RESUMEN
Clearance and relapse characteristics of clobetasol lotion under hydrocolloid occlusion once weekly versus clobetasol ointment twice daily were assessed in a comparative flow cytometric study. Quantitative analysis of markers for epidermal proliferation, differentiation and inflammation was performed on epidermal single cell suspensions prepared from 3-mm punch biopsies taken from 15 patients with psoriasis vulgaris before therapy, at clearance and 6 weeks after clearance. After treatment both therapy regimens resulted in substantial changes of all flow cytometric parameters, but clearance was induced earlier in the corticosteroid under hydrocolloid occlusion-treated group. With respect to the relapse phase no difference was observed between both treatments. Although it is remotely possible that the outcome in the treatment of more extensive psoriatic lesions might be different, the present study suggests that the robust clinical efficacy of the treatment with a topical corticosteroid under hydrocolloid occlusion is not associated with a rebound phenomenon.
Asunto(s)
Antiinflamatorios/administración & dosificación , Clobetasol/administración & dosificación , Coloides/administración & dosificación , Citometría de Flujo , Psoriasis/tratamiento farmacológico , Administración Tópica , Antiinflamatorios/farmacocinética , Vendas Hidrocoloidales , Clobetasol/farmacocinética , Coloides/farmacocinética , Esquema de Medicación , Citometría de Flujo/métodos , Glucocorticoides , Humanos , Pomadas , Psoriasis/metabolismo , Psoriasis/patología , RecurrenciaRESUMEN
Recently a new approach for epidermal cell characterization was developed: three-parameter flow cytometrical analysis of pure and complete epidermal cell suspensions prepared from punch biopsies followed by dermoepidermal separation by thermolysin. The aim of the present communication is the comparison between psoriatic lesional skin and normal skin using this new approach with respect to the percentage of suprabasal keratinocytes (keratin 10+ cells), mesenchymal cells, including the infiltrate cells (vimentin+ cells) and the percentage of basal cells in SG2 M phase, in order to validate this methodology in studies on psoriatic skin. Punch biopsies were taken from 7 healthy volunteers and in 7 psoriatic patients 4 biopsies were taken in each of them from comparable lesions. The present study reconfirmed that the percentage of basal keratinocytes in psoriasis was increased and the percentage of keratin 10+ cells was substantially decreased as compared to normal skin. The new methodology revealed data with a narrow range. In psoriatic lesional skin the intra individual variation was less compared to the inter individual variation.
Asunto(s)
Separación Celular/métodos , Células Epidérmicas , Epidermis/patología , Citometría de Flujo/métodos , Psoriasis/patología , Estudios de Casos y Controles , Recuento de Células , Ciclo Celular , Epidermis/metabolismo , Humanos , Psoriasis/metabolismo , Vimentina/metabolismoRESUMEN
A parallel-group, double-blind, randomised study was performed to establish the effect of the vitamin D3 analogue KH 1060, in an ointment versus vehicle only, on the epidermal cell characteristics of chronic idiopathic lichen planus; KH 1060 also has marked immunosuppressive activity. A group of 10 patients were treated for 8 weeks with either KH 1060 ointment or vehicle only. In addition to the assessment of clinical scores, keratotome biopsies were taken before and after 8 weeks' treatment. Epidermal cell suspensions were prepared with trypsin and the suspensions incubated with TO-PRO-3 (DNA marker), RKSE 60 (marker for keratin 10-positive cells) and antivimentin (marker for all non-keratinocytes). In nine of the 10 patients, keratotome biopsies were obtained both before and after 8 weeks treatment. The vehicle alone had no significant effect on the clinical severity scores or epidermal cell characteristics. In contrast, the KH 1060 ointment resulted in a statistically significant reduction in the percentage of cells in S- and G2M phase and the percentage of vimentin-positive cells, but it did not affect the percentage of keratin 10-positive cells.
Asunto(s)
Calcitriol/análogos & derivados , Inmunosupresores/uso terapéutico , Liquen Plano/tratamiento farmacológico , Calcitriol/uso terapéutico , Método Doble Ciego , Femenino , Humanos , Queratinocitos/patología , Liquen Plano/patología , Masculino , Persona de Mediana Edad , Mitosis , Fase S , Piel/efectos de los fármacosRESUMEN
The aim of the present study has been to analyse remission and relapse characteristics in psoriasis vulgaris. In 15 patients, two different psoriatic lesions (clinical and flow cytometric study) were treated with clobetasol propionate until clearance for maximally 23 days. In the clinical study only cleared lesions were divided into three test sectors with different post-clearance treatment: (1) alcoholic solution under occlusion, (2) occlusion only, and (3) no treatment. In the flow cytometric study, biopsies were taken from the test lesion before clobetasol therapy (i), at clearance (ii), and at relapse from both visibly affected and unaffected skin (iii, iv). Epidermal proliferation, differentiation and inflammation were quantified by multiparameter flow cytometry. The clinical evaluation worked well and could discriminate between the different therapy modalities. After 28 days, 80% of untreated sectors showed a relapse. Occlusion decreased this percentage to 50%. Application of the alcoholic solution further decreased this percentage to 30%. The flow cytometric analysis demonstrated a very low proliferative activity of the basal compartment at clearance. This activity was higher in the visibly unaffected skin at relapse, whereas highest values were assessed in the affected skin at relapse. Interestingly, at relapse the proliferative activity in the suprabasal compartment of the visibly unaffected skin had increased to values identical to the affected skin. The present model allows standardized comparison of different approaches for maintenance therapy in psoriasis vulgaris. We demonstrate that occlusion has an inhibitory effect on the tendency to relapse after successful treatment with clobetasol propionate. Quantitative information on remission and relapse of psoriasis can be obtained by multiparameter flow cytometry.
Asunto(s)
Citometría de Flujo , Psoriasis/prevención & control , Psoriasis/terapia , Administración Tópica , Adolescente , Adulto , Anciano , Alcoholes/uso terapéutico , Antiinflamatorios/uso terapéutico , Biopsia , Clobetasol/uso terapéutico , Terapia Combinada , Femenino , Glucocorticoides , Humanos , Masculino , Persona de Mediana Edad , Apósitos Oclusivos , Psoriasis/patología , Recurrencia , Inducción de Remisión , Índice de Severidad de la Enfermedad , Piel/citología , Piel/efectos de los fármacos , Piel/patologíaRESUMEN
Objective comparison of different antipsoriatic therapies requires quantitative assessment of disease severity. However, clinical assessment with the widely used Psoriasis Area and Severity Index (PASI) introduces inaccuracy. An alternative is the quantitative analysis of different epidermal cell parameters using multiparameter flow cytometry. Our aim in the present study was to compare the clinical and flow cytometric approach to monitor disease activity and to evaluate antipsoriatic efficacy. Clinical scores for erythema, induration and scaling were assessed and biopsies for flow cytometric analysis were obtained from the psoriatic plaques of 89 patients before and after treatment with different therapeutic regimens consisting of vitamin D3 analogues and corticosteroids. In total, 219 epidermal cell suspensions were analysed using triple-labelling, with the simultaneous staining of markers for epidermal proliferation (DNA dye TO-PRO-3), differentiation (antikeratin 10), and inflammation (antivimentin). Correlation analysis was performed on 166 paired values obtained from 83 patients. A highly significant correlation was observed between erythema and the percentage of vimentin-positive cells, between scaling and the percentage of keratin 10 positive keratinocytes, and between induration and the number of basal keratinocytes in the S- and G2M phase, when all 166 biopsies were assessed. The correlation remained in the same range if the analysis was restricted to the 83 pretreatment biopsies. In contrast to the clinical scores, the flow cytometric analysis permitted a clear separation between the antiproliferative and anti-inflammatory or keratinization-enhancing effects of antipsoriatic treatment. The vitamin D3 analogues proved to exert a mainly antiproliferative effect. The combination of calcipotriol and a topical corticosteroid improved all cell biological markers substantially, and clobetasol monotherapy had a powerful effect on these markers. In conclusion, multiparameter flow cytometry has been shown to be a sensitive tool to evaluate the growth inhibiting, anti-inflammatory and keratinization-enhancing effects of antipsoriatic therapies.
Asunto(s)
Citometría de Flujo/métodos , Psoriasis/tratamiento farmacológico , Psoriasis/patología , Índice de Severidad de la Enfermedad , Biomarcadores , Ciclo Celular , Epidermis/patología , Eritema/tratamiento farmacológico , Eritema/patología , Humanos , Queratinas/metabolismo , Psoriasis/metabolismo , Resultado del Tratamiento , Vimentina/metabolismoRESUMEN
Calcipotriol and corticosteroids, two therapy modalities frequently prescribed in the treatment of psoriasis, are often used in combination. The aim of the present study was to determine whether the cell biological response pattern of concurrent use of calcipotriol and corticosteroids is different from calcipotriol monotherapy. Forty patients with chronic plaque psoriasis were divided at random in four parallel groups and treated for 8 weeks with: (1) calcipotriol cream (50 micrograms/g once daily); (2) calcipotriol cream twice daily; (3) calcipotriol and clobetasone 17-butyrate (0.5 mg/g) creams; and (4) calcipotriol and betamethasone 17-valerate (1 mg/g) creams. Before and after treatment keratotome biopsies were taken and single cell suspensions prepared for flow cytometric analysis. Flow cytometric multiparameter quantification of markers for proliferation (TO-PRO-3), differentiation (antikeratin 10) and inflammation (antivimentin) was used to evaluate all four therapy modalities. A statistically significant decrease of the percentage of basal cells in S- and G2M-phase (proliferation) was obtained with all therapy modalities, except for calcipotriol monotherapy applied once daily. A significant reduction of the number of vimentin-positive cells (non-keratinocytes) was observed following combined treatment with calcipotriol and clobetasone butyrate. In contrast, monotherapy with calcipotriol had virtually no effect on the number of vimentin-positive cells. It can be concluded that: (i) calcipotriol monotherapy, applied once daily was less antiproliferative compared with twice daily applications of calcipotriol or the combined treatment with corticosteroids and that (ii) the combination of calcipotriol and corticosteroids proved to have a marked effect on the percentage of non-keratinocytes, in contrast to the modest effect of calcipotriol.
Asunto(s)
Antiinflamatorios/uso terapéutico , Calcitriol/análogos & derivados , Fármacos Dermatológicos/uso terapéutico , Epidermis/efectos de los fármacos , Psoriasis/tratamiento farmacológico , Administración Tópica , Valerato de Betametasona/uso terapéutico , Calcitriol/farmacología , Calcitriol/uso terapéutico , División Celular/efectos de los fármacos , Clobetasol/análogos & derivados , Clobetasol/uso terapéutico , ADN/efectos de los fármacos , Fármacos Dermatológicos/farmacología , Esquema de Medicación , Quimioterapia Combinada , Citometría de Flujo , Glucocorticoides , Humanos , Queratinas/efectos de los fármacos , Psoriasis/patología , Vimentina/efectos de los fármacosRESUMEN
Reliable flow cytometric analysis of normal and diseased skin requires pure epidermal single-cell suspensions. Several methods to separate the dermis from the epidermis are available. The proteolytic enzyme thermolysin separates the epidermis from the dermis at the lamina lucida and therefore permits reliable dermoepidermal separation. In the present study an optimized cell isolation procedure using thermolysin and trypsin is described, which is particularly suitable for punch biopsies. A 16-20-h (overnight) incubation of biopsies taken from normal and hyperproliferative skin with thermolysin (0.5 mg/ml) at 4 degrees C produced a selective separation of the dermis and epidermis. After a 30-min trypsin incubation (0.25 mg/ml) at 37 degrees C a cell suspension was produced which was characterized by minimal cell damage (cellular debris and clumps), a high recovery of basal cells and high quality DNA histograms. Furthermore, dermal contamination was very low. The thermolysin-trypsin separation methodology followed by triple-labelling flow cytometry provided a precise quantification of the percentage of keratin 10-positive cells, vimentin-positive cells and cells in S and G2M phases. Proliferative activity was selectively measured in the basal, the suprabasal and the non-keratinocyte compartment at various time intervals during epidermal regeneration after adhesive tape stripping. In contrast to the non-keratinocytes, the percentage of cells in S and G2M phases in the basal keratinocytes and in the suprabasal compartment increased 44-48 h after stripping. The increased proliferation following tape stripping was paralleled by an increased invasion of vimentin-positive cells into the epidermis and preceded by a decreased number of keratin 10-positive cells. Thermolysin-trypsin separation followed by three-colour flow cytometry permits a highly selective characterization of normal and hyperproliferative epidermis.
Asunto(s)
Técnicas Citológicas , Epidermis/patología , Citometría de Flujo/métodos , Psoriasis/patología , Adolescente , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Valores de Referencia , Manejo de Especímenes/métodos , Termolisina , TripsinaRESUMEN
Leukotriene B4 (LTB4) is a potent chemoattractant and a well-established stimulator of DNA-synthesis in keratinocytes. Previously, repeated applications of LTB4 have been reported to induce a topically defined tachyphylaxis with respect to the extravasation of polymorphonuclear neutrophils. The aim of the present study was to quantify epidermal proliferation (% basal keratinocytes in S- and G2M phase), epidermal keratinization (% keratin 10-positive keratinocytes) and the appearance of "non-keratinocytes", including melanocytes, Langerhans' cells and infiltrate cells (% vimentin-positive cells) in order to further elucidate the effect of chronic exposition of normal skin to LTB4. Using three-colour flow cytometry, we could reconfirm that the response to one single epicutaneous application of LTB4 was characterized by a marked increase of the percentage of basal keratinocytes in S- and G2M phase, and a marked increase of non-keratinocytes. Repeated applications of LTB4 induced a moderate increase of the percentage of cells in S- and G2M phase and a moderate increase of the percentage of keratin 10-positive keratinocytes. Remarkably, the percentage of non-keratinocytes had decreased following repeated applications of LTB4, compared to unchallenged normal skin. The present study suggests that chronic exposure of normal skin to LTB4 induces changes which differ markedly from the histological features of the chronic psoriatic lesion. Therefore, LTB4 is unlikely to be responsible for the perpetuation of the psoriatic plaque.
Asunto(s)
Factores Quimiotácticos/farmacología , Leucotrieno B4/farmacología , Piel/citología , Piel/efectos de los fármacos , Adulto , División Celular , Separación Celular , Factores Quimiotácticos/administración & dosificación , Femenino , Citometría de Flujo , Humanos , Queratinocitos/citología , Leucotrieno B4/administración & dosificación , MasculinoRESUMEN
The clinical efficacy and tolerability of the vitamin D3 analogues calcitriol, calcipotriol and 1 alpha, 24 dihydroxyvitamin D3 in the treatment of psoriasis have been assessed in various clinical studies. In vitro and in vivo investigations have shown interference of these compounds with epidermal growth, keratinisation and inflammation. In this study we quantified the in vivo cell biological effects during treatment of psoriatic plaques with 1 alpha, 24 dihydroxyvitamin D3. By using a flow cytometric triple labelling procedure, we could discriminate different epidermal subpopulations, permitting precise assessment of epidermal cell cycle kinetics. Twenty patients with plaque-type psoriasis were treated in a double-blind placebo-controlled left-right comparative study with 1 alpha, 24 dihydroxyvitamin D3 ointment (4 micrograms/g applied once daily) for 8 weeks. Epidermal cell suspensions prepared from keratotome biopsies taken before and after treatment were stained with TO-PRO-3 iodide (a new DNA fluorochrome) and monoclonal antibodies against keratin 10 (as a marker for differentiation) and vimentin (as a marker for inflammation), simultaneously. The flow cytometric analyses showed a significant decrease of proliferating basal keratinocytes in verum-treated lesions, whereas such a decrease was not observed in placebo-treated lesions. The amount of keratin 10-positive keratinocytes increased and the presence of vimentin-positive cells decreased in cell suspensions derived from both verum- and placebo-treated lesions, but these effects were not significant. We conclude that multiparameter flow cytometry promises to be an adequate approach to assess the interference of antipsoriatic treatments with cutaneous inflammation, epidermal proliferation and keratinisation. Topical 1 alpha, 24 dihydroxyvitamin D3 seems to exert its in vivo antipsoriatic effect mainly through an inhibition of epidermal growth.
Asunto(s)
Fármacos Dermatológicos/uso terapéutico , Dihidroxicolecalciferoles/uso terapéutico , Psoriasis/tratamiento farmacológico , Administración Cutánea , Adulto , Anciano , Biopsia , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , ADN/análisis , Fármacos Dermatológicos/administración & dosificación , Dihidroxicolecalciferoles/administración & dosificación , Método Doble Ciego , Epidermis/efectos de los fármacos , Epidermis/patología , Femenino , Citometría de Flujo , Colorantes Fluorescentes , Humanos , Inflamación , Queratinas/análisis , Masculino , Persona de Mediana Edad , Placebos , Psoriasis/patología , Vimentina/análisisRESUMEN
A new red emitting fluorophore, TO-PRO-3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two-laser system. As an example, we present a three-color flow cytometric application utilizing TP3 in combination with fluorescein-isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper. A subequilibrium concentration of 1 microM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human- and mouse-Balb/MK-keratinocyte populations with different S-phase fractions, PI and TP3 showed a good correlation. Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously.
Asunto(s)
ADN/análisis , Citometría de Flujo/métodos , Coloración y Etiquetado , Animales , Células Cultivadas , Sondas de ADN , Estudios de Evaluación como Asunto , Fluoresceína-5-Isotiocianato , Colorantes Fluorescentes , Humanos , Queratinocitos/química , Rayos Láser , Linfocitos/química , Ratones , Ficoeritrina , PropidioRESUMEN
Published data (mainly from rodent skin) suggest a correlation between compounds which inhibit protein kinase C (PKC), have anti-inflammatory or antitumor characteristics and possess antipsoriatic potential. We have investigated the effects of topical application of sphingosine (a naturally occurring PKC inhibitor), isoquinoline (a component of coal tar which showed antipsoriatic capacities in the mouse tail model) and tannic acid (a plant phenol with antitumor activity) on human skin. In each case we have assessed (a) the level of induction of ornithine decarboxylase (ODC) following Sellotape stripping as an indicator for potential PKC inhibition in vivo, and (b) its effects on the lesions of chronic plaque psoriasis. The control group consisted of 18 healthy volunteers, used for the ODC induction experiments (0.0/0.1/0.2 M sphingosine in ethanol, 100% coal tar and 0/50 mM tannic acid in acetone) and 17 psoriatic patients used for double-blind scoring of two randomly selected lesions (0.0/0.1 M sphingosine in ethanol, 0.0/0.2% isoquinoline in white vaseline/lanette wax cream 50%/50% and 0/10% tannic acid in lanette wax cream) and also for some of the ODC induction experiments (0.0/0.2% isoquinoline and 0/10% tannic acid). Biopsies were taken 8 h after stripping and ODC activity was assessed by measurement of 14CO2 release. Lesions were scored with a modified psoriasis area and severity index on days 0, 7 (isoquinoline and tannic acid), 13 (sphingosine) and 21 (isoquinoline and tannic acid). Application of 0.1 or 0.2 M sphingosine resulted in a decrease of ODC activity of 52% and 66%, respectively (p < 0.01), but histologic sections showed intraepidermal necrosis.(ABSTRACT TRUNCATED AT 250 WORDS)