effect was unrelated to buffering of H+, accompanying cation or changes in luminal solution osmolality. Protection by SO4(2-) was specific since other divalent (HPO4(2-] or impermeant anions (gluconate-) failed to reduce the acid-induced decline in R. Protection was confirmed in vivo by showing that acid-perfused esophagi exposed to SO4(2-) had less morphologic damage, higher R and lower permeability to 14C-mannitol and H+ than controls. These results indicate that SO4(2-) have a unique protective action against acid injury to esophageal epithelia, and this action appears to explain the cytoprotective properties of sucralfate.

[Therapy-resistant depression with fatigue. A case of presumed streptococcal-associated autoimmune disorder]. / Therapieresistente Depression mit Fatigue. Fall einer vermutlichen streptokokkenassoziierten Autoimmunkrankheit.

After a tick bite and erythema chronicum migrans, a 31-year-old patient developed headaches, fatigue, multilocular pain and therapy-resistant depression with cognitive disturbances. Antibodies against Borrelia and Borna disease virus, high antibody titers against streptococci at the point of most severe depression and blood-CSF barrier dysfunction were found. Streptococcal antibody titers were normal 2 years before and 4 years after. With penicillin treatment and tonsillectomy, therapy-resistant depression improved. We suggest that the whole syndrome was streptococcal-associated autoimmune disease.

Mid-infrared spectroscopy on skin using a silver halide fibre probe in vivo.

BACKGROUND/AIM: Mid-infrared spectroscopy is a versatile method for in vivo investigation of skin after topical treatment with skin care products. METHODS: FTIR-spectrometer (Bruker Optics) with a flexible silver halide fibre probe (Infrared Fiber Sensors). RESULTS: Absorbance spectra from 700 to 3000 cm(-1) have been recorded to gain information about proteins (amide-I and amide-II vibrations at 1650 and 1550 cm(-1)), esters (1740 cm(-1)), carboxylic acid (1710 cm(-1)), polyalcohols (1050 cm(-1)) and hydrocarbons (CH(n) vibrations at 2800-3000 cm(-1)). CONCLUSIONS: Using the particular light guide, we were able to measure for the first time the effects of lip care products on lips directly. Furthermore, water binding and glycerol content of the skin could be determined simultaneously, as well as the replenishment of lipids by lipid-enriched bath oil.

[Ca2+]i and contraction of ear artery in response to rapid stimulation by NE: measurements with quin2 and fura-2.

The relationship between the quin2 and fura-2 fluorescence and isovolumetric contraction of smooth muscle cells of everted segments of the rabbit ear artery in response to rapid stimulation by norepinephrine (NE) was investigated. The resting level measured with quin2 (91 +/- 10 nM, n = 4) and with fura-2 (87 +/- 9 nM, n = 5) did not significantly differ. After addition of NE, an initial slow increase (ISI) in emission could be measured with quin2; however, Ca2+ buffering by this indicator slowed the increase of intracellular Ca2+ concentration ([Ca2+]i) and of contraction. When using fura-2, no ISI could be measured, but this indicator did not significantly interfere with the normal time course of contraction. Maximal response in [Ca2+]i to 1 microM NE was 424 +/- 30 nM (with quin2) and 337 +/- 46 nM (with fura-2). Mechanical latency depended not only on the onset but also on the initial rate of the increase in [Ca2+]i. Contractile response was quickest around 0.1 s after the onset of the fura-2 signal. During its rising phase, the rate of pressure development was linearly correlated with the -log molar Ca2+ concentration (pCa) of the cytoplasm (pCai), whereas during slow relaxation, pressure was linearly related to the pCai. These results suggest that contraction may depend not only on [Ca2+]i but also depend on its rate of change.

Intrasplenic venous thrombosis: CT findings.

OBJECTIVE: To describe the CT findings of intrasplenic venous thrombosis in three cases. MATERIALS AND METHODS: Three adult patients had abdominal CT indicative of short-segment thrombosis of an intrasplenic blood vessel. Two patients underwent splenectomy; thrombosis of a segmental splenic vein was confirmed histologically in both cases. RESULTS: Segmental splenic vein thrombosis appeared on CT as a short, 1-2 cm, linear lucency surrounded by normal splenic parenchyma. The probable etiology of intrasplenic venous thrombosis was main splenic vein occlusion related to pancreatitis in two cases and splenic compression and inflammation by perisplenic abscess in the third case. CONCLUSION: Thrombosis of segmental intrasplenic veins is detectable with CT and can be a secondary CT finding of main splenic vein thrombosis.

Hypogammaglobulinemia in McArdle myopathy (glycogenosis type V).

Glycogenosis type V (McArdle) was the first myopathy to be enzymatically defined myopathy and has been found in approximately 120 patients. It is characterized by a myophosphorylase defect. In 2 patients with completely missing phosphorylase activity, muscle fiber necrosis and creatinine kinase elevation, we found reproducibly low gammaglobulins and low immunoglobulin-G. Compared with 124 nonmyopathic control patients with hypogammaglobulinemia, we did not find any established cause for low gammaglobulins in either case of McArdle disease. Myopathies with selected laboratory features or histopathology in common did not show changes in gammaglobulins or immunoglobulins. Unaffected family members had normal gamma-globulins and immunoglobulins. Therefore, gammaglobulins indicate an immunologic involvement in phosphorylase deficiency, and a potential for genomic co-localization.

Corticosteroid-induced atrophy and barrier impairment measured by non-invasive methods in human skin.

BACKGROUND/AIMS: Atrophy is a distressing side effect of potent corticosteroids. After open application of a high potency steroid, we monitored atrophogenicity by a variety of non-invasive methods. METHODS: Volar forearms were treated twice daily for 3 or 4 weeks, with clobetasol propionate cream (Temovate). The following methods were used: 1) confocal microscopy, 2) transepidermal water loss (TEWL), 3) dimethyl sulfoxide whealing, 4) sodium hydroxide erosions, 5) analysis of stratum corneum lipids, and 6) B-scan ultrasound. RESULTS: Confocal microscopy revealed thinning of the epidermis, decreased microvasculature and decreased size of keratinocytes. Evaporimetry demonstrated transepidermal water loss. Whealing to dimethyl sulfoxide was enhanced. Sodium hydroxide erosions formed more quickly. The amount of ceramides, cholesterol, and free fatty acids was reduced. Ultrasound showed thinning of the dermis. CONCLUSION: Non-invasive methods are very useful for quantifying the atrophogenicity of topical corticosteroids.

Autologous blood clot seal to prevent pneumothorax at CT-guided lung biopsy.

PURPOSE: To determine whether the use of autologous blood clot seal (ABCS) after biopsy of lung lesions can reduce or prevent pneumothorax. MATERIALS AND METHODS: The authors evaluated 100 patients (63 men, 37 women; age range, 27-78 years) with pleural (n = 23) or deep (n = 77) lesions. Thirty-eight patients had emphysema. Patients were randomly assigned to one of two groups: those in whom the biopsy track was sealed with autologous blood clot (n = 50) and those who did not receive autologous blood clot (n = 50). Biopsy was performed with computed tomographic (CT) guidance and a 19-gauge coaxial system. The autologous blood clot, which ranged from 0.5 to 4.5 mL, was injected while the sheath was being withdrawn. RESULTS: Pneumothorax developed in four of the 23 patients (17%) with pleural lesions and 19 of the 77 patients (24%) with deep lesions. Pneumothorax occurred in four of the 45 patients (9%) who had deep lesions and received autologous blood clot and in 15 of the 32 patients (47%) who had deep lesions and did not receive autologous blood clot (P <.001). In patients with emphysema, pneumothorax occurred in three of the 20 patients (15%) who received autologous blood clot and 10 of the 14 (71%) who did not (P <.001). There were seven large pneumothoraces necessitating treatment; all occurred in patients who did not receive autologous blood clot. CONCLUSION: Plugging of biopsy tracks with ABCS, particularly after biopsy of deep lung lesions, significantly reduced the frequency of pneumothorax-particularly of large pneumothoraces-and, therefore, the need for treatment and the attendant cost.

Cytoprotective effect of sulfate ions in acid-exposed rabbit esophagus.

Sodium sulfate significantly inhibited the decline in epithelial electrical resistance (R) produced by mucosal acidification (pH 1.4) of rabbit esophagus mounted in the Ussing chamber. This protective effect was not due to the cation, to sodium loading, hyperosmolality, or pH change of the mucosal solution. Protection was specific for sulfate ions (SO2-4), since other divalent (HPO2-4) or impermeant anions (gluconate-) failed to prevent the acid-induced decline in R. In vivo studies in HCl-perfused rabbit esophagi confirmed protection by SO2-4. Tissues exposed to SO2-4 and HCl had higher R, lower permeability to H+ and mannitol, and less morphologic damage than controls exposed to HCl. These results suggest that SO2-4 have a unique protective action against acid injury to esophageal epithelia, and this action appears to explain the cytoprotective properties of sucralfate, a clinically effective agent for treating acid-peptic disease in humans.

Active electrolyte transport in mammalian buccal mucosa.

The transmural electrical potential difference (PD) was measured in vivo across the buccal mucosa of humans and experimental animals. Mean PD was -31 +/- 2 mV in humans, -34 +/- 2 mV in dogs, -39 +/- 2 mV in rabbits, and -18 +/- 1 mV in hamsters. The mechanisms responsible for this PD were explored in Ussing chambers using dog buccal mucosa. After equilibration, mean PD was -16 +/- 2 mV, short-circuit current (Isc) was 15 +/- 1 microA/cm2, and resistance was 1,090 +/- 100 omega.cm2, the latter indicating an electrically "tight" tissue. Fluxes of [14C]mannitol, a marker of paracellular permeability, varied directly with tissue conductance. The net fluxes of 22Na and 36Cl were +0.21 +/- 0.05 and -0.04 +/- 0.02 mueq/h.cm2, respectively, but only the Na+ flux differed significantly from zero. Isc was reduced by luminal amiloride, serosal ouabain, or by reducing luminal Na+ below 20 mM. This indicated that the Isc was determined primarily by active Na+ absorption and that Na+ traverses the apical membrane at least partly through amiloride-sensitive channels and exits across the basolateral membrane through Na+-K+-ATPase activity. We conclude that buccal mucosa is capable of active electrolyte transport and that this capacity contributes to generation of the buccal PD in vivo.

Serosal bicarbonate protects against acid injury to rabbit esophagus.

The role of serosal bicarbonate ions (HCO3-) in protection against acid injury was investigated in rabbit esophageal mucosa mounted in Ussing chambers. Luminal acidification reduced potential difference and resistance in tissues exposed serosally to HCO3- or (unbuffered) HCO3-free solution. Whereas resistance declined similarly in both groups, potential difference declined less in HCO3- solution. After washout, HCO3-bathed tissues also had a greater increase in resistance, lower permeability to mannitol, and less histologic damage. Furthermore, as protection by HCO3- was not blocked by pretreatment with either the anion exchange blocker, 4 acetamido-4'-isothiocyanatostilbene 2-2'-disulfonic acid, or the carbonic anhydrase inhibitor, acetazolamide, and replacement of HCO3- with N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid, a buffer impermeant to cells, was protective, an extracellular site for protection by HCO3- was likely. Where in the extracellular space HCO3- buffers H+ is unclear, but the absence of change in luminal pH and the inability to prevent the acid-induced increase in permeability in HCO3-bathed tissues argue against a luminal (preepithelial) site. Also, rapid repair was not demonstrated, indicating that a luminal site for protection after surface cell damage was unlikely. We conclude that serosal HCO3- is important in esophageal protection against acid damage by buffering H+ within the intercellular compartment of the extracellular space.