Expression of transforming growth factor type beta on human epidermal dendritic cells.

Our study demonstrates that epidermal Langerhans cells (LC) of healthy human skin produce Transforming growth factor-beta (TGF-beta). This multipotent mediator might keep the immunocompetent LC in an "immature" stage, in which they are able to capture and process antigen. Development to potent antigen-presenting cells would become possible after migration from the epidermal network to the draining lymph node to stimulate (auto-)reactive T-cell clones.

Skin reactions and itch sensation induced by epicutaneous histamine application in atopic dermatitis and controls.

Itch sensations and skin reactions induced by histamine iontophoresis at six different current intensities were studied in 27 atopic dermatitis (AD) patients and 20 healthy controls. Subjective itch ratings were assessed on a visual analogue scale (VAS) for 8-min periods after 10-sec histamine application, while changes of skin blood flow were simultaneously measured using two Laser Doppler flowmeters. Ten minutes after each histamine application, the areas of wheal and flare reactions were planimetrically evaluated. When no or weak current was applied, AD patients revealed stronger wheal and flare reactions than controls, possibly due to disturbed skin barrier function. Higher histamine doses, however, produced weaker subjective and vascular reactions in AD patients. In contrast to the controls, AD patients were unable to distinguish between weak and strong histamine stimulation, as shown by their VAS ratings. These results imply that AD patients have an altered histamine response. In particular, their afferent cutaneous nerve fibers show a decreased ability to signal itching to the central nervous system and to release vasoactive neuropeptides upon histamine stimulation.

Segmental cell kinetics of the human anagen hair: a DNA-flow cytometric analysis.

The cell kinetics of anagen scalp hair taken by punch biopsies from 70 healthy male volunteers were determined at nine different defined bulbar and follicular hair segments using microdissection and DNA-flow cytometry. The highest mean proliferative activity (S-phase) was measured within the lowermost bulbar segment (14.0%), but decreased to 7.6% at Auber's segment and to 5.9% at the follicle isthmus. Notably, the S-phase data of the upper follicular segments (subdermal 2.4%, infundibular 2.4%) were found to be similar to those of the epidermis (2.5%). This study supporting and supplementing former autoradiographic investigations on human hair matrix epithelium clearly demonstrates the main proliferative activity of the anagen hair follicle being localized in the bulbar segments below Auber's level. Moreover, the method described is well suited for studying the effects of agents influencing cell growth (e.g., hormones or drugs) on the cell kinetics of different anagen hair compartments.

Microcirculatory functions in systemic sclerosis: additional parameters for therapeutic concepts?

To study the functional reactivity of the cutaneous microcirculation in progressive systemic sclerosis (PSS), hyperemic responses after arterial occlusion (3 min) and during local heating (42 degrees C) were investigated with simultaneous measurements of red blood cell flux and cutaneous oxygen tension (pcuO2) of the skin in female patients (n = 19) with PSS and in healthy female controls (n = 15). Additionally, serum levels of 6-keto-prostaglandin 1 alpha (PGF1 alpha), a stable metabolite of prostacyclin, were compared to the microcirculatory data, and both were used to evaluate further a standardized therapy with 10-d intravenous calcitonin (100 IU/d) infusion in six PSS patients. In PSS, the initial mean pcuO2 value was significantly reduced and was inversely proportional to flux and to PGF1 alpha levels, whereas the flux and pcuO2 responses to the above hyperemic stimuli showed significant reductions, revealing a pattern of "hyperemic hypoxia" probably due to exhausted functional reserves of cutaneous perfusion. During calcitonin infusion significant rises in pcuO2 and temporarily in PGF1 alpha and flux were found. After 10 d of therapy, increased pcuO2 was associated significantly with decreased flux, indicating a shifting of blood from deeper regulatory vessels to the subepidermal capillaries. Both clinical improvement and the results of microcirculatory measurements demonstrate a beneficial effect of calcitonin on the cutaneous microcirculation in PSS patients, possibly due in part to a short-term increase in release of endogenous prostacyclin from the vascular endothelium during the infusion. The disturbed reactivity of the dermal vessels in PSS is important for the evaluation of therapeutic concepts and stresses, together with the elevated PGF1 alpha plasma levels, vascular factors in the pathogenesis of PSS.

Growth and cell kinetics of human hair papilla cells in vitro. An autoradiographic and flow cytometric study.

Hair papilla, a distinct specialized dermal compartment, plays a fundamental role in the biology of hair growth. Recently some attention has been focused on hair papilla cells (HPC) as possible targets for drugs influencing the hair growth. Isolation and cultivation of the HPC facilitates screening for such drugs. In the present work, growth and cell kinetics of human occipital scalp follicle HPC have been studied in vitro. HPC grow according to a Gompertz function, i.e. with considerable growth delay long before becoming confluent cultures, due probably to elongation of the potential doubling time (Tpot) and to a parallel increasing cell loss rate. The [3H]dT labelling index of the HPC strongly depends on the age of the subculture; the cycle time being about 4 days. A potential doubling time of about 93 h, indicative of growth fraction (GF) = 1, and a duration of S phase and G2 + M phase of about 8 h each were found by the combined application of continuous labelling with [3H]dT and DNA flow cytometry.

Cortisol, corticotropin, and beta-endorphin responses to corticotropin-releasing hormone in patients with atopic eczema.

Alterations in the hypothalamic-pituitary-adrenal (HPA) system are well documented in affective disorders. In depression these include increased secretion of cortisol, an insufficient suppressibility of cortisol by dexamethasone, a blunted corticotropin (ACTH) response to corticotropin-releasing hormone (CRH) and a dysfunction of the glucocorticoid receptor. Patients with atopic eczema, a common chronic skin disease, show seasonal variations in disease activity, symptoms of minor depression and immunological disturbances similar to those seen in patients with depression. To explore the integrity of the HPA system integrity in individuals with atopic eczema we studied the 24-h cortisol secretion and the cortisol, ACTH and beta-endorphin responses to CRH in such individuals and in healthy controls matched for sex and age. The 24-h secretion of cortisol did not differ between the patients with atopic eczema and the controls. The net response to CRH administered as a 100 micrograms i.v. bolus was significantly attenuated for both cortisol (24,235 +/- 12,443 vs. 47,019 +/- 34,515 nmol.min/dl; p < .03) and for ACTH (546 +/- 205 vs. 727 +/- 310 pmol.min/l; p < .05) in the patient group, whereas the beta-endorphin response did not differ between the groups (1072 +/- 448 vs. 1603 +/- 421 nmol.min/l). The blunted response of cortisol and ACTH cannot be explained by hypercortisolism as it is the case in major depression. Rather, it may be related to a prolonged underexposure to hypothalamic CRH or to an increased sensitivity of glucocorticoid feedback inhibition.

In situ [3H]thymidine labelling of human hair papilla: an in vitro autoradiographic study.

The labelling index (LI) and the location of labelled cells in the papilla of human anagen scalp hair have been studied after in vitro [3H]thymidine (3H-TdR) pulse labelling. Forty-three anagen stage VI papillae from 2 individuals (both females, 25 and 55 years) obtained by micropreparation from deep scalp biopsies were analysed. Labelled cells were found only paraaxially in the basal third of the papilla, i.e. below Auber's line. The 3H-TdR-LI of the papilla cells (0.13 and 0.16%) is about 200-times lower than that of the hair matrix cells below the line of Auber (25.5 and 29.5%). The biological significance of this low proliferation rate in the anagen stage VI hair papilla of healthy human should be defined.

Physical stress-induced secretion of adrenal and pituitary hormones in patients with atopic eczema compared with normal controls.

Atopic eczema is a chronic inflammatory skin disease which shares some psychological and neuroendocrine disturbances with patients suffering from depression. In view of recent findings of an attenuated response of the hypothalamic-pituitary-adrenal (HPA) system in patients with atopic eczema during a human corticotropin-releasing hormone (hCRH) challenge paradigm fourteen consecutive non-specifically trained in-patients with atopic eczema (8 men, 6 women) and an age-matched control group (8 men, 6 women) performed exhausting incremental graded bicycle exercise to evaluate cortisol, adrenocorticotropin (ACTH), beta-endorphin, epinephrine and norepinephrine releases induced by physical stress. The exercise yielded significant increases in cortisol, ACTH, beta-endorphin, epinephrine and norepinephrine concentrations in both groups. Patients with severe eczema displayed a significantly lower increase in norepinephrine levels when compared with the less affected patient group. In contrast to the challenge with exogenous hCRH no substantial difference in the net responses of ACTH and cortisol could be detected between patients with atopic eczema and controls using the physical stress paradigm. These substantial differences in the net outcome between both challenges may be related to the potential synergizing effects of various neuropeptides, e.g. CRH and vasopressin, when activating the HPA system by challenges at a suprapituitary site which may override subtle disturbances in the responsivity of the HPA system as revealed by CRH challenge alone in patients with atopic eczema.

Autoradiographic study on some proliferative properties of human buccal mucosa.

This study was designed to investigate the proliferative activity of the buccal mucosa in healthy adult persons with regard to individual age. Punch biopsy specimens were taken from the buccal mucosa of 19 individuals (10 females, 9 males) ranging in age from 24 to 80 years, and were then examined autoradiographically, applying nuclear 3H-thymidine labelling in vitro under oxygen pressure of 2.2 atm. The mean total labelling index in the progenitor compartment amounted to 26.0 +/- 2.6% including 6.2 +/- 2.0% labelled basal nuclei and 19.8 +/- 2.6% labelled suprabasal nuclei. Related to 1,000 mu surface length, the mean number of basal cells runs up to 270.7 +/- 38.6 and the total nuclear labelling index to 70.9 +/- 10.4. This high rate of S phase nuclei, exceeding that of the epidermis several times, depends on the ample progenitor compartment as well as on the basal epithelium length which, as compared to the epithelium surface length, was shown to be extended by a mean proportion of 1.75. None of the autoradiographic results obtained in the present study revealed an age dependency of the buccal mucosa proliferative activity. The magnitude of the proliferative pool constitutes the matrix of the high renewal rate of the healthy buccal mucosa and provides a steady state of superficial cell loss and basal cell regeneration, that was proved, at least in the normal mucosa, to continue until senile age.

Effects of plucking on the anatomy of the anagen hair bulb. A light microscopic study.

The effects of mechanical plucking on the anatomy of human anagen hair bulbs were studied histologically in biopsy specimens taken from scalp areas of ten volunteers immediately after plucking. Anagen hair bulbs were shown to tear off not arbitrarily but in reproducible patterns which include, apart from the "typical" break conically surrounding the dermal papilla, three additional break forms: (1) rupture of the hair around the upper third of the papilla resulting in so-called dysplastic anagen hairs of the trichogram, (2) rupture of the hair well above the dermal papilla resulting in "broken" anagen hairs, (3) total removal of the proximal follicle epithelium with removal of the dermal papilla resulting in so-called papilla hairs of the trichogram. Plucking also gives rise to alterations of the mesenchymal sheath of the hair follicle mainly leading to hemorrhages and a distinct edema entailing an increase in the volume of both the dermal papilla and the underlying "papilla cushion" of Pinkus. The different break types can be due to inappropriate plucking techniques or may depend on different subphases of the anagen stage.

In vitro studies on microphage functions in chronic pyoderma vegetans.

The function of microphages has been studied in two patients with chronic pyoderma vegetans by in vitro determination of phagocytosis as well as chemotaxis. The results showed a striking decrease in the chemotactic activity of the neutrophil granulocytes, a reduced phagocytosis of Candida albicans and Staphylococcus aureus, and a weakness of the intracellular killing of these microorganisms. However, the NADH-dependent oxidase activity appeared to be intact. No defect was found in the specific cellular or humoral immune system in either patient.

In vitro studies on microphage functions in chronic pyoderma vegetans.

The function of microphages has been studied in two patients with chronic pyoderma vegetans by in vitro determination of phagocytosis as well as chemotaxis. The results showed a striking decrease in the chemotactic activity of the neutrophil granulocytes, a reduced phagocytosis of Candida albicans and Staphylococcus aureus, and a weakness of the intracellular killing of these microorganisms. However, the NADH-dependent oxidase activity appeared to be intact. No defect was found in the specific cellular or humoral immune system in either patient.

Flow cytometric measurement of ploidy and proliferative activity of carcinomas of the oropharyngeal mucosa.

The most important indicator of ploidy and cell cycle stage in ortho- and pathologic tissues is the nuclear DNA content. To study this parameter of malignancy in squamous carcinomas of the oral, pharyngeal, or laryngeal mucosa we analyzed DNA histograms by aid of flow cytophotometry after using pepsin digestion of tumor specimens for cell dispersal and fluorochromation with combined ethidium bromide and mithramycin. In 30 of 36 tumor tissue specimens aneuploid states of nuclear DNA content (83%) were recognized, in some cases only by comparing tumor cells with admixed diploid human reference cells. All but two tumors (with hypodiploid DNA pattern) exhibited hyperdiploid stem line abnormalities, one specimen even exhibited them with triclonal DNA distribution pattern. The degree of ploidy (DNA index), defined as the ratio of peak modal channel number for the G1/0 proportion of tumor cells to that of normal cells, ranged from 0.59 to 3.24 (mean 1.58). Cell cycle analysis of untreated squamous carcinomas, calculated by the relative DNA distribution pattern in histograms, also offered a considerable variation in proliferative activity.

Human papillomavirus 25-related DNA in solitary keratoacanthoma.

Solitary keratoacanthomas of 32 patients were screened for the presence of human papillomavirus (HPV) 25 DNA, which was originally isolated and molecularly cloned from warts of an epidermodysplasia verruciformis (Ev) patient. Biotinylated virus DNA was hybridized in situ to thin sections obtained from paraffin-embedded material. HPV DNA was detected in 12 of 32 tumors under stringent conditions, and in 2 additional tumors under relaxed conditions.

Plasma levels of cyclic nucleotides are elevated in atopic eczema.

Plasma cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) taken at distinct times of the day were measured in 45 inpatients with generalized atopic eczema (AE). The results were compared with 34 healthy controls. Female patients not taking oral contraceptives (OC) and male patients revealed significantly higher median levels of cAMP in comparison with the controls (p less than 0.002 for males; p less than 0.001 for females). No significant cAMP differences were found between patients of either sex and female controls taking OC. The median cGMP plasma levels did not differ between patients and controls. Both cyclic nucleotides are discussed under the aspect of cAMP as a second messenger of various hormones, reflecting and integrating different responses of the body to AE as a distressing systemic skin disorder.

Enhancement of granulocyte chemotaxis in Behcet's disease.

The humoral and cellular immune status, the cutaneous pathergy, PMNL in vitro function tests as well as clinical and general laboratory examination were performed and evaluated on ten patients suffering from Behcet's disease (BD) and ten other patients with benign recurrent aphthosis (RA). The same set of in vitro tests (except PMNL exposure to solutions of copper sulfate or DDT) was carried out on a control group consisting of more than 100 healthy male and female individuals of various ages. Apart from general signs of inflammatory activity (leukocytosis, elevation of ESR, increased IgG serum levels), cutaneous pathergy to mild local highly injuries and marked enhancement of PMNL chemotaxis proved to be highly significant symptoms in acute phases of BD in contrast to benign RA. Both symptoms, in particular the hyperchemotaxis of PMNL, can be regarded as valuable diagnostic means in the early detection of beginning or atypical BD.

Impairment of chemotactic activity of microphages in chronic mucocutaneous candidosis.

The chemotactic microphage function in combating and eliminating microorganisms is one of the most important features of the cellular immune system. Using a modified Boyden method we studied in vitro the chemotactic activity of the granulocytes in 5 patients suffering from chronic muco-cutaneous candidosis (CMCC), 3 of the familial and 2 of the non-familial type. To obtain quantitative results comparable to those of a control group, we investigated the granulocytes of 55 clinically and immunologically healthy persons by the same way. So we found, in comparison with the results of the controls, a striking reduction of the chemotactic granulocyte activity in all CMCC patients. This impairment of chemotaxis is apparently due to an inborn defect of the microphages. It is pointed out, however, that a non-specific stimulation of the immune system might be a promising way in the treatment of patients affected by CMCC.

Collagen-type synthesis in human-hair papilla cells in culture.

Hair-papilla derived cells were grown in monolayer culture and revealed the typical morphology and growth pattern which was similar but not identical to control fibroblasts. Hair-papilla cells were found to produce considerable amounts of collagen type I and type III and fibronectin. Type IV collagen production could not be detected. The ratio of collagen type III and type I clearly differed from the pattern observed in normal fibroblasts, being much higher in hair-papilla cells, where type III accounted for more than 20% of total collagen synthesis. These data show that hair-papilla derived cells have biosynthetic capacities similar to those of human skin fibroblasts as well as characteristic differences, indicating that they represent a specialized fibroblast subpopulation.

Comparative morphological and growth kinetics studies of human hair bulb papilla cells and root sheath fibroblasts in vitro.

Dermal papillae isolated from anagen hair bulbs obtained from biopsy specimens from five subjects with normal hair pattern, and fibroblasts derived from the mesenchymal root sheaths (RSF) of the same hair follicles were separately grown in culture and the cell-cycle distribution pattern on different days was analyzed by applying DNA flow cytometry (FCM). Papilla cells (PC) exhibited distinctive morphological features by forming cell aggregates differing from RSF with respect to cell shape and growth pattern. They also proliferated remarkably more slowly than RSF. DNA-FCM analysis showed that both PC and RSF demonstrated synchronous fluctuations in the percentage of cells in G1/0, S and G2 + M phases during the period of subculture.

Effects of epidermal growth factor, fibroblast growth factor, minoxidil and hydrocortisone on growth kinetics in human hair bulb papilla cells and root sheath fibroblasts cultured in vitro.

Comparative studies on growth kinetics of cultivated human hair bulb papilla cells (PCs) and hair root sheath fibroblasts (RSFs) yielded evidence of some peculiarities of PCs in both proliferative behavior and morphological growth pattern. As the dermal papilla, essentially supporting the nutrition of matrix epithelium, can be considered a target tissue for agents influencing maintenance of hair growth, we studied the effects of epidermal growth factor (EGF), fibroblast growth factor (FGF), minoxidil (Mino), and hydrocortisone (HC) on the proliferation of PCs and RSFs, both gained from dissected hair follicles of scalp biopsy specimens of two male adults and separately cultured in vitro. EGF and FGF proved to increase proliferation of both PCs and RSFs most, yet at a different intensity for each cell group. HC slowed proliferation, and Mino failed to influence growth of PCs and RSFs.