Preferential expression of a high number of ATP binding cassette transporters in both normal and leukemic CD34+CD38- cells.
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ADP-ribosyl Cyclase 1↗
Biomedical subjects
J B Boezeman
Publications and source records attributed to J B Boezeman.
Multiparameter flow cytometry as a tool to evaluate antipsoriatic therapy.
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.
Biomarkers↗
Cell kinetic characterization of cultured human keratinocytes from normal and psoriatic individuals.
Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, disturbed differentiation, and inflammation. It is still a matter of debate whether the pathogenesis of psoriasis is based on immunological mechanisms, on defective growth control mechanisms, or possibly on a combination of both. Several in vivo cell biological differences between psoriatic lesional epidermis and normal epidermis have been reported. However, it is not clear whether these changes are causal or consequential. In case that keratinocytes from psoriatic patients have genetically determined deficiencies or polymorphisms with respect to autocrine growth regulation and the response to inflammatory cytokines, we hypothesize that these differences should be maintained in culture. Here we have started a systematic comparison of first passage keratinocytes cultured from normal skin and uninvolved psoriatic skin to address the question whether there are intrinsic differences in basic cell cycle parameters. In an established, defined culture system using keratinocyte growth medium (KGM) we have determined: (i) cell cycle parameters of exponentially growing keratinocytes, (ii) induction of quiescence by transforming growth factor beta 1 (TGF-beta 1) and (iii) restimulation from the G0-phase of the cell cycle. Bivariate analysis of lodo-deoxyuridine incorporation and relative DNA content was performed by flow cytometry. Within the limitations of this model no gross differences were found between normal and psoriatic keratinocytes with respect to S-phase duration (Ts), total cell cycle duration (Tc), responsiveness to TGF-beta 1 and the kinetics for recruitment from G0. In psoriatic keratinocytes we found a lower amount of cell in S-phase and a shorter duration of G1, compared to normal keratinocytes. The methodology developed here provides us with a model for further studies on differences between normal and psoriatic keratinocytes in their response to immunological and inflammatory mediators.
Cell Cycle↗
Bias in nucleotide composition of antisense oligonucleotides.
In this study we investigated if specific sequence motifs occur with a higher frequency in antisense oligonucleotides than can be expected on the basis of the mRNA composition to get an impression of the importance of these motifs for antisense effects. Computer analysis of 206 antisense oligonucleotides extracted from the literature and from sequence databases, all targeted against human mRNA, was performed. We compared the sequence composition of these oligonucleotides with the average of 100 equally large and randomly selected sequences from sequence databases and of their target mRNA. We found that the frequency of sequence motifs containing GG, CCC, CC, GAC, and CG is significantly higher and TT and that TCC is significantly lower in antisense oligonucleotide sequences than in the randomly selected mRNA sequences. We conclude that there is a bias in the nucleotide composition of antisense oligonucleotides. Some of these biased sequence motifs have been reported to induce nonantisense effects mediated by protein binding. Further analysis of the biologic function of these motifs is necessary to investigate if they should be avoided or incorporated into future designs of therapeutic effective oligonucleotides.
Base Composition↗
Psoriasis of the nails associated with disability in a large number of patients: results of a recent interview with 1,728 patients.
BACKGROUND AND OBJECTIVE: Occurrence rates of clinical features of nail psoriasis vary considerably in the literature. Little information is available on subjective complaints of patients affected by psoriasis of the nails. METHOD: Interviews with 1,728 psoriatic patients concerning their nail changes and complaints are reviewed. RESULTS: The results indicate that pitting and deformation are the most common clinical aberrations in psoriatic nails, with a positive association between the duration of skin lesions and nail psoriasis. No relation was found between age and nail psoriasis in this group. Remarkably, 51.8% of patients suffered from pain-caused by the nail changes, and a large group of patients was restricted in their daily activities, housekeeping and/or profession (58.9, 56.1, 47.9%). Treatment was disappointing: only 19.3% showed marked improvement during treatment. CONCLUSION: This indicates that psoriasis of the nails is a more important individual and social-economic problem than previously assumed and that development of new treatments is needed.
Adolescent↗
Cell cycle kinetics in normal human skin by in vivo administration of iododeoxyuridine and application of a differentiation marker--implications for cell cycle kinetics in psoriatic skin.
Renewal of epidermal cells is a highly coordinated process in which terminal differentiation balances the proliferative rate in the germinative compartment. Exact quantitative data on cell cycle parameters of normal human epidermis are fragmentary, and do not allow firm conclusions on issues such as cell cycle time, duration of the various cell cycle phases, and the pool sizes of the different cellular populations. As part of a study on bone marrow cell cycle kinetics, 14 lymphoma patients were infused with the thymidine analogue iododeoxyuridine (IdUrd). This provided us with the unique opportunity to study the cell cycle kinetics in normal epidermis obtained from these patients. Single epidermal cell suspensions were prepared from skin, stained with propidium iodide (PI) for relative DNA content, and simultaneously labeled with an anti-IdUrd antibody to detect DNA-synthesizing cells. In parallel samples suprabasal cells were analyzed by labeling with an anti-cytokeratin 10 antibody. Analysis was performed using bivariate flow cytometry. The results showed that 3.5% of the total epidermal cell population was in S-phase. An S-phase duration of 9.7 +/- 0.6 h and a cytokeratin 10-positive pool size of 59.6 +/- 4.6% were obtained. The duration of the G1-phase and the G2M-phase were calculated to be 7.6 +/- 2.0 h and 11.1 +/- 2.0 h, respectively. From these data a total cell cycle time can be calculated of 28.4 h. Combining this data with previous findings we were able to determine a similar cell cycle time of 27.8 h, and pool sizes of the epidermal cells: 30% quiescent (resting, G0) and 10% cycling cells. The implications of these findings for the interpretation of deviations in growth control as found in hyperproliferative skin diseases (e.g. psoriasis) are discussed.
Antibodies, Monoclonal↗
The growth and differentiation of human keratinocytes in vitro: a combined immunohistochemical and flow cytometric study.
In this study we performed a cell kinetic characterization of the growth and differentiation of human keratinocytes. Using a combination of immunohistochemical and flow cytometric techniques it was possible to obtain a detailed description of these processes. The proliferative activity of the cell cultures was analysed with flow cytometric techniques, measuring relative DNA content, iododeoxyuridine incorporation and the expression of the antigen recognized by Ki-67. In addition to a standard monolayer culture technique, cells were maintained in suspension. Under these conditions these cells were not capable of dividing, started to lose their nuclei, and the expression of differentiation-related proteins such as involucrin and filaggrin was induced, suggesting that the cells changed towards a differentiated phenotype. Binding of the antibody Ks8.12, recognizing keratins 13 and 16, occurred under all culture conditions, independent of cell density, and also in suspension, suggesting that it is a marker for abnormal differentiation rather than for hyperproliferation.
Animals↗
Cell cycle kinetics of hematopoiesis before and after in vivo administration of GM-CSF in refractory anemia: evidence for a shortening of the granulocyte release time.
GM-CSF administration to patients with refractory anemia (RA) induces an increase in neutrophils and eosinophils. We studied cell kinetic mechanisms underlying this observation using clonogenic assays and in vivo iododeoxyuridine labeling of bone marrow cells. Cell cycle kinetics were studied in three patients before and during GM-CSF administration (two daily subcutaneous injections of 54 or 108 micrograms). No consistent effect on the relative number of bone marrow CFU-GM was noticed. The DNA synthesis time and potential doubling time of low-density bone marrow cells remained essentially the same. A slight decrease (1.5-3.7%) in labeling index was found, originating from the myelo(-mono)cytic lineage. In all three patients the release time of labeled granulocytes from the bone marrow into the peripheral blood was shortened (before GM-CSF treatment 5-7 days and during GM-CSF 3-4 days). Cell cycle kinetics of CD34+ cells were studied in order to obtain kinetic information on immature precursor and progenitor cells. The DNA synthesis time of the CD34+ cells was shortened during GM-CSF therapy, resulting in a shorter potential doubling time. GM-CSF administration to patients with RA results in a rise in granulocytes that might be due partly to an accelerated release of granulocytes from the bone marrow compartment into the circulating blood and partly to an increased proliferative activity of the immature precursor and progenitor cells.
Anemia, Refractory↗
The effect of tacalcitol (1,24(OH)2D3) on cutaneous inflammation, epidermal proliferation and keratinization in psoriasis: a placebo-controlled, double-blind study.
The aim of the present study was to discover to what extent 1,24(OH)2D3 ointment (tacalcitol; 4 micrograms/g) can modulate epidermal proliferation and keratinization, and several aspects of inflammation. Ten patients with psoriasis vulgaris were included in a placebo-controlled, double-blind study, using 1,24(OH)2D3 ointment (4 micrograms/g). Before, and after 8 weeks of treatment, punch biopsies were taken from lesions treated with the active agent and placebo-treated lesions. An immunohistochemical study was carried out using monoclonal antibodies against the hyperproliferation-associated keratin 16, against cycling nuclei, filaggrin, involucrin, T lymphocytes, Langerhans cells, CD14 and polymorphonuclear leucocytes (PMN). The Wilcoxon test for matched pairs was used for statistical analysis of results. The biopsies from the lesions treated with the active agent showed a statistically significant change towards normalization of all aspects of inflammation studied, and of epidermal proliferation and keratinization, but there did not appear to be any effect on Langerhans cells. The only parameter which showed a significant alteration in the placebo-treated lesions was the number of cycling nuclei in the epidermis (P < or = 0.02). However, the biopsies from the plaques treated with the active agent showed a greater decrease of cycling cells (decrease: Mactive = 70, Mplacebo = 53) and a lower P-value (< or = 0.01). We therefore conclude that at the cell biological level 1,24(OH)2D3 ointment (4 micrograms/g) has a substantial effect on several cell types, with regard to inflammation, epidermal proliferation and keratinization, with the exception of Langerhans cells.
Cell Division↗
Cell kinetic characterization of growth arrest in cultured human keratinocytes.
In this study we have performed a cell kinetic characterization of growth and growth arrest of keratinocytes derived from normal human skin. Proliferative activity of the cell cultures was analysed with a flow cytometric technique, measuring relative DNA content and iododeoxyuridine (IdUrd) incorporation simultaneously. Normal human keratinocytes were grown in keratinocyte growth medium (KGM) and growth arrest was induced by using either keratinocyte basal medium (KBM) or KGM supplemented with TGF-beta 1. It was found that human keratinocytes grown in KGM plus TGF-beta 1 were growth-arrested within 52 hours. The rate of IdUrd incorporation into DNA decreased by more than 95% after 52 hours and paralleled the decrease of cells in S-phase. Within 52 hours after addition of TGF-beta 1, 79% of the growth-arrested cells were in the G0/G1-phase of the cell cycle, a situation that approaches that of the normal epidermis. Growth arrest of human keratinocytes in KBM showed a similar decrease in the rate of IdUrd incorporation. However, the decrease in IdUrd incorporation was not reflected in a decrease in cells in S-phase, suggesting that the cells were blocked in G0/G1, S or G2/M-phase rather than selectively in the physiological growth arrest state of G0/G1. Secondly, we investigated the kinetics of the cells when they were restimulated after growth arrest. We found that after termination of the growth arrest in KGM supplemented with TGF-beta 1 the cells require 6 to 8 hours to initiate DNA synthesis, with a continued decrease in the G0/G1 population, suggesting that the cells are recruited as a cohort. After growth arrest induced by KBM, cells also require 6 to 8 hours in KGM to initiate DNA synthesis, but the cells are not recruited as a cohort. We conclude that growth arrest induced by TGF-beta 1 is the preferred system in which to study induction of keratinocyte proliferation, since it induces a state of quiescence that approaches that of normal human epidermis.
Cell Cycle↗
Weekly treatment of psoriasis with a hydrocolloid dressing in combination with triamcinolone acetonide. A controlled comparative study.
In a within subject comparison, the efficacy and the tolerability of topical treatment with triamcinolone acetonide under a hydrocolloid dressing were compared with triamcinolone acetonide monotherapy, monotherapy with hydrocolloid dressing and triamcinolone acetonide under plastic occlusion. The clinical efficacy of hydrocolloid dressing as a monotherapy and the pronounced clinical efficacy of this occlusive in combination with triamcinolone acetonide were confirmed. Comparing the hydrocolloid dressing (Duoderm E) and the plastic semi-occlusive (Opsite IV 3000), we concluded that the tolerability of both approaches was similar. However, Duoderm E in conjunction with triamcinolone acetonide was highly effective, whereas Opsite IV 3000 in combination with this steroid only had an inconspicuous antipsoriatic potential.
Adult↗
Proliferation patterns in acute myeloid leukemia: leukemic clonogenic growth and in vivo cell cycle kinetics.
In a prospective study of 33 newly diagnosed patients with acute myeloid leukemia (AML), we analyzed the relationship of proliferation parameters with clinical parameters, response to induction therapy, and survival. The median follow-up was 26 months. The proliferative capacity of the leukemic progenitor cells was studied using colony-forming assays (number of colony-forming units, growth pattern, and spontaneous clonogenic growth capacity). The cell kinetic parameters of the bone marrow blasts were determined by in vivo labeling with iododeoxyuridine and subsequent flow cytometry: labeling index (LI), DNA synthesis time (Ts), potential doubling time. No or only weak relationships were observed between the experimental and clinical parameters such as age, sex, % blasts, white blood cell count, FAB subtype, cytogenetics, and % CD 34+ cells. This suggests that clonogenic growth and cell cycle kinetics of bone marrow blasts are independent cell biologic properties of AML. No association between the proliferation parameters and induction response rate was noticed. Analysis of the overall survival and event-free survival revealed trends to longer survival rates in patients with a below-median LI (< or = 7.6%) and below-median Ts value (< or = 14.3 h). These trends were more pronounced in the group of de novo AML (n = 23), where the prolonged event-free survival in patients with below-median Ts reached statistical significance (p = 0.02). None of the other parameters appeared significantly correlated with survival, although there was a trend to longer survival rates in patients who had no spontaneous clonogenic growth capacity (p = 0.13). In conclusion, proliferation parameters in leukemic cells provide additional information on the cell biologic characteristics of AML, and these parameters may have prognostic value for response and duration of survival in AML.
Adult↗
The immunohistochemical effect of a hydrocolloid occlusive dressing (DuoDERM E) in psoriasis vulgaris.
The topical application of a hydrocolloid occlusive dressing (HCD) has been shown in various studies to have an antipsoriatic effect as monotherapy but especially in combination with a topical corticosteroid. The aim of the present study was to assess the effect of 3 weeks of HCD monotherapy at the immunohistochemical level. Ten patients were treated. Before and after treatment, a biopsy was taken, and immunohistochemical stainings were carried out with markers for epidermal growth, keratinization, inflammation and endothelium. Suprabasal expression of keratin 16, the number of cycling epidermal cells and the number of polymorphonuclear leucocytes and T lymphocytes tended to decrease during treatment. The endothelial markers did not change during HCD treatment. This study confirms the antipsoriatic effect of HCD and demonstrates that its effect upon some markers of inflammation, epidermal proliferation and keratinization is modest.
Adult↗
Cell cycle kinetics in malignant lymphoma studied with in vivo iododeoxyuridine administration, nuclear Ki-67 staining, and flow cytometry.
Cell cycle kinetics of malignant lymphoma were investigated using in vivo labeling with iododeoxyuridine (IdUrd) and subsequent flow cytometry (FCM) of IdUrd/DNA and Ki-67/DNA. This approach provides an extensive cell kinetic profile from only one single tumor biopsy, including data upon the percentage of S-phase cells, the IdUrd labeling index (LI), Ki-67-derived growth fraction, duration of the S-phase, duration of the G1-phase, potential doubling time, cell production rate, and total cell cycle time. Tissue samples from 33 patients were studied: non-Hodgkin's lymphoma (NHL; n = 22), Hodgkin's disease (HD; n = 7), and reactive hyperplasia (n = 4). In NHL, the percentage of S-phase cells, LI, growth fraction, duration of the S-phase, and cell production rate were significantly correlated with the histologic malignancy grade according to the Working Formulation (P < or = .02). Data found in HD were not essentially different from those in low-grade NHL and reactive hyperplasia. Remarkably, the duration of the S-phase, the duration of the G1-phase, and the total cell cycle time appeared to be rather independent of histologic malignancy grade within the NHL category. A significant correlation was observed between the IdUrd LI and the percentage of S-phase cells, the growth fraction, the potential doubling time, and the cell production rate (P < .001), but not with the duration of the separate cell cycle phases (P > .05). Our data show (1) that it is feasible to obtain detailed information on the in vivo growth characteristics of malignant lymphoma; and (2) that the transition time through the different cell cycle phases widely varies, even within distinct histologic subgroups.
Adult↗
Epidermal proliferation and keratinization following standardized elicitation with diphenylcyclopropenone.
Diphenylcyclopropenone (DCP) was applied to the upper arms of five alopecia areata patients using 10% of the concentration that had been applied previously to the scalp during topical immunotherapy. DCP applied in this concentration evoked a mild eczematous reaction. Biopsies were taken before DCP application and after 24, 48 and 96 h. A large increase in T-lymphocytes and CD14-positive cells in the dermis was seen after 24 h. Migration of these cells into the epidermis was mainly observed during the first 48 h. This was followed by epidermal proliferation as assessed by the number of Ki-67-positive nuclei and the degree of Ks8.12-binding. Both showed their main increase after 48 h; but after 24 h the increase of Ki-67-positive nuclei was significant (P < 0.04). Involucrin and filaggrin showed a gradual increase which became substantial after 96 h (both P < 0.04). As the invasion of inflammatory cells into the epidermis preceded the main increase in epidermal proliferation, cytokines are suggested as possible mediators for the initial phase of the proliferative response after DCP application.
Cell Division↗
Topical immunotherapy for alopecia areata: re-evaluation of 139 cases after an additional follow-up period of 19 months.
Within a group of 139 patients previously studied during treatment for alopecia areata with diphenylcyclopropenone (DCP), hair growth was re-evaluated after a period of 19 months following completion of our previous study. Fifty-four patients treated with DCP had total and 6 had partial but cosmetically acceptable regrowth. Twenty-five patients with total regrowth had stopped DCP treatment for a mean period of 15 months and had not relapsed. Nineteen of 28 patients who still applied DCP were in the process of stepwise discontinuation of treatment. Fifteen patients had subsequently been treated with squaric acid dibutylester (SADBE) after having acquired 'tolerance' to DCP; at the time of re-evaluation 3 of these patients had complete regrowth, and 4 patients had partial but cosmetically acceptable regrowth. Topical immunotherapy with DCP and SADBE had resulted in total regrowth in 57/139 patients (41.0%) and in partial but cosmetically satisfactory regrowth in 10/139 patients (7.2%). The type of involvement and duration of alopecia areata were factors of prognostic significance.
Administration, Topical↗
Quantification of mitochondrial proteins in cultured cells by immuno-flow cytometry.
Immuno-flow cytometry was tested as a tool to estimate the cellular concentration of mitochondrial proteins in cultured cells, using cytochrome c oxidase as a model enzyme. Cells labelled with antibodies against cytochrome c oxidase, in which the amount of the enzyme was reduced by various extents, showed a linear relationship between the size of the signal obtained by immuno-flow cytometry and the amount of the enzyme. The determination by immuno-flow cytometry resulted in data comparable to the results obtained by immunoprecipitation and activity measurements. Since immuno-flow cytometry requires only limited numbers of cells, the method could especially be of value for diagnostic purposes. This is illustrated by the results obtained by comparing activity measurements and immuno-flow cytometry in the initial screening of cell lines derived from patients with deficiencies in the activity of cytochrome c oxidase.
Cells, Cultured↗
Ratiometric measurement of intracellular pH in cultured human keratinocytes using carboxy-SNARF-1 and flow cytometry.
In this study we describe a method to measure intracellular pH in cultured human keratinocytes using flow cytometry. Keratinocytes pose a technical problem because the population is heterogeneous with respect to size and metabolic activity (nonspecific esterase activity), resulting in variability in dye uptake. In order to compensate for this, dyes were selected that change colour with pH. The ratio of fluorescence intensities at two wavelengths was recorded and used as a measure of intracellular pH by reference to the pH in the presence of the proton ionophore nigericin. However, methods published till now do not routinely combine the ratiometric technique and excitation with an argon ion laser set at 488 nm. Therefore we have tested the recently developed pH-sensitive dye carboxyseminaphthorhodafluor-1 (SNARF-1) as a possible candidate for flow cytometric pH measurements and compared it with 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and 2,3-dicyanohydroquinone (DCH) with respect to emission spectra, resolution, range, and stability of cellular fluorescence. SNARF-1 had a practical and stable excitation wavelength of 488 nm rather than UV, it offered the possibility of ratiometric measurements on the basis of a real emission shift, and had superior resolution for the pH range 7-8. With SNARF-1 we found that keratinocytes cultured under low serum conditions (0.2%) contain a higher proportion of cells with relatively low intracellular pH compared to high serum cultures (6%). Furthermore, pH changes were followed by changes in relative DNA content. These findings suggest that intracellular pH can be an early functional proliferation marker for human keratinocytes.
Animals↗