bluuebunny/crossref_metadata_2025 huggingface dataset:
Sirolimus steady‐state trough concentrations are not affected by bolus methylprednisolone therapy in renal allograft recipients
Authors: Bäckman L. , Kreis H. , Morales J. M. , Wilczek H. , Taylor R. , Burke J. T.
Published: March 2003

Aims  To determine whether bolus doses of methylprednisolone affect the steady‐state trough concentrations of sirolimus.

Methods  Fourteen renal transplant recipients received concentration‐controlled sirolimus therapy in combination with azathioprine and steroids (n=8) or mycophenolate mofetil and steroids (n=6). Bolus doses of methylprednisolone (mean total dose over 1–5 days, 1694 mg; range, 500–3000 mg) were given for the treatment of acute rejection. For each patient, the sirolimus dose (mean, 24.1 mg; range, 3.3–52.5 mg) was the same before and during methylprednisolone therapy.

Results  Mean sirolimus whole blood trough concentrations before and after treatment with methylprednisolone were 28.8 ng ml−1 (range, 13.9–45.3 ng ml−1), and 28.5 ng ml−1 (range, 13.0–47.9 ng ml−1), respectively (P=0.85; 95% confidence interval on the difference −3.3, 4.0 ng ml−1).

Conclusions  Bolus methylprednisolone treatment does not affect steady‐state sirolimus trough concentrations.
Relationships between body composition parameters and fluorouracil pharmacokinetics
Authors: Gusella Milena , Toso Sivia , Ferrazzi Eros , Ferrari Mariano , Padrini Roberto
Published: March 2003

Aims  To verify whether fluorouracil (FU) clearance (CL) and volume of distribution (Vss) are better correlated with specific body compartments, such as body cell mass (BCM), total body water (TBW) or fat free mass (FFM), rather than with body surface area (BSA) or total body weight (BW).

Methods  Thirty‐four patients (13 females and 21 males) affected by colorectal cancer and receiving FU as adjuvant therapy entered the study. CL and Vss were determined after a 2 min i.v. injection of FU (425 mg m−2) and leucovorin (20 mg m−2). Body composition, in terms of BCM, TBW and FFM, was evaluated non‐invasively by bioelectrical impedance analysis (BIA).

Results  Significant but poor correlations were found between CL or Vss and most anthropometric parameters, including BIA‐derived measures (r2 range=0.10–0.21). However, when multiple regression analysis was performed with sex, TBW and FFM as independent variables, the correlations improved greatly. The best correlation was obtained between CL and sex (r2=0.44) and between Vss and sex (r2=0.36). FFM‐normalized CL was significantly higher in women than in men (0.030±0.008 vs 0.022±0.005 l min−1 kg−1; 95% CI of difference 0.012, 0.003; P=0.003), suggesting that FU metabolism is more rapid in females. Surprisingly, Vss was highly correlated with CL (r2=0.67; CL=0.52+Vss×0.040). This finding may either be explained by extensive drug metabolism in extra‐hepatic organs or by variable inactivation on first‐pass through the lung. Both these hypotheses need experimental validation.

Conclusions  The pharmacokinetics of FU are better predicted by FFM and TBW than by standard anthropometric parameters and predictions are sex‐dependent. The use of BIA may lead to improved dosing with FU.
Intestinal first pass metabolism of midazolam in liver cirrhosis – effect of grapefruit juice
Authors: Andersen Vibeke , Pedersen Natalie , Larsen Niels‐Erik , Sonne Jesper , Larsen Steen
Published: March 2003

Aims  Grapefruit juice inhibits CYP3A4 in the intestinal wall leading to a reduced intestinal first pass metabolism and thereby an increased oral bioavailability of certain drugs. For example, it has been shown that the oral bioavailability of midazolam, a CYP3A4 substrate, increased by 52% in healthy subjects after ingestion of grapefruit juice. However, this interaction has not been studied in patients with impaired liver function. Accordingly, the effect of grapefruit juice on the AUC of midazolam and the metabolite α‐hydroxymidazolam was studied in patients with cirrhosis of the liver.

Methods  An open randomized two‐way crossover study was performed. Ten patients (3 females, 7 males) with liver cirrhosis based on biopsy or clinical criteria participated. Six patients had a Child‐Pugh score of A, one B and three C. Tap water (200 ml) or grapefruit juice were consumed 60 and 15 min before midazolam (15 mg) was administered orally. Plasma samples were analysed for midazolam and α‐hydroxymidazolam.

Results  Grapefruit juice increased the AUC of midazolam by 106% (16, 197%) (mean (95% confidence interval)) and the AUC of the metabolite α‐hydroxymidazolam decreased to 25% (12, 37%) (P<0.05 for both). The ratio of the AUCs of the metabolite α‐hydroxymidazolam to midazolam decreased from 0.77 (0.46, 1.07) to 0.11 (0.05, 0.19) (P<0.05). t½ remained unaltered for both drug and metabolite. Midazolam Cmax, tmax, and α‐hydroxymidazolam tmax increased, but these changes were not statistically significant, whereas Cmax of the metabolite decreased to 30% (14, 47%) (P<0.05).

Conclusions  A marked interaction between oral midazolam and grapefruit juice was found and the data are consistent with a reduced first‐pass metabolism of midazolam. This is likely to occur at the intestinal wall inhibition of CYP3A4 activity by grapefruit juice. These results indicate that patients with liver cirrhosis are more dependent on the intestine for metabolism of CYP3A4 substrates than subjects with normal liver function.
Pharmacokinetics and effects of propofol 6% for short‐term sedation in paediatric patients following cardiac surgery
Authors: Knibbe Catherijne A. J. , Melenhorst‐de Jong Gitte , Mestrom Maaike , Rademaker Carin M. A. , Reijnvaan Allart F. A. , Zuideveld Klaas P. , Kuks Paul F. M. , Van Vught Hans , Danhof Meindert
Published: March 2003

Aims
This paper describes the pharmacokinetics and effects of propofol in short‐term sedated paediatric patients.

Methods
Six mechanically ventilated children aged 1–5 years received a 6 h continuous infusion of propofol 6% at the rate of 2 or 3 mg kg−1 h−1 for sedation following cardiac surgery. A total of seven arterial blood samples was collected at various time points during and after the infusion in each patient. Pharmacokinetic modelling was performed using NONMEM. Effects were assessed on the basis of the Ramsay sedation score as well as a subjective sedation scale.

Results
The data were best described by a two‐compartment pharmacokinetic model. In the model, body weight was a significant covariate for clearance. Pharmacokinetic  parameters in the weight‐proportional model were clearance (CL) = 35 ml kg−1 min−1, volume of central compartment (V1) = 12 l, intercompartmental clearance (Q) = 0.35 l min−1 and volume of peripheral compartment (V2) = 24 l. The interindividual variabilities for these parameters were 8%, < 1%, 11% and 35%, respectively. Compared with the population pharmacokinetics in adults following   cardiac   surgery   and   when   normalized   for   body   weight,   statistically   significant    differences   were   observed   for   the   parameters   CL   and  V1   (35  vs 29 ml kg−1 min−1 and 0.78 vs 0.26 l kg−1P < 0.05), whereas the values for Q and V2 were similar (23 vs 18 ml kg−1 min−1 and 1.6 vs 1.8 l kg−1, P > 0.05). In children, the   percentage   of   adequately   sedated   patients   was   similar   compared   with   adults (50% vs 67%) despite considerably higher propofol concentrations (1.3 ± 0.10 vs 0.51 ± 0.035 mg l−1, mean ± s.e. mean), suggesting a lower pharmacodynamic sensitivity  to propofol in children.

Conclusions
In children aged 1–5 years, a pharmacokinetic model for propofol was described using sparse data. In contrast to adults, body weight was a significant covariate for clearance in children. The model may serve as a useful basis to study the role of covariates in the pharmacokinetics and pharmacodynamics of propofol in paediatric patients of different ages.
The prevalence of CYP2C19 genotypes in a population of healthy Dutch volunteers and apparent gender differences in phenotypes
Authors: Tamminga Wim J. , Wemer Johan , Oosterhuis Berend , De Zeeuw Rokus A. , De Leij Lou F. M. H. , Jonkman Jan H. G.
Published: March 2003

We reported the prevalence of poor metabolizers of CYP2C19 to be 1.8% in a sample of the Dutch population (n=4301; [1]). These data were obtained by phenotyping. In that study pronounced oral contraceptive related gender differences for CYP2C19 activity were reported. The present study was performed to estimate the prevalence of non‐coding mutations of CYP2C19 leading to the poor metabolizer phenotype. Furthermore, the data were analysed for gender differences.

Seven hundred and sixty‐five unrelated healthy volunteers were evaluated for their CYP2C19 status. Subjects were phenotyped using mephenytoin. Genotyping was performed by the polymerase chain reaction (PCR) and restriction analysis for the most common null alleles.

For CYP2C19*2 and CYP2C19*3 a frequency of 13.3% and 0.2%, respectively, was observed. The S/R‐ratio was significantly higher in heterozygous subjects (S/R‐ratio=0.22) compared with homozygous wild type subjects (S/R‐ratio=0.11). Comparison of all subjects below 45 years showed a significantly higher S/R‐ratio in females compared with males especially in heterozygous subjects (S/R‐ratio: 0.39 vs 0.19; P<0.001).

The frequencies of CYP2C19 allelic variants in a population of Dutch healthy volunteers were in accordance with data on other European populations. Assessment of *2 and *3 for CYP2C19 predicted the phenotype with an accuracy of over 98.6%. Gene‐dose effect existed for CYP2C19. CYP2C19 heterozygous females had a decreased CYP2C19 activity that may be at least partly due to the use of oral contraceptives.
Does the change of omeprazole to pantoprazole affect clozapine plasma concentrations?
Authors: Mookhoek E. J. , Loonen A. J. M.
Published: March 2003

Many chronic psychiatric inpatients suffer from schizophrenia and gastric disorders. Some of them are therefore treated with a combination of clozapine and an acid pump inhibitor. The atypical antipsychotic clozapine is metabolized by CYP1A2, CYP3A4 and CYP2D6. The acid pump inhibitor omeprazole can inhibit the activity of CYP2C19 and induce the activity of CYP2C8‐9, CYP3A4 and CYP1A2. The acid pump inhibitor pantoprazole is largely devoid of these activities. The possibility of pharmacokinetic drug drug interactions with psychotropic drugs lead in Spring 2000 to the replacement of omeprazole by pantoprazole in the drug formulary of Delta Psychiatric Hospital. In this hospital the plasma concentrations of clozapine and its primary metabolite are measured at regular intervals on a routine basis. This offers the opportunity to investigate the possible consequences of the change of acid pump inhibitor.

The aim was to retrospectively assess the possible effect of the change of omeprazole to pantoprazole on the plasma concentrations of clozapine.

The medication records of all long‐stay psychiatric in‐patients were checked and those patients were selected who used omeprazole and clozapine on April 4, 2000 and who were changed to pantoprazole for the rest of the year. The medical records of these patients were searched for data on clozapine plasma concentrations before and at least 6 weeks after the change, necessary changes of the clozapine dosage due to a changed clinical condition, and possibly reported adverse events. The data were controlled for co‐medication that influences the activity of CYP isoforms and for smoking habits.

Out of 430 long‐stay patients 49 used omeprazole and 15 also used clozapine. Of these, 13 were finally evaluable. The 13 patients used 423±153.6 mg clozapine per day (mean±s.d.). Before the change, the clozapine concentrations measured 382±190.6 µg l−1. After the change, these concentrations measured 382±158.9 µg l−1. In five patients an increase in plasma concentrations was observed. Three of them were non‐smokers. A significant correlation (Pearson Rank, −0.615, P=0.025) was found between the second clozapine concentration and the amount of tobacco smoked per day. There existed no reports of changes in the medical condition, particularly the occurrence of relevant adverse events, that could be attributed to a change in clozapine plasma concentrations.

No relevant changes were observed retrospectively in the plasma concentrations and clinical effects of clozapine due to the alteration of omeprazole to pantoprazole in this small patient population. However, there appears to exist a relationship between the smoking habits and the sensitivity to this change. This might be due to the CYP inducing activity of tobacco smoke, which may be more important than the CYP inducing activity of omeprazole.
In vitro permeation of β‐lactam antibiotics across rat jejunum and its correlation with oral bioavailability in humans
Authors: Saitoh Hiroshi , Aungst Bruce J. , Tohyama Masashi , Hatakeyama Yuko , Ohwada Keiko , Kobayashi Michiya , Fujisaki Hiroko , Miyazaki Katsumi
Published: March 2003

Aims
To investigate the correlation between in vitro permeation of 11 β‐lactam antibiotics across rat jejunum and their oral bioavailability in humans.

Methods
The absorptive and secretory permeation across rat jejunum was evaluated and apparent permeability coefficients (Papp) were determined.

Results
A steep, sigmoid‐type curve was obtained for the relationship between Papp in the absorptive permeation and human oral bioavailability. When the ratios of Papp in the absorptive direction to Papp in the secretory direction were plotted against human oral bioavailability, a much improved correlation was obtained (r = 0.98, P < 0.001). The addition of glycylglycine to both mucosal and serosal media modi­fied the permeation of ceftibuten and cephalexin from the absorptive to the secretory direction.

Conclusions
For 11 β‐lactam antibiotics rat intestinal permeation correlated well with human oral bioavailability, especially when corrected for secretory transport.
A comparison of relative abundance, activity factor and inhibitory monoclonal antibody approaches in the characterization of human CYP enzymology
Authors: Soars Matthew G. , Gelboin Harry V. , Krausz Kristopher W. , Riley Robert J.
Published: March 2003

Aims The objective of this study was to evaluate the potential uses of relative abundance, relative activity approaches and inhibitory monoclonal antibodies (mAbs) in the characterization of CYP enzymology in early drug discovery.

Methods Intrinsic clearance estimates for the oxidation of ethoxyresorufin (a selective probe of CYP1A2 activity), tolbutamide (CYP2C9), S‐mephenytoin (CYPC19), dextromethorphan (CYP2D6) and testosterone (CYP3A4) were used to determine relative activity factors (RAFs). CLint values were determined for the metabolism of 14 drugs in human liver microsomes (HLM) and for these major CYPs. The relative contribution of each individual CYP to the oxidation of each drug was then assessed using relative abundance and activity techniques in addition to inhibitory mAbs.

Results Relative abundance and activity methods as well as inhibitory mAbs qualitatively assigned the same CYP isoform as predominantly responsible for the clearance of each drug by HLM. Metabolism catalysed by CYP1A2, 2C9, 2D6 and 3A4 was also predicted to be quantitatively similar using both abundance and activity techniques. However, the relative contribution of the polymorphic CYP2C19 appeared to be over‐estimated approximately two‐fold using recombinant CYP compared with that from the HLM and mAb approach.

Conclusions All three methods investigated in this study appear suitable for use in the characterization of the CYP metabolism of new chemical entities produced during early drug discovery.
Blood pressure response to conventional and low‐dose enalapril in chronic renal failure
Authors: Elung‐Jensen Thomas , Heisterberg Jens , Kamper Anne‐Lise , Sonne Jesper , Strandgaard Svend
Published: March 2003

Aims In chronic renal failure, the clearance of most ACE inhibitors including enalapril is reduced. Hence, with conventional dosage, plasma enalaprilat may be markedly elevated. It is unclear whether this excess of drug exposure affords an improved control of blood pressure. The aim of the present study was to evaluate short‐term blood pressure response to two different plasma levels of enalaprilat.

Methods As part of an open, randomized, controlled trial of the effect of high and low dosage of enalapril on the progression of renal failure, short‐term blood pressure response was evaluated. Data were analysed in all patients completing 3 months of follow‐up. The patients were allocated to two trough plasma concentrations of enalaprilat, either above 50 ng ml−1 (high) (n = 17) or below 10 ng ml−1 (low) (n = 18), and the daily dose of enalapril titrated accordingly.

Results Median (range) glomerular filtration rate (GFR) at baseline was 18 (7.9) in the high enalaprilat concentration group and 17 (7.3) ml min−1 1.73 m2 in the low concentration group (NS). Nine patients in each group were on treatment with enalapril at baseline with a median daily dose of 5 mg in both the high (5–10) and low (2.5–20) concentration group. At 3 months’ follow‐up, the dose was 10 (2.5–30) and 1.9 (1.25–5) mg (P < 0.0001), respectively. After 3 months median trough concentrations of enalaprilat were 82.5 (22–244) ng ml−1 and 9.1 (2.5–74.8) ng ml−1 (P < 0.002). At baseline the median systolic blood pressures in the two groups were 140 (110–200) and 133 (110–165), in the high and low enalaprilat concentration groups, respectively, and after 3 months they were 135 (105–170) and 130 (105–170) mmHg (NS). Median diastolic blood pressure was 80 mmHg in each group both at baseline (65–100) and at follow‐up (60–95) (NS). There was no difference between the groups in concomitant antihypertensive treatment (number of patients treated, mean daily dose) during the observation period. Proteinuria remained stable during the study period in both groups; patients in the high concentration group had higher plasma potassium concentrations at day 90 and patients in the low group experienced a slight increase in GFR.

Conclusions  In moderate to severe chronic renal insufficiency the same degree of blood pressure control was achieved on low as well as moderate daily doses of enalapril. This was irrespective of concomitant antihypertensive treatment.
Low‐dose atorvastatin therapy does not augment endothelial function in active hypercholesterolaemic males
Authors: Parnell Melinda M. , Chin‐Dusting Jaye P. F. , Starr Jennifer , Kaye David M.
Published: March 2003

Aims As statin therapy has been demonstrated to augment endothelial function in sedentary hypercholesterolaemia (HC), we aimed to investigate the effects of atorvastatin therapy on endothelial function in physically active, HC men.

Method and results Eleven physically active, HC males were recruited. Endothelial function [forearm blood flow response to brachial artery infusion of acetylcholine (Ach)] was assessed twice in each subject following atorvastatin or no therapy in a randomized crossover design. In addition, endothelial function was compared with an active, normolipidaemic control group (C). Atorvastatin therapy reduced total and LDL cholesterol, but had no effect on basal blood flow or endothelial function (peak ACh mean difference ± standard error 0.75 ± 1.75 ml min−1 per 100 ml tissue) [95% confidence interval (CI) −3.1, 4.6]. In addition, there was no difference in endothelial function between the HC and C groups (−1.14 ± 2.60 ml min−1 per 100 ml tissue; CI −6.53, 4.25).

Conclusion Statin therapy in HC patients with normal endothelial function does not augment endothelial function.
Changes in collagen I and collagen III metabolism in patients with generalized atopic eczema undergoing medium-dose ultraviolet A1 phototherapy
Authors: Mempel M. , Schmidt T. , Boeck K. , Brockow K. , Stachowitz S. , Fesq H. , Schäfer T. , Thomsen S. , Schnopp C. , Ring J. , Probst R. , Luppa P. , Abeck D.
Published: March 2003

Fourteen patients suffering from acute, exacerbated atopic eczema were screened for changes in collagen I and collagen III metabolism in serum (n = 11), urine (n = 11) and skin biopsies (n = 9) before and after medium-dose ultraviolet (UV) A1 phototherapy (15 exposures of 50 J/cm2 over a 3-week period, total dose 750 J/cm2). Mature collagen I and, to a lesser extent, mature collagen III were found to be decreased after the therapy in skin samples from the irradiated patients. As markers of collagen I degradation, the cross-links pyridoline and deoxypyridoline were analysed in urine using high-performance liquid chromatography. Both cross-links were found to be mildly increased after UVA1 phototherapy, without reaching statistical significance. As markers of de novo collagen synthesis we screened for the procollagen I-carboxyterminal peptide (PICP) and procollagen III-aminoterminal peptide (PIIINP) levels in serum and skin. The ratio of PICP to PIIINP in serum dropped significantly after the UVA1 phototherapy, suggesting a different impact of UVA1 on the two collagens. These findings were paralleled by a diminished ratio of PICP to PIIINP in tissue samples. Staining for matrix metalloproteinase 1 (MMP-1) and its specific counterpart, tissue inhibitor of MMP-1 (TIMP-1), showed slight increases for both proteins by therapeutic UVA1; this was also seen in serum for TIMP-1 but not MMP-1. In our study, high-energy UVA1 doses induced changes of the skin collagens in patients with atopic eczema which are measurable by their metabolites in serum and urine.
The treatment of atopic dermatitis with adjunctive high-dose intravenous immunoglobulin: a report of three patients and review of the literature
Authors: Jolles S. , Hughes J. , Rustin M.
Published: March 2003

There are few reports of the use of high-dose intravenous immunoglobulin (hdIVIg) in the treatment of atopic dermatitis (AD). We describe our experience using this therapy in three patients with severe AD, all of whom had steroid-related side-effects. These patients received either Alphaglobin® or Sandoglobulin® 2 g/kg monthly: all had improved skin scores, allowing reduction of their steroid dose. Total IgE fell in one of three patients. We discuss the side-effects of hdIVIg and their management, and detail the differences between the available immunoglobulin products available in the U.K. There are several proposed mechanisms of action of this therapy which may be operative, and those most important in AD are discussed. In view of the time and expense involved in the treatment of patients with hdIVIg, careful patient assessment is vital. We describe dose reduction strategies and methods for cost containment. In addition, one of the patients has embarked on IVIg home therapy training. This will be the first time this has been attempted for a dermatological indication. Training of this type may be available through an immunotherapy service such as exists for patients with primary immunodeficiencies.
Regulation of the uPAR/uPA system expressed on monocytes by the deactivating cytokines, IL‐4, IL‐10 and IL‐13: consequences on cell adhesion to vitronectin and fibrinogen
Authors: Paysant Jérôme , Vasse Marc , Soria Jeannette , Lenormand Bernard , Pourtau Jérôme , Vannier Jean‐Pierre , Soria Claudine
Published: March 2003

Urokinase (uPA) and its receptor (uPAR) have been proposed to be involved in monocyte migration by inducing degradation of matrix proteins. In addition, uPAR is also implicated in cell adhesion to the vascular wall. The adhesive function of uPAR depends on a direct interaction with vitronectin which is increased by uPA and by modification of cell surface integrin (such as CD11b–CD18) when associated to uPAR. In this study we analysed the role of three deactivating cytokines, IL‐4, IL‐10 and IL‐13, on the surface expression of uPA, uPAR and CD11b by monocytes and their consequences on monocyte adhesion to immobilized fibrinogen and vitronectin.

IL‐10 induced a decrease in uPA and CD11b after 18 h incubation and a delayed decrease in uPAR which was only significant after 48 h incubation. These results may explain the decrease in monocyte adhesion, which was observed after an 18 h incubation with IL‐10, on immobilized vitronectin and fibrinogen. In contrast, IL‐4 and IL‐13 induced a decrease in uPAR after 18 h and a significant increase in uPA both in the cell lysates and at the cell surface, as well as an increase in cell surface associated CD11b. These cytokines did not modify cell adhesiveness to vitronectin or fibrinogen despite the increase in CD11b–CD18. This could be due to the decrease in uPAR because CD11b–CD18/uPAR forms a cell adhesion complex. In addition, the increase in uPA induced by IL‐4 could counterbalance the direct interaction of uPAR with vitronectin. The increase in uPA suggests that IL‐4 and IL‐13 could induce plaque fissuring by monocytes, whereas IL‐10 may induce protection against matrix protein degradation by decreasing uPA.
Comparative adhesion of human haemopoietic cell lines to extracellular matrix components, bone marrow stromal and endothelial cultures
Authors: Turner Marc L. , Masek Lisa C. , Hardy Cheryl L. , Parker Alistair C. , Sweetenham John W.
Published: March 2003

We used flow cytometry to characterize cell adhesion molecule expression of the human haemopoietic cell lines KG1a, K562, HL‐60, NALM‐6 and CEM. A 51chromium labelling assay was used to study the adhesion of these cell lines to extracellular matrix components and to bone marrow stromal and endothelial cultures. Both adhesion molecule expression and functional binding behaviour varied between cell lines. All five cell lines expressed the integrins α4β1 and α5β1 and all adhered to fibronectin. However, differences in intensity of expression of these integrins failed to correlate with extent of fibronectin adhesion. Inhibition experiments demonstrated that adhesion of KG1a to fibronectin was completely inhibited by divalent cation chelation and partially inhibited by RGDS peptides and chondroitinase ABC, suggesting that both α4β1 and α5β1 as well as CD44 were responsible for this interaction. Adhesion to bone marrow stromal and endothelial layers was superior to that to purified extracellular matrix components and was partially inhibited by divalent cation chelation. RGD peptides and anti‐α4 monoclonal antibody also partially inhibited KG1a adhesion to bone marrow endothelium.

Discordance between cell adhesion molecule expression and adhesive behaviour suggest that current phenotypic descriptions remain incomplete and reinforce the need for complementary functional binding studies.
Large granular lymphocytic leukaemia with a mixed T‐cell/B‐cell phenotype
Authors: Akashi Koichi , Shibuya Tsunefumi , Nakamura Minoru , Oogami Akiko , Harada Mine , Niho Yoshiyuki
Published: March 2003

We report a case of large granular lymphocytic leukaemia (LGLL) with mixed T‐cell/B‐cell phenotypes. The LGLL cells expressed T‐cell markers such as CD1, CD2, CD3, CD5, CD7, CD8 and CD57. The CD8+ LGLL cells coexpressed B‐cell markers including CD20 and PCA‐1, and a fraction of purified CD8+ LGLL cells secreted double isotypes of immunoglobulins (IgG‐κ and IgA‐κ). Both TCRB and IGH genes were clonally rearranged. The LGLL cells could be divided into at least three subpopulations that were cytogenetically distinct, and all subpopulations involved the 11q23. The expression of both T‐ and B‐cell markers on the LGLL cells suggests the involvement of a putative common lymphoid progenitor in leukaemic transformation.
Hereditary sideroblastic anaemia due to a mutation in exon 10 of the erythroid 5‐aminolaevulinate synthase gene
Authors: Edgar  , Wickramasinghe
Published: March 2003

DNA sequencing of the coding region of the erythroid 5‐aminolaevulinate synthase (ALAS2) cDNA from a male with pyridoxine‐responsive sideroblastic anaemia revealed a missense mutation C1622G and a closely linked polymorphism C1612A in exon 10 of the gene. Sequence analysis of the genomic DNA from other family members revealed that the proband's mother and daughter were heterozygous carriers of the mutation, consistent with the X‐linked inheritance. The C1622G mutation results in a histidine to aspartic acid substitution at amino acid residue 524. The histidine residue is conserved in both the erythroid and housekeeping ALAS proteins in vertebrates, all other known ALAS proteins and other oxamine synthases that have pyridoxal 5′‐phosphate as a co‐factor. This histidine is located in a predicted loop, preceding a long alpha‐helix region near the carboxy‐terminus.
Successful excision of a pseudotumour in a congenitally factor V deficient patient
Authors: Tanis  , van der Meer  , Bloem  , Vlasveld
Published: March 2003

We report a patient with congenital homozygous factor V deficiency in whom a large pseudotumour in the right upper leg was successfully surgically excised under continuous substitution with fresh‐frozen plasma.
Minimal residual disease analysis for the prediction of relapse in children with standard‐risk acute lymphoblastic leukaemia
Authors: Goulden Nicholas J. , Knechtli Christopher J. C. , Garland Russell J. , Langlands Kenneth , Hancock Jeremy P. , Potter Michael N. , Steward Colin G. , Oakhill Anthony
Published: March 2003

We report a largely retrospective analysis of minimal residual disease (MRD) in a cohort of 66 children suffering from acute lymphoblastic leukaemia (ALL). All patients lacked high‐risk features at diagnosis, i.e. the presenting white cell count was <50 × 109/l, age 1–16 years and translocations t(9;22) and t(4;11) were not present. All were treated according to either the MRC protocols UKALL X or XI. PCR of IgH, TCRδ and TCRγ gene rearrangements and allele‐specific oligoprobing were employed for the detection of MRD. Sensitivity was at least 10−4 in 78/82 (93%) probes examined.

A total of 33 patients relapsed (seven on therapy and 26 off) and 33 remain in continuing complete remission (CCR) (median follow‐up 69 months from diagnosis). Of those who remain in CCR, MRD was present in the bone marrow in 32%, 10% and 0% at 1, 3 and 5 months into therapy respectively. This is in marked contrast to the presence of MRD at these times in 82%, 60% and 41% of patients who relapsed (P<0.001, P<0.005 and P<0.005). These results provide further evidence of a strong correlation between clearance of MRD early in therapy and clinical outcome in childhood ALL.
Flow cytometric identification of candidate normal stem cell populations in CD45‐negative B‐cell precursor acute lymphoblastic leukaemia
Authors: Vormoor  , Baersch  , Baumann  , Ritter  , Jürgens
Published: March 2003

CD45‐negative B‐cell precursor acute lymphoblastic leukaemia (ALL) provides a unique model to study the stem cell compartment in ALL as leukaemic CD34‐positive cells, unlike their normal counterparts, do not express CD45. By increasing the number of events analysed to 106, storing only the events in the region of interest (storage gate), using appropriate isotype controls and stringent washing procedures, a flow cytometric protocol was established to characterize rare CD34+CD19− events. In eight of 12 patients (67%) with CD45‐negative B‐cell precursor ALL, a distinct CD34+CD19−CD45+ candidate normal stem cell population could be detected. In one patient analysed by four‐colour staining, the CD34+CD19−CD45+ cells, unlike the CD45‐negative leukaemic cells, expressed CD117 (c‐kit), providing further evidence that these cells represent residual non‐leukaemic normal cells. By multiparameter analysis, this population of candidate normal stem cells could be separated from contaminating leukaemic CD34+CD19−CD45−cells, which were detected in 11 of the 12 patients within the CD34+CD19− compartment.
Association of myelodysplastic changes with purine analogues
Authors: Orchard  , Bolam  , Oscier
Published: March 2003

We describe the occurrence of myelodysplastic changes (hypogranular myeloid series and Pelger cells, dyserythropoiesis with ring sideroblasts) in five of 31 patients with chronic lymphoproliferative disorders after treatment with purine analogues. The bone marrows of 31 patients with chronic lymphoproliferative disorders before and after treatment with purine analogues were reviewed. The majority of patients had received extensive prior treatment, but none had dysplastic changes prior to treatment with purine analogues. We suggest that a purine analogue may have been responsible for dysplastic change and that further follow‐up of this phenomenon is warranted.
Factors which affect the CFU‐GM content of the peripheral blood haemopoietic progenitor cell harvests in patients with acute myeloid leukaemia
Authors: Jowitt  , Chang  , Morgenstern  , Howe  , Ryder  , Testa  , Scarffe
Published: March 2003

Autologous peripheral blood haemopoietic stem cells (PBSC) were harvested from 30 patients withde novoacute leukaemia, 29 of whom had entered remission following standard chemotherapy. Correlation of CD34+cells/kg to CFU‐GM/kg in the harvests was good (correlation coefficient=0.72,P<0.001). We demonstrated significant associations between the CFU‐GM content of the harvest and the following: time to platelets >50×109/l post final induction course (P<0.0001), days to harvest from day 1 of intensification/mobilization (correlation coefficient=−0.73,P<0.001), platelets >20×109/l at time of harvest (P=0.02), time to WBC >1.0×109/l post intensification/mobilization (correlation coefficient=−0.70,P<0.001), and WBC on day of harvest (correlation coefficient=0.60,P<0.001). In contrast, we found no relationship between the CFU‐GM content of the harvest and patient age up to 65 years, presence of absence of coexistent features of trilineage myelodysplasia at diagnosis, number of induction courses to remission or total number of courses of chemotherapy prior to intensification/mobilization. Haemopoietic recovery after reinfusion of PBSC was highly correlated to the number of CFU‐GM infused (neutrophils >0.5×109/lrs=−0.72,P=0.001; platelets >20×109/l unsupportedrs=−0.71,P=0.001).

Our results show that the number of induction courses received, and thus exposure to cytotoxic agents received, made no significant difference to subsequent CFU‐GM harvest content. We collected superior harvests from those patients with faster platelet recovery following mobilization therapy. We also found that faster platelet recovery following the final induction therapy was a better predictor of the CFU‐GM harvest following mobilization than was the neutrophil recovery following final induction.
Serum erythropoietin concentration in anaemia of visceral leishmaniasis (kala‐azar) before and during antimonial therapy
Authors: Saeed Amal M. , Khalil Eltahir A. G. , Elhassan Afaf M. A. , Hashim Faisal A. , Elhassan Ahmed M. , Fandrey Joachim , Jelkmann Wolfgang
Published: March 2003

Serum erythropoietin (Epo) concentrations and variables of red cell and iron status were studied in 27 Sudanese patients who were treated with sodium stibogluconate for visceral leishmaniasis (kala‐azar). Blood haemoglobin increased from 6.4 (±1.7 SD) to 9.5 (±1.4) g/dl during treatment. Serum ferritin decreased concomitantly. Serum iron levels were unchanged whereas the total iron binding capacity increased slightly. The pre‐treatment serum Epo concentration in relation to the blood haemoglobin concentration was not as high as expected from the one in primary haematological diseases, indicating that there is a relative lack of Epo in anaemic kala‐azar patients. Serum Epo further decreased during stibogluconate therapy. The normal dependence of the serum Epo level on the blood haemoglobin concentration was lost during mid‐term antimonial treatment, but it recovered thereafter. Cell culture studies with the human hepatoma cells HepG2 showed that stibogluconate ( 30 μg/ml) inhibited Epo gene expression. Thus, effective treatment of kala‐azar with stibogluconate results in improvement of anaemia, although the drug itself may impair Epo production.
Cytotoxic effectors activated by low‐dose IL‐2 plus IL‐12 lyse IL‐2‐resistant autologous acute myeloid leukaemia blasts
Authors: Vitale Antonella , Guarini Anna , Latagliata Roberto , Cignetti Alessandro , Foa Robert
Published: March 2003

We have investigated the susceptibility of primary acute myeloid leukaemia (AML) samples to the lytic action of normal peripheral blood mononuclear cells (PBMC), as well as of the patients' autologous and allogeneic PBMC collected at the time of remission following stimulation with different concentrations of interleukin (IL)‐2 and IL‐12, both alone and in variable combinations. Primary AML blasts were resistant to IL‐2‐activated PBMC effectors generated from normal individuals, allogeneic and autologous patients in five, six and eight of the 10 AML samples tested, respectively. IL‐12 alone proved ineffective in generating anti‐leukaemic activity, whereas, in combination, the two cytokines induced anti‐leukaemic killing in all five cases resistant to normal PBMC, in 4/6 samples resistant to allogeneic AML effectors and in 5/8 cases resistant to autologous effectors. In each case the lytic effect was maintained at the lowest cytokine combination (10 + 10 IU/ml) utilized. The suggestion of a synergistic effect was further strengthened by the evidence that at the lowest doses of IL‐2 and IL‐12 the degree of killing was greater than that promoted by each cytokine independently.

The results of this study suggest that two major limitations associated with the administration of IL‐2 to AML patients, the resistance of the blasts to IL‐2‐generated killing and the toxicity associated with high‐dose IL‐2, may be overcome by the combined use of very low doses of IL‐2 and IL‐12. As IL‐2‐resistant blasts may be lysed by a low‐dose combination of IL‐2/IL‐12, feasibility studies with this cytokine combination are worthy of exploration in vivo.
Selective expansion of normal haemopoietic progenitors from chronic myelogenous leukaemia marrow
Authors: Fogli Miriam , Amabile Marilina , Martinelli Giovanni , Fortuna Alessandra , Rondelli Damiano , Ratta Marina , Curti Antonio , Tura Sante , Lemoli Roberto M.
Published: March 2003

CD34+ and CD34+ DR− cells from the bone marrow (BM) of chronic‐phase chronic myelogenous leukaemia (CML) patients at diagnosis were tested for their colony‐forming ability in response to early and intermediate‐late colony stimulating factors (CSFs). Molecular analysis revealed that 55.6 ± 9% SD of CD34+DR− colonies, in which actin and ABL mRNA were detectable, expressed the product of the BCR‐ABL gene. The percentage and the clonogenic efficiency of CML DR− cells were significantly lower than those of comparable DR− cells from normal donors. However, clonogenic assays using recombinant human CSFs demonstrated a remarkable proliferation of CML cells when stimulated by SCF, IL‐11 and IL‐3, used as single factors in the presence of erythropoietin (EPO) and was almost entirely due to erythroid progenitors. Conversely, optimal stimulation of CD34+DR− cells from normal donors required co‐incubation with three or more CSFs. Stroma‐noncontact long‐term cultures were then established in the presence of exogenous CSFs and human irradiated allogeneic stromal layers or the murine stromal cell line M2‐10B4, engineered to produce G‐CSF and IL‐3. In these cultures the combination of SCF and IL‐3 induced a 25.4 ± 5 SD, 40 ± 6 SD and 20.5 ± 6 SD fold increase of colony‐forming unit cells (CFU‐C), at weeks 2, 4 and 5, respectively. At the same time‐points the number of primitive long‐term culture initiating cells (LTC‐IC) showed a 4 ± 2 SD, 3.3 ± 1.5 SD and 2.3 ± 1 SD fold increase compared to baseline values. BCR‐ABL mRNA analysis of single colonies demonstrated that 27 ± 9% SD and 7 ± 3% SD CFU‐C at weeks 4 and 5, respectively, expressed the fusion gene, whereas leukaemic LTC‐IC disappeared from the culture by week 2. These results suggest that leukaemic CD34+DR− cells have a different pattern of response to CSFs than normal cells. In addition, we established culture conditions which allow selective expansion of benign haemopoietic cells coexisting with leukaemic progenitors.
Mobilization of Philadelphia‐negative peripheral blood progenitor cells with chemotherapy with rhuG‐CSF in chronic myelogenous leukaemia patients with a poor response to interferon‐alpha
Authors: Carella  , Simonsson  , Link  , Lennard  , Boogaerts  , Gorin  , Tomas‐Martinez  , Dabouz‐Harrouche  , Gautier  , Badri
Published: March 2003

The purpose of this cooperative study was to evaluate the quantity and quality of Ph1‐negative progenitor cells mobilized in the peripheral blood of patients with chronic myelogenous leukaemia soon after aplasia induced by chemotherapy. 32 patients ineligible for allografting who were cytogenetically refractory to interferon‐alpha (IFN‐α) were entered into this study. The chronic phase varied widely, with a median duration of 17 months (range 3–90 months). All patients were treated with intensive conventional chemotherapy regimens and recombinant human granulocyte colony‐stimulating factor (rhuG‐CSF, lenograstim). Peripheral blood progenitor cells (PBPC) were harvested by leukaphereses during early recovery from chemotherapy‐induced aplasia. A total of 119 leukaphereses were performed. Median numbers of CD34+ cells and CFU‐GM collected were 2.04 × 106/kg and 2.9 × 104/kg, respectively. There was a significant correlation between white cell count and number of CD34+ cells in the leukaphereses (P = 0.0001, r2 = 0.41, n = 104). A strict correlation between the number of CD34+ cells and CFU‐GM in the leukapheretic product (P = 0.0001, r2 = 0.39, n = 110) was observed. 21% of evaluable patients (6/29) achieved a complete cytogenetic remission in the leukapheretic product and the other four patients achieved a major cytogenetic response for an overall response of 35% (10/22 patients). To date, 16 patients have been autografted and are alive. Five of them are Ph1‐negative (three patients) or partially Ph1‐negative (two patients). In conclusion, despite the high‐risk characteristics of this study population, Ph1‐negative PBPC were successfully mobilized in more than one‐quarter of patients using a chemotherapy plus rhuG‐CSF regimen. The importance of this achievement is increased by the current lack of other practical methods of rescuing Ph1‐negative cells in such patients.
Rheological properties and function of blood cells in stored bank blood and salvaged blood
Authors: Kirkpatrick  , Adams  , Lardi  , Mccollum
Published: March 2003

The technique involving filtration of diluted blood enables the separate analysis of the flow properties of different cell subpopulations. This study was designed to assess the changes occurring in the flow properties and function of blood cells in stored bank blood and salvaged blood compared to patient blood in a given clinical situation. We measured hydrogen peroxide production by neutrophils and the filterability, through 5 μm Nucleopore filters, of isolated red blood cells and of diluted blood. Samples were obtained from patients undergoing aortic surgery and blood intended for transfusion: either salvaged during surgery or stored bank blood. Both salvaged and bank blood were much less filterable than patient blood, with reduced deformability of both red and white blood cells. However, salvaged blood contained highly activated neutrophils with a prolonged transit time of the ‘fast‐flowing’ cells in the analysis compared to bank blood. Bank blood contained significantly more particles which acted as pore‐blockers. Cells in bank and salvaged blood therefore have markedly abnormal flow and biochemical properties compared to patient blood.
Mutation analysis by a non‐radioactive single‐strand conformation polymorphism assay in nine families with X‐linked severe combined immunodeficiency (SCIDX1)
Authors: Wengler Georg S. , Giliani Silvia , Fiorini Maurilia , Mella Patrizia , Mantuano Elide , Zanola Alessandra , Pollonini Gabriella , Eibl Martha M. , Ugazio Alberto G. , Notarangelo Luigi D. , Parolini Ornella
Published: March 2003

X‐linked severe combined immunodeficiency (SCIDX1) is an inherited disease characterized by profound abnormalities of cell‐mediated and humoral immunity. Patients with SCIDX1 have defects in the common cytokine receptor gamma chain gene (IL2RG) that encodes a shared, essential component of the receptors for interleukin‐2 (IL‐2), IL‐4, IL‐7, IL‐9 and IL‐15. We have characterized nine SCIDX1 families by using a DNA‐based, non‐radioactive screening method and DNA sequencing. Nine different mutations were found, scattered from exon 1 to exon 5 of the IL2RG gene. Two of these mutations have been previously identified in other unrelated patients; the other seven are novel mutations that differ from all of the 95 already reported in the IL2RG mutation data base.

In addition to describing novel mutations in the IL2RG gene, this study shows that the knowledge of the genetic defect and the use of an efficient, non‐radioactive, and rapid screening approach have important implications for prenatal and postnatal diagnosis, carrier female identification, and possibly prenatal therapy.
Haemopoietic defect and decreased expansion potential of bone marrow autografts from patients with acute myeloid leukaemia in first remission
Authors: Straetmans  , Herman  , Van Bockstaele  , Michaux  , Hagemeijer  , Ferrant
Published: March 2003

Autologous bone marrow (BM) transplantation for acute myeloid leukaemia (AML) in complete remission (CR) is frequently followed by a slow haemopoietic recovery. We assessed the haemopoietic capacity of purified BM stem cell (CD34+DR−) and progenitor cell (CD34+DR+) populations from patients with AML in CR, and compared these data with those of normal BM. The feasibility of ex vivo expansion in stroma‐conditioned medium supplemented with cytokines was also investigated.

The number of CFU‐GM produced by initial patient CD34+DR− cells was decreased compared to normal, whereas these values were similar to normal for CD34+DR+ cells. BFU‐E, HPP‐CFC and LTC‐IC were reduced for both patient CD34+DR− and CD34+DR+ subpopulations. In contrast to normal, the patient CD34+DR− fraction was not enriched in LTC‐IC.

CFU‐GM expansion from patient CD34+DR− cells was poor and decreased after 14 d of culture. No HPP‐CFC expansion could be observed for patient cells. LTC‐IC were below the level of detection after 14–21 d of expansion culture of CD34+DR− patient cells, whereas they were variably maintained or expanded for normal cells. After expansion culture, cytogenetic and/or FISH analyses did not reveal the anomalies present at diagnosis, regardless of the cell subpopulation analysed.

In conclusion, BM cells of patients with AML in CR show a profound defect at the level of a stem cell enriched population. No meaningful ex vivo expansion could be obtained in culture conditions allowing for a significant expansion from a normal stem cell population.
Oligoclonal proliferation of human T‐cell leukaemia virus type 1 bearing T cells in adult T‐cell leukaemia/lymphoma without deletion of the 3′ provirus integration sites
Authors: Leclercq India , Cavrois Marielle , Mortreux Franck , Hermine Olivier , Gessain Antoine , Morschhauser Franck , Wattel Eric
Published: March 2003

We report a new case of an asymptomatic carrier with a deletion of a 3′ HTLV‐1 integration site. We further investigated whether these 3′ deletions of flanking sequences may explain the oligoclonal pattern of HTLV‐1 replication, evidenced by inverse PCR (IPCR) analysis of tumourous samples from patients with adult T‐cell leukaemia (ATLL). 48 HTLV‐1 3′ integration sites, derived from tumourous DNA of five ATLL patients were sequenced. One dominant flanking sequence was obtained in the four samples harbouring a unique band after Southern‐blotting. In one sample, which harboured two signals after Southern‐blotting, IPCR amplification of diluted tumourous DNA revealed that these two sequences corresponded to one clone harbouring two integrated proviruses rather than to two distinct cellular clones, a result consistent with superinfection of the tumourous sample. In addition to integration sites corresponding to malignant clones, two to six oligoclonal forms were sequenced in four samples. No flanking sequence homology was found between clones derived from each patient, indicating that integration sites deletion in the vicinity of the provirus is a rare event in ATLL. The oligoclonal pattern of HTLV‐1 replication in ATLL may result from clonal expansion of non‐malignant HTLV‐1‐bearing clones within the sample and partly from HTLV‐1 superinfection of monoclonal tumour cells.