SAFESTEREO: phase II randomized trial to compare stereotactic radiosurgery with fractionated stereotactic radiosurgery for brain metastases.

BACKGROUND: Stereotactic radiosurgery (SRS) is a frequently chosen treatment for patients with brain metastases and the number of long-term survivors is increasing. Brain necrosis (e.g. radionecrosis) is the most important long-term side effect of the treatment. Retrospective studies show a lower risk of radionecrosis and local tumor recurrence after fractionated stereotactic radiosurgery (fSRS, e.g. five fractions) compared with stereotactic radiosurgery in one or three fractions. This is especially true for patients with large brain metastases. As such, the 2022 ASTRO guideline of radiotherapy for brain metastases recommends more research to fSRS to reduce the risk of radionecrosis. This multicenter prospective randomized study aims to determine whether the incidence of adverse local events (either local failure or radionecrosis) can be reduced using fSRS versus SRS in one or three fractions in patients with brain metastases. METHODS: Patients are eligible with one or more brain metastases from a solid primary tumor, age of 18 years or older, and a Karnofsky Performance Status ≥ 70. Exclusion criteria include patients with small cell lung cancer, germinoma or lymphoma, leptomeningeal metastases, a contraindication for MRI, prior inclusion in this study, prior surgery for brain metastases, prior radiotherapy for the same brain metastases (in-field re-irradiation). Participants will be randomized between SRS with a dose of 15-24 Gy in 1 or 3 fractions (standard arm) or fSRS 35 Gy in five fractions (experimental arm). The primary endpoint is the incidence of a local adverse event (local tumor failure or radionecrosis identified on MRI scans) at two years after treatment. Secondary endpoints are salvage treatment and the use of corticosteroids, bevacizumab, or antiepileptic drugs, survival, distant brain recurrences, toxicity, and quality of life. DISCUSSION: Currently, limiting the risk of adverse events such as radionecrosis is a major challenge in the treatment of brain metastases. fSRS potentially reduces this risk of radionecrosis and local tumor failure. TRIAL REGISTRATION: ClincalTrials.gov, trial registration number: NCT05346367 , trial registration date: 26 April 2022.

The time to progression ratio: a new individualized volumetric parameter for the early detection of clinical benefit of targeted therapies.

BACKGROUND: Early signs of efficacy are critical in drug development. Response Evaluation Criteria in Solid Tumors (RECIST) are commonly used to determine the efficacy of anti-cancer therapy in clinical trials. RECIST, however, emphasizes the value of tumor shrinkage, while many targeted agents induce prolonged tumor growth arrest. This limits its use for the detection of treatment efficacy for these more cytostatic regimens. Therefore, we designed an individualized variant of a time to progression (TTP) end point based on prospective volumetric measurements and an intra-patient control, the TTP ratio. PATIENTS AND METHODS: Patients with any metastatic malignancy, without regular treatment options, were treated with the mTOR inhibitor everolimus. Treatment response was determined using both RECIST and the TTP ratio. The TTP ratio was defined as the volumetric pretreatment TTP divided by the volumetric on-treatment TTP. A patient was classified as a responder if the TTP ratio was <0.7. Consistency and reproducibility of volumetric measurements were determined. RESULTS: Seventy-three patients were included of whom 59 started treatment. A TTP ratio could be established in 73% (n = 43) of the treated patients. The inter-observer agreement for volumetric progression was 0.78 (95% confidence interval 0.70-0.87) (Krippendorff's α-coefficient). According to RECIST, 35 patients (59%) had stable disease (SD) and 1 patient demonstrated a partial response (PR), whereas only 21 patients (36%) met the prespecified criteria for treatment efficacy according to the TTP ratio. Treatment response according to both the TTP ratio and RECIST (SD + PR) correlated with overall survival (OS) [P(log-rank) < 0.001]. The TTP ratio, however, was also able to differentiate which patients had a better OS within the RECIST SD group [P(log-rank) = 0.0496]. CONCLUSION: The TTP ratio had a high inter-observer agreement, correlated with OS and identified which patients within the RECIST SD group had a longer OS. CLINICALTRIALSGOV IDENTIFIER: NCT01566279.

Pharmacokinetically guided sunitinib dosing: a feasibility study in patients with advanced solid tumours.

BACKGROUND: Plasma exposure of sunitinib shows large inter-individual variation. Therefore, a pharmacokinetic (PK) study was performed to determine safety and feasibility of sunitinib dosing based on PK levels. METHODS: Patients were treated with sunitinib 37.5 mg once daily. At days 15 and 29 of treatment, plasma trough levels of sunitinib and N-desethyl sunitinib were measured. If the total trough level (TTL) was <50 ng ml(-1) and the patient did not show any grade ⩾3 toxicity, the daily sunitinib dose was increased by 12.5 mg. If the patient suffered from grade ⩾3 toxicity, the sunitinib dose was lowered by 12.5 mg. RESULTS: Twenty-nine out of 43 patients were evaluable for PK assessments. Grade ⩾3 adverse events were experienced in seven patients (24%) at the starting dose and in nine patients (31%) after dose escalation. TTLs were below target in 15 patients (52%) at the starting dose. Of these, five patients (17%) reached target TTL after dose escalation without additional toxicity. CONCLUSIONS: In a third of the patients that were below target TTL at standard dose, the sunitinib dose could be increased without additional toxicities. This could be the basis for future studies and the implementation of a PK-guided dosing strategy in clinical practice.

Environmental attributes related to walking and bicycling at the individual and contextual level.

OBJECTIVE: The aim of the present paper was to give insight into the practical consequences of using either single-level or multilevel regression analyses on data from research on environmental determinants of physical activity. METHODS: For this purpose, results from single-level and multilevel regression analyses on comparable attributes of the environment were compared using a combination of individual and aggregated data, merged at the neighbourhood level. RESULTS: Using only individual level data, applying multilevel instead of single-level analyses did not substantially influence the results. However, ignoring the multilevel structure of our data by applying single-level in stead of multilevel analyses resulted in statistically significant associations for all the environmental attributes under study. Moreover, using information on environmental attributes at both the individual and the contextual level to adjust associations at one level for the other level showed that associated environmental attributes tend to be associated either at the individual or at the contextual level. CONCLUSIONS: These results stress the importance for reviews and meta-analyses of recording type of measurement and type of analytical strategy used and incorporating them in the review process. Using advanced multilevel designs will still only partly solve the methodological issues involved in studying environmental attributes associated with physical activity, but it will help in disentangling this complex relationship. Therefore, it is recommended that, whenever there is a presumably relevant grouping (context; eg neighbourhoods) in a study, a multilevel approach should at least be considered.

Higher mortality in urban neighbourhoods in The Netherlands: who is at risk?

BACKGROUND: Urban residents have higher mortality risks than rural residents. These urban-rural differences might be more pronounced within certain demographic subpopulations. AIM: To determine urban-rural differences in all-cause and cause-specific mortality within specific demographic subpopulations of the Dutch population. METHOD: Mortality records with information on gender, age, marital status, region of origin and place of residence were available for 1995 through 2000. Neighbourhood data on address density and socioeconomic level were linked through postcode information. Variations in all-cause and cause-specific mortality between urban and rural neighbourhoods were estimated through Poisson regression. Additionally, analyses were stratified according to demographic subpopulation. RESULT: After adjustments for population composition, urban neighbourhoods have higher all-cause mortality risks than rural neighbourhoods (RR = 1.05; CI 1.04 to 1.05), but this pattern reverses after adjustment for neighbourhood socioeconomic level (RR = 0.98; CI 0.97 to 0.99). The beneficial effect of living in an urban environment applies particularly to individuals aged 10-40 years and 80 years and above, people who never married and residents from non-Western ethnic origins. The beneficial effect of urban residence for non-married people is related to their lower cancer and heart disease mortality. The beneficial effect of urban residence for people of non-Western ethnic origin is related to their lower cancer and suicide mortality. CONCLUSION: In The Netherlands, living in an urban environment is not consistently related to higher mortality risks. Young adults, elderly, single and non-Western residents, especially, benefit from living in an urban environment. The urban environment seems to offer these subgroups better opportunities for a healthy life.

Adalimumab, a fully human anti-TNF-alpha monoclonal antibody, treatment does not influence experimental UV response in the skin of rheumatoid arthritis patients.

TNF-alpha is known to play an important role in UV-induced immunomodulation and photodamage. It plays a role in UVB-mediated induction of apoptosis and is a strong inducer of the c-Jun N-terminal kinase (JNK) pathway, which eventually leads to the loss of dermal collagen and elastin content. Recently chimeric anti-TNF-alpha has been introduced as a therapy for rheumatoid arthritis. The aim of the present study was to investigate the effect of anti-TNF-alpha treatment on UV-induced DNA damage, apoptosis, and induction of matrix metallo proteinases. Twelve patients with rheumatoid arthritis were included and irradiated with 2 MED broadband UVB before and after administration of 0.5 mg/kg anti-TNF-alpha monoclonal antibody. Twenty-four hours after irradiation biopsies were taken. Frozen and paraffin sections were stained for p53, c-Jun, phosphorylated c-Jun, sunburn cells and MMP-1. No significant changes were observed in the expression of p53 and sunburn cells and MMP-1 content after treatment with anti-TNF-alpha, whereas a slight but significant decrease in c-Jun and phosphorylated c-Jun expression was noted (P = 0.0250 and P = 0.0431, respectively). Our results showed no influence of anti-TNF-alpha on UV response at therapeutic doses in patients with rheumatoid arthritis.

The effect of a liquid nutrition supplement on body composition and physical functioning in elderly people.

BACKGROUND & AIMS: The elderly are at an increased risk of poor nutritional status which is mutually interacting with functional status. We evaluated the effects of a liquid nutrition supplement on anthropometric and functional indices in elderly people. METHODS: Subjects (n=68; mean age=82+/-7 years) with body mass index

Flow-cytometric characterization of normal versus psoriatic epidermis using improved cell separation methodology.

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.

Pharmacological effects of a specific leukotriene B(4) receptor antagonist (VML 295) on blood leukocytes, cutaneous inflammation and epidermal proliferation.

VML 295 (LY 293111) is a potent and specific leukotriene(4) receptor antagonist. It has previously been shown in human volunteers that VML 295 at a dosage of 48 mg twice daily inhibits the ex vivo leukotriene B(4) (LTB(4))-induced upregulation of CD11b on peripheral blood neutrophils. A clear dose-response relatinship was shown. In addition, VML 295 inhibits various inflammatory aspects resulting from LTB(4) challenge of the skin, again showing a dose-response relationship. In view of the large variation in the elimination half-life of VML 295 (25-88.5 h) in individual human subjects, the present pharmacological study was designed to provide information on the pharmacodynamics of the drug by the assessment of VML 295 plasma concentrations, ex vivo LTB(4)-induced CD11b upregulation of neutrophils, neutrophil accumulation in the skin following epicutaneous application of LTB(4) and epidermal regeneration following standardized surface trauma. A group of 36 healthy volunteers were treated in a double-blind study with VML 295 at 200 mg twice daily, VML 295 at 200 mg once daily or placebo for 7 days. Before treatment, at the end of treatment and following discontinuation of treatment, VML 295 plasma concentrations and CD11b upregulation of blood neutrophils were assessed. In 18 subjects, the effects of the three treatments on LTB(4)-induced inflammatory were assessed before and at the end of treatment, and in the remaining 18 subjects the effects of these treatments on epidermal regeneration were assessed similarly. VML 295 at 200 mg either twice or once daily has a profound inhibitory effect on ex vivo LTB(4)-induced CD11b upregulation of blood neutrophils, LTB(4)-induced neutrophil accumulation in the skin, trauma-induced hyperproliferation of the epidermis and regenerative keratinization. The twice daily dose schedule was significantly more effective than the once daily regimen in reducing ex vivo CD11b stimulation of neutrophils, in blood samples collected 24 h after discontinuation of VML 295 treatment. The twice daily schedule tended to be more efficient in skin biopsies, although this difference was not statistically significant in the number of subjects investigated. A plasma concentration of 100 ng/ml proved to be the threshold for these effects. The profound biological effects, both systemically and cutaneously, as well as the safety profile, make VML 295 a promising drug for skin disorders characterized by epidermal proliferation and neutrophil accumulation.

Sequence-specific inhibition of gene expression in intact human skin by epicutaneous application of chimeric antisense oligodeoxynucleotides.

Targeted and selective inhibition of keratinocyte gene expression in human epidermis could be an efficient and safe pharmacologic approach in many skin diseases. In this study we investigated whether topical application of antisense oligodeoxynucleotides (ODN) on intact human skin can be used to inhibit expression of a gene in the differentiated compartment of the epidermis. We applied a variety of 20-mer antisense and control ODN designed to hybridize to different regions on the mRNA of the inducible epidermal proteinase inhibitor skin-derived antileukoproteinase (SKALP)/elafin that was used as a model target gene. When nuclease-resistant fully phosphorothioate ODN were applied to explant cultures of human skin, they were found to be either ineffective at low doses or severely toxic at higher doses which could be attributed to the extremely high degree of protein binding found with this type of ODN. When chimeric ODN with a phosphodiester core and phosphorothioate 5' and 3' ends were applied to intact skin, no toxicity was noted. One of the tested chimeric ODN, that exhibit only minor protein binding, was found to inhibit SKALP expression at the protein level in a dose-dependent manner. The observed inhibition on SKALP expression levels was specific as evaluated by application of strict criteria. Sequence specificity was assessed by the addition of sense and scrambled ODN which were ineffective. Furthermore the expression levels of three other differentiation-related genes (involucrin, cytokeratin 16, and secretory leukocyte proteinase inhibitor) were not affected, indicating that the inhibition was gene specific. Confocal laser scanning analysis of fluorescently labeled ODN confirmed that these molecules can easily penetrate the epidermis and localize in the cytoplasm of differentiated keratinocytes. We conclude that topical application of antisense ODN can be used to modulate epidermal gene expression, and could potentially be useful to inhibit expression of genes that are relevant in skin diseases.

The response of distant uninvolved psoriatic skin to standardised injury is not different from that in normal skin.

The epidermis of uninvolved psoriatic skin is characterised by a slight hyperproliferation and an increase in inflammatory parameters, whereas no differentiation abnormalities are seen. Data with respect to the response of distant uninvolved psoriatic skin to standardised injury are not uniform. In this study, a recently developed multiparameter flow cytometric assay was used to compare the response to tape stripping of uninvolved psoriatic and normal skin. With this method, a parameter for proliferation, differentiation and inflammation was measured simultaneously. Concerning these parameters, no statistically significant differences were found between uninvolved psoriatic skin and normal skin. The mechanism that underlies hyperproliferation in distant uninvolved psoriatic skin does not indicate an intrinsic abnormality in keratinocytes. Inflammatory signals might play a role in this process.

Quantification of epidermal cell populations in the centre and margin of stable psoriatic plaques.

The histological picture of psoriasis has been studied extensively. Several authors have investigated the differences between the centre and the margin of spreading plaques, because the margin is of great pathogenic interest as lesions enlarge by centrifugal expansion. However, little is known about the differences between the centre and the margin of stable plaques. In the present study we investigated quantitatively the differences between the centre and margin of stable psoriatic plaques with respect to differentiation, inflammation and proliferation. To quantify these parameters, we used flow cytometry. From nine patients with nonspreading, stable psoriasis, we obtained punch biopsies from the centre and from the lesional margin of a plaque, and performed a flow cytometric assessment, using the markers keratin 10 for differentiation, vimentin for inflammation, and TO-PRO-3 iodide for proliferation. We found that the quantitative parameters showed a large interindividual variability, and that there was no significant difference in the quantitative parameters for inflammation and proliferation between the centre and margin of stable plaques. However, the percentage of differentiated cells was significantly higher in the margin than in the centre. We conclude that there is a great heterogeneity within stable psoriatic plaques with respect to differentiation, inflammation and proliferation, but further quantitative studies are needed to substantiate the pathogenic relevance of the significant difference in keratinization between the centre and the margin of stable psoriatic plaques.

Investigation on a novel and specific leukotriene B4 receptor antagonist in the treatment of stable plaque psoriasis.

The aim of the present study was to investigate the efficacy and clinical tolerability of the specific leukotriene B4 receptor antagonist VML295 in the treatment of stable plaque psoriasis. Immunohistochemical and flow cytometrical methods were used to assess the effects on inflammation and epidermal proliferation. VML295 in the treatment of chronic plaque psoriasis was shown to be safe and well tolerated. After treatment, there was a statistically significant difference between patients treated with VML295 and patients treated with placebo with respect to the leukotriene B4-induced CD11b up-regulation on the cell surface of polymorphonuclear leukocytes derived from peripheral blood. Ex vivo CD11b up-regulation in the VML295-treated group was completely inhibited after 7 days of treatment (P = 0.001). This effect persisted until the end of the treatment period (P = 0.004 on day 15 and P < 0.0001 after 4 weeks), whereas CD11b up-regulation in the placebo group remained unaffected. There was no statistically significant difference in the median psoriasis area and severity index between the treatment groups at the end of the treatment period. During treatment, no significant histological changes were observed in the markers for cutaneous inflammation and epidermal proliferation. Although not statistically significant, a tendency for the increased expression of some markers of cutaneous inflammation and epidermal proliferation was observed after 1 week of treatment with VML295, and a decreased expression of these markers was seen after 4 weeks of treatment with VML295. This observation could indicate anti-inflammatory effects of VML295 appearing between 2 and 4 weeks after the start of treatment.

The effect of long-term treatment with tacalcitol on the psoriatic epidermis. A flow cytometric analysis.

During the last decade, novel analogues of 1alpha,25-dihydroxy vitamin D3 have been developed for the treatment of psoriasis. Recently, the efficacy of short-term treatment with the novel derivative tacalcitol (1alpha,24-dihydroxy vitamin D3) has been documented. However, data on the long-term effect of tacalcitol on psoriatic skin are sparse. In this study, we assessed the cell characteristics of the psoriatic epidermis after treatment with tacalcitol for up to 24 weeks. We investigated how long-term treatment with tacalcitol modulates the percentages of differentiated keratinocytes, inflammation cells and basal keratinocytes, and the percentage of cells in the SG2M phase in the basal cell population. From 11 patients who were treated with tacalcitol for up to 18 months, we obtained single-cell suspensions of a representative psoriatic lesion after 0, 8, 12, 18 and 24 weeks of treatment. A Psoriasis Area and Severity Index was performed at each visit as well. Cell suspensions were stained with markers for inflammation (Vim3B4), differentiation (RKSE60) and proliferation (TO-PRO-3 iodide) and analysed flow cytometrically. Clinically, patients improved significantly after 8 weeks of treatment. This clinical effect was preserved for the rest of the period of treatment with no further significant improvement. Proliferative activity also decreased significantly after 8 weeks of treatment. Proliferation did not show further significant decreases or habituation after 12, 18 and 24 weeks. For inflammation, no statistically reliable trends could be seen. Differentiation improved significantly after 8 weeks of treatment, but decreased again significantly after 12 weeks. In the period from 12 to 24 weeks, no further significant change was observed. We conclude that tacalcitol is an effective antipsoriatic drug. Prolonged treatment with tacalcitol will generally maintain improvement at the level reached after 8 weeks. Owing to the beneficial effect on both clinical state and proliferation, tacalcitol is likely to be an adequate maintenance therapy.

Flow cytometric and microscopic characterization of the uptake and distribution of phosphorothioate oligonucleotides in human keratinocytes.

Gene-specific inhibition by antisense oligonucleotides has been successful in a large number of systems. In an attempt to use this strategy for the modulation of skin disease-specific gene expression, we studied oligonucleotide uptake in cultured human keratinocytes. This study revealed a heterogeneous uptake of fluorescently labeled phosphorothioate oligonucleotides. Flow cytometric and microscopic analysis showed two fluorescent cell populations with differences in intensity: a 'bright' population of highly fluorescent small cells and a 'dim' population of less fluorescent but larger cells. The heterogeneity in uptake between these two populations was not a result of differences in cell cycle phases of the keratinocytes, as shown by flow cytometric sorting and measurements of relative DNA content. In both populations the oligonucleotides were transported intracellularly and were mainly located in the cytoplasm. A typically speckled localization pattern was demonstrated by confocal laser scanning microscopy. We used propidium iodide (PI) to assess viability, and showed that in nonviable (PI-permeable) keratinocytes the oligonucleotides accumulated in the nucleus. The use of a lipidfection reagent also changed the intracellular distribution of oligonucleotides from a punctate cytoplasmic pattern to an intense nuclear localization. The process of uptake by the viable keratinocytes was dependent on oligonucleotide concentration, incubation time and temperature. This study underlines the importance of kinetic studies on oligonucleotide uptake in human keratinocytes which must be considered when specific oligonucleotides are used against skin disease-specific genes.

Multi parameter flow cytometric assessment of regenerative epidermis with special reference to the antiproliferative effect of occlusion.

Multi parameter flow cytometrical assays permit simultaneous assessment of proliferation, differentiation, and inflammation parameters. In this study, the validation of TO-PRO-3 iodide (TP3) compared to propidium iodide (PI) and DE-K10 compared to RKSE60 were evaluated in tape stripping induced hyperproliferation. No occlusion, Duoderm (intermediate occlusion) and Blenderm (maximal occlusion) were used as a model to evaluate the effect of occlusion on epidermal regeneration. Proliferation in the keratin 10-negative compartment measured with TP3 proved to be a good approximation of proliferation measured with PI. Other epidermal subpopulations (keratin 10-dim and -bright cells) did not make a relevant contribution to hyperproliferation. DE-K10 is probable more sensitive than RKSE60 to distinguish populations that differ in degree of differentiation. Occlusion of tape stripped skin resulted in decreased proliferation and increased differentiation. This effect was most pronounced with maximal occlusion. This study showed that occlusion is a therapy, which realises normalisation of hyperproliferative skin disorders.

Demonstration of an Na+/H+ exchanger in mouse keratinocytes measured by the novel pH-sensitive fluorochrome SNARF-calcein.

In many cell types cytoplasmic alkalization is an early marker for cell activation. An amiloride-sensitive Na+/H+ exchanger is an important regulator of this process. However, in keratinocytes the existence of a Na+/H+ exchanger nor a proliferation-associated increase in intracellular pH (pHi) has been demonstrated. The aim of this study was to investigate whether or not keratinocytes, derived from the BALB/MK cell line, contain a Na+/H+ exchanger and whether cytoplasmic alkalization is proliferation-associated in these cells. This mouse keratinocyte cell line can easily be switched between a proliferative and a quiescent state under defined culture conditions. The novel pH-sensitive dye seminaphthorhodafluor (SNARF)-calcein proved to be very suitable for flow cytometric pHi measurements in BALB/MK cells. Initial measurements of the pHi using a cocktail of the established fluorochromes 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and SNARF-1 failed because of the differential uptake and binding kinetics of these pH-sensitive dyes. Using SNARF-calcein we were able to show proliferation to be associated with increased pHi. However, culture conditions were critical for these measurements. Our data indicate that the Na+/H+ exchanger is involved in this process, since acid load and pHi-recovery experiments showed the alkalization to be amiloride-sensitive.

The regulation of CD11b integrin levels on human blood leukocytes and leukotriene B4-stimulated skin by a specific leukotriene B4 receptor antagonist (LY293111).

CD11b is part of the beta2-integrin Mac-1 and plays an important role in neutrophil adhesion. Leukotriene B4 (LTB4) is an active upregulator of neutrophil CD11b-expression, acts as a potent chemoattractant to neutrophils and is also known to upmodulate epidermal proliferation. We performed a placebo-controlled study on LY293111, an oral LTB4 receptor antagonist. Twenty healthy male volunteers were randomised over three treatment groups that received placebo, 48 mg, or 200 mg drug twice daily for 10 days. Before and after treatment, flow cytometrical CD11b assessment was performed on in vitro LTB4-stimulated peripheral blood neutrophils. Additionally, skin biopsies were taken at 24 and 72 h after epicutaneous LTB4 application, before and after treatment. The effects on skin were assessed immunohistochemically using various markers. All observed effects were dose related. CD11b upregulation on blood neutrophils was significantly suppressed in both treatment groups compared to placebo. In skin, a significant suppression of inflammation and hyperproliferation occurred. Pronounced inhibition was observed on neutrophil migration into the epidermis and the inflammatory infiltrate was decreased. A similar but weaker response was seen in the dermis. The number of cycling cells as well as suprabasal keratin-16 expression were decreased in both treatment groups. LY293111 proved to be a potent inhibitor of LTB4-induced cutaneous inflammation and hyperproliferation. The potent antiinflammatory effect in vivo and the fact that in the present study the compound showed no clinically significant side effects make it an interesting drug in the future treatment of inflammatory conditions predominated by neutrophils.

Protein-tyrosine phosphatases expressed in mouse epidermal keratinocytes.

The importance of growth factors acting via receptor-type protein-tyrosine kinases in the continuous renewal of the epidermis from the keratinocyte stem cell population has been well established. Protein-tyrosine phosphatases (PTPases), which dephosphorylate phosphotyrosine-containing proteins, may therefore be expected to play an equally important role in the control of epidermal growth and differentiation. In this study, we have made an inventory of the various PTPases that are expressed during mouse keratinocyte proliferation and maturation. A panel of 13 different PTPases probes was obtained by combining a set of PTPase cDNAs previously cloned from mouse brain and a set of PTPase probes obtained from a normalized keratinocyte PTPase cDNA library. This PTPase cDNA panel, spanning probes for receptor-type as well as cytoplasmic-type family members, was used to monitor RNA expression levels in keratinocyte fractions isolated from murine epidermis and in keratinocyte cell cultures. No overt changes were observed in PTPase mRNA levels in all strata of mouse epidermis, but comparison of cultured cells with freshly isolated keratinocytes revealed several conspicuous differences. In the cultured Balb/MK cell line, absence of PTP delta expression and upregulation of PTP kappa and, to a lesser extent, PTP gamma mRNA ratios were observed compared to the freshly isolated cells. These results provide a basis for further research on the impact of PTPase activity on epidermal growth control.

Multiparameter flow cytometric characterization of epidermal cell suspensions prepared from normal and hyperproliferative human skin using an optimized thermolysin-trypsin protocol.

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.