Adalimumab therapy for psoriasis in real-world practice: efficacy, safety and results in biologic-naïve vs. non-naïve patients.

BACKGROUND: Patients and the course of treatment in daily practice are different from randomized controlled trials (RCTs). PRIMARY OBJECTIVE: to analyse the percentage of patients achieving PASI 75. SECONDARY OBJECTIVES: PASI 50, PASI 90, PASI 100 responses, the percentage of patients experiencing at least one serious adverse event (SAE) and the response in biologic-naïve vs. non-naïve patients. METHODS: Prospectively collected efficacy and safety data of a cohort of psoriasis patients treated with adalimumab in daily practice between May 2007 and July 2011 were analyzed. Efficacy was determined using an intention-to-treat analysis and an as treated analysis, in comparison with the course baseline PASI before the start of adalimumab and the original baseline PASI before the start of any biologic therapy. RESULTS: Eighty-five patients received adalimumab therapy with a mean treatment duration of 1.4 (range 0.02-3.1) years. Compared with the original baseline PASI, PASI 75 response rates at week 12 and 24 were 34% and 38% (ITT). PASI 75 responses were well maintained until week 132. Only the PASI 75 response rate at week 12 differed significantly between biologic-naïve (56%) and non-naïve patients (29%). Sixteen patients (19%) experienced 28 SAEs. Seven patients (8%) experienced SAEs considered possibly or probably related to adalimumab. CONCLUSIONS: In this cohort, PASI75 responses were substantial but lower than in RCTs and other daily practice studies. Efficacy was well maintained during more than 2 years of follow-up and differed only between biologic-naïve and non-naïve patients at week 12. The incidence of SAEs was low but seems higher than observed in RCTs.

Safety of treatment with biologics for psoriasis in daily practice: 5-year data.

BACKGROUND: The cumulative exposition to biologics is increasing with prolonged treatment with a certain biologic or consecutive biological treatment. However, long-term safety data are limited available. OBJECTIVES: The aim of this study was to prospectively evaluate the 5-year safety of biological treatment for psoriasis in daily practice. METHODS: A cohort of 173 psoriasis patients on biologics was prospectively followed for 5 years. All adverse events reported were documented and analysed. Primary endpoint was the percentage of patients reporting at least one serious adverse event. The rate of malignancies, serious infections and serious cardiovascular events was compared with the general population incidence rate. The nature and rate of dermatological adverse events was compared with a group of prospectively followed rheumatoid arthritis patients on TNF-α blocking therapy. RESULTS: Between February 2005 and April 2010, 173 patients were enrolled in the registry and went through a total number of 263 treatment episodes. The total number of patient-years of follow-up in the registry was 409. The number of patient-years was the highest for etanercept. Forty-nine patients (28%) reported 88 serious adverse events. Only one serious adverse event was certainly causally related to the biologic and 21 events (24% of SAEs) were considered possibly related. The incidence of malignancies, serious infections and serious cardiovascular events was comparable with the population incidence rate, except for skin malignancies. The incidence of skin malignancies was significantly higher than the general population incidence rate. The nature and rate of dermatological adverse events differed from the rheumatoid arthritis cohort. CONCLUSIONS: In this cohort, the safety of biological therapies for psoriasis was favourable with a low incidence of therapy-related serious adverse events.

Relevance of laboratory investigations in monitoring patients with psoriasis on etanercept or adalimumab.

BACKGROUND: Guidelines concerning biological treatment of patients with psoriasis recommend different pretreatment and monitoring laboratory panels in variable frequencies to monitor treatment. OBJECTIVES: To investigate the relevance of laboratory investigations in monitoring patients with psoriasis on etanercept or adalimumab. METHODS: A prospective cohort study over 5 years was conducted in all consecutive patients with psoriasis on etanercept or adalimumab. All laboratory investigations performed for monitoring treatment were analysed. Laboratory abnormalities were graded according to the Common Terminology Criteria for Adverse Events v4.03. The primary endpoint was the percentage of patients with a grade 3 or grade 4 laboratory abnormality. The secondary endpoints were defined as: (i) significant changes in laboratory parameters during etanercept or adalimumab treatment and (ii) the percentage of patients having a laboratory abnormality requiring discontinuation of etanercept or adalimumab treatment. RESULTS: Laboratory parameters were available for 162 patients treated with etanercept and/or adalimumab. The number of treatment episodes was 155 for etanercept and 58 for adalimumab. Follow-up was 316 patient-years for etanercept and 54 patient-years for adalimumab. Thirty-eight of 146 patients treated with etanercept (26%) had one or more grade 3 and/or grade 4 laboratory abnormalities. For adalimumab, this was eight of 58 (14%). These were predominantly considered unrelated to biologic therapy. For both biologics, significant changes were observed in mean laboratory parameters during treatment compared with pretreatment as well as significant trends. However, mean values during treatment remained within normal ranges. Laboratory abnormalities did not lead to permanent discontinuation of biologic treatment in any patient. CONCLUSIONS: In this cohort, the incidence of biologic therapy-related serious laboratory abnormalities was low. Our findings do not support a need for routine laboratory testing in patients with psoriasis on etanercept or adalimumab beyond the laboratory testing required for concomitant therapies or comorbidities.

Switching from etanercept to adalimumab is effective and safe: results in 30 patients with psoriasis with primary failure, secondary failure or intolerance to etanercept.

BACKGROUND: Knowledge on the sequential treatment of psoriasis with biologics with regard to efficacy and safety is sparse. This also applies to the efficacy and safety of adalimumab in patients previously treated with etanercept. The relationship between the reasons for discontinuation of etanercept and the response to adalimumab is not clear in psoriasis. OBJECTIVES: To evaluate the efficacy and safety of adalimumab in patients with psoriasis with primary failure, secondary failure or intolerance to etanercept in daily practice. METHODS: Data were extracted from two prospective registries from all patients with psoriasis with failure on etanercept treatment, who switched to adalimumab therapy. Thirty patients fulfilled these criteria. All patients were naive to biologics when etanercept was initiated. Primary endpoints were the percentage of patients achieving a 50% or 75% improvement of the baseline Psoriasis Area and Severity Index (PASI) score (PASI 50 and PASI 75, respectively) at weeks 12, 24 and 48. Secondary endpoints were the percentage of patients achieving PASI 90, the mean percentage improvement in the PASI score from baseline and the adverse event rate. RESULTS: Compared with the baseline PASI score before the start of etanercept, the mean percentage improvement in PASI and the PASI 50/75/90 response rates to adalimumab until week 48 were comparable to those achieved with etanercept. In the patients failing on etanercept, PASI 75 was achieved by 27%, 36% and 54% at weeks 12, 24 and 48 of adalimumab treatment, respectively. The majority of patients showed a beneficial response to adalimumab, irrespective of the reason for discontinuation of etanercept. Previous treatment with etanercept did not increase the adverse event rate nor change the nature of the side-effects. CONCLUSIONS: Adalimumab seems to be an effective and safe treatment option for patients with psoriasis who failed on etanercept treatment irrespective of the reason for discontinuation.

Itch and scratching as predictors of time to clearance of psoriasis with narrow-band ultraviolet B therapy.

BACKGROUND: Narrow-band ultraviolet (UV) B phototherapy is an effective treatment for psoriasis. However, there is considerable variability in the number of treatment sessions needed to achieve psoriasis clearance. While several clinical and treatment-related factors predict time to clearance, the effect of itching and scratching on the number of irradiation sessions is insufficiently understood. OBJECTIVE: Predictors of the time to clearance were assessed in patients with psoriasis who were referred for UVB treatment in a randomized double-blind comparison of irradiation regimens for UVB phototherapy. METHODS: After randomization to either UVB irradiation with a suberythematogenic or an erythematogenic regimen, patients were irradiated with 20% and 40% incremental doses, respectively, three times weekly. The Psoriasis Area and Severity Index (PASI) score was measured at baseline and every 4 weeks, and itching and habitual scratching were measured at baseline. RESULTS: Among the 77 patients who achieved psoriasis clearance (90% reduction of PASI), itching and scratching were correlated with the number of irradiation sessions needed to achieve clearance, with higher levels of itch and scratching predicting more sessions. These effects remained significant after controlling for the initial PASI score, irradiation schemes, minimal erythema dose (MED), skin type, cumulative dose, protocol adjustments and lifestyle factors (smoking habits and alcohol consumption). CONCLUSIONS: Patients with higher levels of itch and scratching need more irradiation sessions to achieve clearance of psoriasis with UVB phototherapy. Systematic assessment of the severity of itch and scratching, followed by short-term itch-coping programmes for patients at risk, might be a cost-effective, adjunct to UVB therapy.

Narrowband ultraviolet B therapy in psoriasis: randomized double-blind comparison of high-dose and low-dose irradiation regimens.

BACKGROUND: Ultraviolet (UV) B phototherapy is an established treatment option for psoriasis. The optimum dosage regimen still has to be determined. Within-subject comparisons do not take into account the systemic effects of UVB phototherapy. The area of the body treated with low-dose UVB may benefit from the systemic effects of the site treated with a higher UVB dose. OBJECTIVES: To study the time to clearance in patients with psoriasis in a randomized controlled trial, in which patients were treated with narrowband UVB in either a high-dose or a low-dose regimen. METHODS: One hundred and nine patients were randomized to a high-dose regimen (group 1) or to a low-dose regimen (group 2). Patients of group 1 and 2 were irradiated with 40% and with 20% incremental doses, respectively, three times weekly. Psoriasis Area and Severity Index (PASI) was measured at baseline and at every 4-week control visit. Treatment was stopped in cases of clearance (90% reduction of baseline PASI). RESULTS: No significant differences were found in the number of patients achieving clearance. The high-dosage scheme resulted in four fewer treatments with no significant differences in cumulative UV dose, although more protocol adjustments were required in the beginning of the study because of erythema. After 3 months a significantly better clinical outcome was seen after high-dose UVB therapy. CONCLUSIONS: High-dose UVB therapy results in fewer treatments with better long-term efficacy, with cost-effective benefits for hospital and patients. Therefore UVB phototherapy in a high-dose regimen for psoriasis is recommended. However, a protocol adjustment in the second week with a high-dose regimen is desirable to prevent erythema.

Three-year registry data on biological treatment for psoriasis: the influence of patient characteristics on treatment outcome.

BACKGROUND: The course of biological treatment in clinical practice may be highly different from treatment schedules in clinical trials. Treatment modifications and patient characteristics may influence treatment safety and efficacy. So far, long-term results from the use of biological treatment in clinical practice are lacking. OBJECTIVES: To report short- and long-term efficacy and safety data on biologics, especially etanercept, used in daily clinical practice. Special attention has been paid to patient characteristics that may have influenced the response to therapy. METHODS: Prospectively collected registry data of all patients with psoriasis treated with biologics in the Radboud University Nijmegen Medical Centre outpatient clinic were used for analysis. Patient and treatment characteristics were surveyed. Efficacy and safety of etanercept for up to 3 years were analysed. Moreover, the influence of patient characteristics on etanercept treatment response was studied. RESULTS: The analysed cohort, consisting of 118 patients, went through 142 treatment episodes in total. Patients treated with biologics had an extensive medical history. Optimization of biological treatment was established in various ways, including treatment switches and introduction of concomitant therapies. Short-term etanercept efficacy analysis showed a mean Psoriasis Area and Severity Index (PASI) improvement at week 24 of 59.7%. No significant influence of gender, age, baseline PASI, body mass index, number of previous systemic therapies or duration of psoriasis was found on week 24 efficacy results, although trends were discernible. The efficacy of etanercept remained stable for up to 156 weeks. Long-term daily practice treatment with etanercept was only occasionally accompanied by major safety concerns. CONCLUSIONS: The current study demonstrates that etanercept is able to improve psoriasis symptoms for a considerable time, and that serious side-effects are infrequent. The influence of patient characteristics on treatment response is limited.

Effectiveness, side-effects and period of remission after treatment with methotrexate in localized scleroderma and related sclerotic skin diseases: an inception cohort study.

BACKGROUND: Detailed information is lacking on effectiveness of methotrexate (MTX) in sclerotic skin diseases, side-effects, and duration of remission after discontinuation. OBJECTIVES: To determine effectiveness, side-effects and period of remission gained by use of MTX in sclerotic skin diseases. METHODS: All patients with a sclerotic skin disease who were treated with MTX (group A) or MTX with corticosteroids (CS) (group B) between 1995 and 2007 were evaluated. Detailed information was collected on dosage and duration of MTX treatment, concomitant immunosuppressive medication and CS treatment, effectiveness, side-effects, duration of the remission period, and time until restart. RESULTS: Fifty-eight patients (A, n = 47; B, n = 11) were evaluated. Clinical assessment revealed that 38 patients (81%) treated with MTX and 11 patients (100%) treated with MTX + CS showed improvement of sclerotic skin. After one treatment course 51% of the patients treated with MTX and 73% treated with MTX + CS reached remission status with a median follow-up time of 55 and 58 months. Patients showing relapse still responded to a second and even to a third course of MTX. Patients who showed a relapse had received a lower cumulative dose, due to a shorter period of treatment with MTX in the first course. Serious side-effects were seen in six patients (10%). CONCLUSIONS: MTX was an effective treatment for various sclerotic skin diseases with a long period of remission and relatively low toxicity. Patients showing relapse still responded to a second and third course of MTX.

Relevance of compartmentalization of T-cell subsets for clinical improvement in psoriasis: effect of immune-targeted antipsoriatic therapies.

BACKGROUND: Therapies targeting the T cell-mediated pathology of psoriasis have been found to achieve remarkable clinical improvement and have confirmed the crucial role of the immune system either in peripheral blood (PB) or in skin. No analyses of T-cell counts in both compartments have been conducted in order to confirm or refute the hypothesized shifts between them. OBJECTIVES: To gain more insight in the dynamics of compartmentalization of T cells between PB and lesional skin of patients with psoriasis, in response to immune-targeted antipsoriatic therapies. METHODS: Eighteen patients with psoriasis received either efalizumab (n = 9) or etanercept (n = 9) for 12 weeks. Biopsies were taken for immunohistochemical analysis of T-cell subsets and simultaneously T-cell subsets were isolated from PB specimens by flow cytometry. RESULTS: The Psoriasis Area and Severity Index declined significantly after 12 weeks of etanercept, but not for efalizumab. After treatment with efalizumab, a significantly decreased number of all T-cell subsets was found in the dermis. In the epidermis, CD4+, CD8+, CD25+, CD45RO+ and CD161+ T-cell subsets were significantly decreased. With respect to etanercept, few significant changes in T-cell subsets were found. The percentage of lymphocytes in PB was significantly elevated after efalizumab treatment regardless of responder status. CONCLUSIONS: Treatment with efalizumab establishes successful recompartmentalization of T-cell subsets with modest clinical efficacy after 12 weeks, whereas in etanercept-treated patients, a significant clinical response is no guarantee for significant changes in T-cell subsets in the different compartments. Reductions in T-cell subsets cannot be used as predictive markers for the clinical response to therapy. Interference with the studied T-cell populations in its own right seems not to be responsible for the clinical efficacy of efalizumab and etanercept.

Psoriasis treatment with etanercept and efalizumab: clinical strategies influencing treatment outcome.

BACKGROUND: Multiple trials have been conducted in which the safety and efficacy of different biological therapies for psoriasis have been studied. However, the treatment course in clinical practice is different from the setting in which trials are conducted. OBJECTIVES: The evaluation of the efficacy, safety and adverse events of etanercept and efalizumab treatment in daily practice and the investigation of interfering clinical strategies that could be of influence on treatment outcome. METHODS: A prospective cohort consisting of 101 patients with high-need psoriasis was followed for 2 years and analysed. Patients were treated with etanercept or efalizumab between February 2005 and May 2007. Efficacy, safety and adverse events were investigated. Furthermore, all accompanying factors of which an influence on treatment efficacy outcome was suspected were registered, including treatment interruptions, dosage adjustments and combinations of therapies. RESULTS: Etanercept and efalizumab treatment was effective and safe in most patients. However, in many cases the treatment course was characterized by unsatisfactory efficacy (83%), necessitating combination therapies or dosage adjustments. Treatment interruptions occurred in 56% (etanercept 2x50 mg group), 84% (etanercept 2x25 mg group) and 10% (efalizumab-treated patients). CONCLUSIONS: Treatment of high-need psoriasis patients in daily practice is highly different from treatment courses in clinical trials. Frequently applied clinical strategies such as treatment interruptions, dosage adjustments and combinations of treatments influence treatment outcome in routine treatment in comparison with randomized controlled trials. Information about treatment with these new drugs in daily clinical practice is important for adjusting treatment schedules and guidelines.

A double-blind, randomized quantitative comparison of calcitriol ointment and calcipotriol ointment on epidermal cell populations, proliferation and differentiation.

BACKGROUND: Calcitriol and calcipotriol are widely used in the topical treatment of psoriasis. However, studies comparing both treatment modalities are scarce. Especially, there are almost no studies comparing the effects on epidermal cell populations in a quantitative manner. OBJECTIVES: The aim of this study was to quantitatively compare the effects of topical calcitriol and topical calcipotriol on clinical scores and epidermal subpopulations. PATIENTS AND METHODS: From five patients with stable plaque psoriasis, skin biopsies were taken from two symmetrical regions on the trunk or extremities before and after treatment with either calcitriol or calcipotriol. Frozen sections were labelled immunofluorescently using direct immunofluorescence for beta-1 integrin and the Zenon labelling technique for keratin (K) 6, K10 and K15. The digital photographs of the stained sections were quantitatively analysed and the results of both treatments were compared. RESULTS: The clinical SUM-score improved significantly for both the calcitriol- and the calcipotriol-treated lesions. In the calcipotriol-treated group the expression of K10 and K15 increased and the expression of K6 decreased significantly. No changes were seen for the marker beta-1 integrin. In the calcitriol-treated group none of the markers changed significantly. A tendency towards significance was seen for the changes in the expression of K6 and K15 in favour of calcipotriol. CONCLUSIONS: Both calcitriol and calcipotriol gave a significant improvement in clinical scores. However, treatment with calcipotriol resulted in a normalization of K6, K10 and K15, whereas treatment with calcitriol did not. Comparison of both treatments showed a tendency towards significance for the above-mentioned markers for calcipotriol only.

Monitoring hyperproliferative disorders in human skin: flow cytometry of changing cytokeratin expression.

BACKGROUND: Monitoring dynamics of different cell populations in solid tissues using flow cytometry has several limitations. The interaction and changes in epidermal subpopulations in hyperproliferative skin disorders such as psoriasis, a very common chronic inflammatory skin disease, may, however, elucidate the role of different cell populations in the pathogenesis and the effect of therapy in these disorders. We describe a new, simple method for identifying and quantifying different epidermal subpopulations in hyperproliferative skin disorders and an in vivo model for epidermal hyperproliferation by using six-parameter assessment and three-color flow cytometry. METHODS: Epidermal single-cell suspensions were derived from normal human skin before and after standardized injury and from untreated and treated psoriatic lesions. Samples were stained with anti-cytokeratins 6 (K6) and 10 (K10), anti-vimentin, and 4',6-diamidino-2-phenylindole. With three-color flow cytometry, different epidermal subpopulations were further defined by six different parameters. RESULTS: Correlations were found for mild psoriasis and K10(+)K6(-) cells and for moderate psoriasis and K10(-)K6(+) cells. Treatment of mild psoriasis resulted in a 70% increase of the K10(+)K6(-) cells and an 18% decrease of the K10(-)K6(-) cells, whereas treatment of more severe psoriasis resulted in a 77% increase of the K10(+)K6(-) cells and a 33% decrease of the K10(-)K6(+) cells. The tape-stripping model showed changes in suprabasal keratin expression before proliferative activity. CONCLUSIONS: The present flow cytometric assay permits simultaneous quantification of epidermal differentiation, inflammation, proliferation, and hyperproliferation-associated keratinization and provides information on pathogenesis and therapy of hyperproliferative skin disorders.

Single-cell image analysis to assess ABC-transporter-mediated efflux in highly purified hematopoietic progenitors.

BACKGROUND: Normal and malignant hematopoietic stem cells are characterized by their capacity to actively extrude fluorescent dyes. The contribution of different ATP-binding cassette (ABC) transporters to this phenomenon is largely unknown due to the small stem cell numbers limiting the use of standard methods to assess functional efflux. METHODS: We used epifluorescence microscopy (EFM) in combination with single-cell image analysis to study ABC-transporter-mediated efflux in highly purified, viable, CD34+CD38- cells sorted on an adhesive biolayer. P-glycoprotein and multidrug-resistant protein (MRP)-mediated efflux were quantitated using fluorescent substrates (rhodamine-123 and calcein acetoxymethyl ester [calcein-AM]) and specific inhibitors (verapamil and probenecid, respectively). RESULTS: The feasibility, sensitivity, and reproducibility of rhodamine-123 efflux quantitation using single-cell EFM was shown in cell lines and compared with standard flow cytometric assessment. P-glycoprotein-mediated transport was higher in CD34+CD38- cells than in more differentiated progenitors (mean efflux index = 2.24 +/- 0.35 and 1.14 +/- 0.11, respectively; P = 0.01). P-glycoprotein-mediated transport was the main determinant of the rhodamine "dull" phenotype of these cells. In addition, significant MRP-mediated efflux was demonstrated in CD34+CD38- and CD38+ cells (mean efflux index = 1.42 +/- 0.19 and 1.28 +/- 0.18, respectively). CONCLUSION: The described method is a valuable tool for assessing ABC-transporter-mediated efflux in highly purified single cells. Both P-glycoprotein and MRP-mediated efflux are present in human CD34+CD38- hematopoietic stem cells.

Long-term follow-up of persisting mixed chimerism after partially T cell-depleted allogeneic stem cell transplantation.

Using red cell phenotyping (RCP) and/or cytogenetics (CYT) we identified 19 patients with persisting mixed chimerism (MC) among 231 patients transplanted with partially T cell-depleted stem cell grafts from HLA-identical siblings. Persisting MC is defined as MC for more than 2 years in patients without any evidence of relapse. Median leukemia-free survival in these patients was 150 (range, 50-218) months. Diagnoses were ALL (n= 10); AML (n = 2); CML (n = 2); NHL (n = 2); MDS (n= 1); MM (n = 1) and SAA (n = 1). Purpose of this study was the long-term follow-up of MC and definition of patterns of chimerism in the various subsets of PBMCs and granulocytes. Using a PCR-STR technique CD3(+)/CD4(+) (T4 lymphocytes), CD3(+)/CD8(+) (T8 lymphocytes), CD45(+)/CD19(+) (B lymphocytes), CD45(+)/CD14(+) (monocytes), CD45(+)/CD15(+) (granulocytes) and CD3(-)/CD56(+) (NK-cells) were analyzed. The majority of patients with persisting MC were conditioned with a less intensive conditioning regimen and had little GVHD. Sequential monitoring of the chimerism resulted in a group of patients (n = 7) with very slow transient mixed chimerism that resulted in complete DC after median 7 years. Another nine patients had a relatively high percentage of persisting autologous cells for a median of 12 years and in three patients we observed a stable low percentage of autologous cells. Only two out of 19 patients (AML-CR1, CML-CP1) relapsed during follow-up. Both patients had a relatively high percentage of autologous cells. Chimerism in granulocytes and PBMC subsets was analyzed at a median of 8 years after SCT in nine patients. In five patients mixed chimerism simultaneously detected by RCP and CYT was associated with MC in all subsets. Within each individual patient the percentages of donor and recipient cells were very different between the different subsets. Two CML-CP1 patients were mixed chimera in only two subsets and in one patient these subsets represented pending relapse. In another two patients mixed chimerism with a very low number of autologous red cells was not found in the PBMCs because of the different sensitivity level of the RCP and the PCR-STR technique. We conclude that in patients with persisting mixed chimerism after partially T cell-depleted SCT a remarkable number of patients had lymphoid malignancies, the majority of the patients were conditioned with less intensive conditioning regimens and the mixed chimerism was not correlated with relapse. Chimerism in granulocytes and PBMC subsets did show great intra-individual differences in the subsets and these data correlated well with RCP and CYT data with the exception of the NK cells.

The dynamic process of apoptosis analyzed by flow cytometry using Annexin-V/propidium iodide and a modified in situ end labeling technique.

BACKGROUND: To study the apoptotic process in time, we used the following flow cytometric (FCM) techniques: phosphatidylserine (PS) translocation by Annexin-V (AnV), DNA fragmentation by in situ end labeling (ISEL), and propidium iodide (PI) staining. Because PS translocation is assumed to be an early feature of programmed cell death (PCD), we questioned if AnV positivity implies inevitable cell death. METHODS: Apoptosis was induced in Jurkat cells by gamma-irradiation, incubation with camptothecin (CPT), or cytosine beta-D-arabinofuranoside (Ara-C). At different time intervals, PCD was quantified by AnV/PI and ISEL. To analyze the influence of cell handling procedures on PCD, we applied these three FCM techniques on CD34+ bone marrow (BM) stem cells after selection and after a freeze-thaw procedure. Various AnV/PI- CD34+ fractions were cultured in a single-cell single-well (SCSW) assay. RESULTS: Jurkat cells under three different detrimental conditions showed essentially the same pattern of apoptosis in time. Initially developed AnV+/PI- cells subsequently (within 1 h) showed ISEL positivity, after which they turned into AnV+/PI++ cells with even higher levels of ISEL positivity (80-90%). Eventually, they lost some of their PI and ISEL positivity and formed the AnV+/PI+ fraction. Cell handling of CD34+ cells caused high and variable AnV+/PI- fractions (overall range 23-62%). Within total AnV+ and AnV+/PI- populations, only a minority of CD34+ cells showed ISEL positivity (range 4-8% and 0.8-6%, respectively). Different fractions of AnV+/PI- CD34+ cells did have clonogenic capacity. CONCLUSIONS: PCD of cell suspensions in vitro can be followed accurately in time by these three FCM techniques. PS translocation is followed rapidly (within 1 h) by oligo-nucleosomal DNA fragmentation, after which cell (and nuclear) membrane leakage occurs. Detection of PS asymmetry by AnV-fluorescein isothiocyanate (FITC) is not always associated with (inevitable) apoptosis, as can be concluded from the proliferative capacity of AnV+ /PI- CD34+ cells in the SCSW assay.

Idarubicin DNA intercalation is reduced by MRP1 and not Pgp.

Currently available data regarding the substrate specificity of the multi-drug resistance (MDR) mechanisms P-glycoprotein (Pgp) and MDR-associated protein (MRP1) for idarubicin are inconclusive. A multiparameter flow cytometry method was developed which allows simultaneous quantitative measurement of total cellular fluorescence and the amount of anthracyclines intercalated into the DNA. Anthracycline DNA intercalation was measured by fluorescence resonance energy transfer (FRET) between Hoechst 33342 and anthracyclines. Daunorubicin and idarubicin accumulation were studied and compared in established cell lines expressing Pgp and MRP1. The data demonstrate that daunorubicin DNA intercalation is affected by both Pgp and MRP1 whereas idarubicin DNA intercalation is affected only by MRP1. MRP1 and Pgp function could be blocked completely by 5 microM PAK 104P, while higher concentrations of verapamil, PSC 833 and cyclosporin A were necessary to attain complete blocking of MRP1 compared to Pgp. Daunorubicin DNA intercalation correlates better with cell survival and is more sensitive at physiological MDR expression as observed in hematopoietic progenitors than daunorubicin levels measured by total cellular fluorescence. In conclusion, idarubicin DNA intercalation is reduced by MRP1 but not by Pgp. PAK-104P is an effective modulator for both Pgp and MRP1 and may further improve idarubicin efficacy.

Dithranol embedded in crystalline monoglycerides combined with phototherapy (UVB): a new approach in the treatment of psoriasis.

Treatment of psoriasis with dithranol as monotherapy or dithranol in combination with UVB phototherapy is an effective and safe approach for the management of psoriasis. Recently a new formulation of dithranol embedded in crystalline monoglycerides (Micanol) has become available. It was shown that this formulation combines adequate efficacy with low irritation and staining properties. The aim of the present study was to compare and contrast three treatment schedules with respect to clinical efficacy and tolerability: Micanol monotherapy, Micanol in combination with UVB phototherapy and placebo combined with UVB phototherapy. The study design was a partly open, partly double-blind, randomized, left-right comparison. In total 36 patients were included and 24 body halves were available for each of the three treatments. The combination of Micanol with UVB resulted in clearance of lesions in 54% of the patients (body halves). Combination therapies with Micanol and either of the two other therapies were highly effective. However, with the number of patients investigated, a statistically significant difference between the three therapeutic approaches with respect to efficacy could not be shown. The three treatments resulted in a grosso modo comparable clinical improvement. Severe irritation was observed in 8% and staining of the skin in 29% of the patients treated with the combination therapy Micanol/UVB, which is far less compared to the irritation and staining by the conventional short contact approaches. The efficacy and tolerability of Micanol make this active substance an important tool in the management of psoriasis.

Automatic analysis of growth onset, growth rate and colony size of individual bone marrow progenitors.

A method for automatic enumeration of proliferating bone marrow progenitors after single cell sorting is described. The system is based on regular inverse microscopy, recording with a video camera, and image analysis using dedicated software on an Apple computer. Single CD34+ progenitor cells were sorted in 96-well plates. Three times weekly phase-contrast video images of each well were stored and analyzed for the actual number of cells. From the subsequent counts growth curves were plotted for each individual progenitor. Enumeration by image analysis correlated very well with manual cell counting (r = 0.99, P < 0.0001). To show the capability of the method to analyze growth rate and growth delay, more differentiated (CD34+/CD13+/CD33+) progenitors were compared with more primitive (CD34+/CD13+/CD33g-) progenitors. Differences in the timing of colony outgrowth were shown to be based on delay in growth initiation. Initiation of growth was delayed 2.6-3.1 days in CD34+/CD13+/ CD33- fraction of 3 different donors (P < 0.0001). The growth rates of the progenitors in both fractions were not significantly different. The described method seems important to more accurately evaluate subpopulations of progenitors, the effect of growth promoting or inhibiting factors, and effects of cytotoxic drugs and irradiation.