bluuebunny/crossref_metadata_2025 huggingface dataset:
Should a platelet limit of 600 × 109/l be used as a diagnostic criterion in essential thrombocythaemia? An analysis of the natural course including early stages
Authors: Lengfelder Eva , Hochhaus Andreas , Kronawitter Ursula , Höche Dietrich , Queisser Wolfgang , Jahn‐Eder Michaela , Burkhardt Rolf , Reiter Andreas , Ansari Hassan , Hehlmann Rüdiger
Published: March 2003

In order to evaluate the natural history of essential thrombocythaemia (ET), clinical data and prognostic factors of 143 patients with ET were retrospectively analysed (mean observation time 6.1 ± 4.6 years). In 42 patients the early phase of the disease with initial platelet counts between 250 and 600 × 109/l was assessed. In most early cases, ET was suggested by clinical symptoms (79%) and increased megakaryopoiesis (95%) with abnormal megakaryocytes in bone marrow histology (n = 34) and cytology (n = 5). Other myeloproliferative disorders and reactive thrombocytosis were excluded according to the diagnostic criteria of the Polycythemia Vera Study Group. During follow‐up of the 38 early cases not treated cytoreductively at diagnosis, the platelet counts increased to >600 × 109/l in 28 patients (74%) and remained between 450 and 600 × 109/l in 10 patients (26%). In primarily asymptomatic patients (n = 46) with initial platelet counts above (n = 37) and below 600 × 109/l (n = 9) the rates of increase of symptomatic patients were similar at about 7% per year. No influence of the initial platelet count on survival was seen in multivariate analysis of prognostic factors which included all 143 cases. Survival was mainly influenced by the rate of ET‐related complications during follow‐up (P = 0.002). Analysing the influence of cytoreductive therapy on symptom‐free survival, platelet reduction benefited patients under 60 years (19 cytoreductively treated v 65 untreated patients, P = 0.075). The results demonstrate the possible clinical relevance of the early stages of ET and suggest that the features of pathologic megakaryopoiesis in the bone marrow are a more reliable diagnostic criterion than a definite platelet limit. Therefore, further therapeutic studies should include all stages of the disease and all age groups.
Factors affecting cardiolipin antibody assays: modification with polyethylene glycol compound
Authors: Kilpatrick David C.
Published: March 2003

Anti‐cardiolipin antibody (aCL) measurement is only semi‐reproducible, and current assays detect irrelevant as well as clinically significant antibodies. Factors found to influence results included the source of the enzyme‐linked immunoabsorbent assay (ELISA) plate, and its pre‐treatment with solvents; the nature of the blocking solution; and the composition of the diluent used for reagents. Fetal calf serum (FCS) in the diluent appeared to reduce non‐specific (clinically irrelevant) binding and was not simply a source of β2‐glycoprotein‐1 (β2GP1). A polyethylene glycol compound was found to be an effective blocking agent, and its use enhanced the discrimination between positive and negative samples. A high level of variation may be inherent in aCL‐ELISA methodology, but the use of polyethylene glycol compound appeared to be a helpful modification.
Increased number of peripheral blood CD34+ cells in lithium‐treated patients
Authors: Ballin Ami , Lehman Dan , Sirota Pinkhas , Litvinjuk Ulia , Meytes Dina
Published: March 2003

Eight adult patients with bipolar disorder were prospectively examined to find whether lithium carbonate increased their peripheral blood CD34+ haemopoietic stem cells. Following lithium therapy for 3–4 weeks their neutrophil counts increased by a mean of 88% (from 4625 ± 1350 × 109/l, mean ± SD pretreatment, to a peak of 8300 ± 3910 × 109/l). Concommitantly, there was a significant increment in their CD34+ cells (from 0.11 ± 0.01% to a peak of 0.18 ± 0.08%). There was a significant correlation between the rise in neutrophil count and that of the CD34+ cells (r = 0.795, P = 0.019). Lithium therapy may be used to mobilize peripheral blood CD34+ cells for marrow transplantation.
Improved outcome of adult acute lymphoblastic leukaemia by moderately intensified chemotherapy which includes a ‘pre‐induction’ course for rapid tumour reduction: preliminary results on 66 patients
Authors: Daenen  , van Imhoff  , van den Berg  , de Kam  , Haaxma‐Reiche  , Vellenga  , Smit  , Halie
Published: March 2003

Sixty‐six consecutive adult patients with acute lymphoblastic leukaemia (ALL) were treated with intensified chemotherapy which included a ‘pre‐induction’ course of cytarabine (AraC) and etoposide (VP16) when the white blood cell count (WBC) was ≥30 × 109/l (18 patients), and maintenance chemotherapy with regular intensifications for a total treatment duration of 3 years. Patients with a mediastinal mass (17) received consolidation courses with intermediate‐dose AraC and VP16 followed by mediastinal irradiation. 11 patients underwent allogeneic bone marrow transplantation in first complete remission (CR). 58 patients (87.9%, CI 77.5–94.6) attained CR; with a median follow‐up of 7 years, 35 of them (60.3%, CI 46.6–73.0) remain in CR. Toxicity was mild, although three patients died during remission induction, including two who were over 70 years of age. 23 patients (39.7%, CI 27.1–53.4) relapsed, seven of them primarily in the central nervous system (CNS), necessitating intensification of CNS‐directed therapy. Only one of 13 patients with WBC 30–100 × 109/l, but eight of nine with WBC >100 × 109/l, relapsed. The survival of older patients in CR did not differ from younger patients. The outcome of ALL in adult patients could thus be improved by slight intensification of treatment whilst keeping the toxicity within acceptable limits. ‘Pre‐induction’ with AraC and VP16 might improve the prognosis, especially in patients with WBC <100 × 109/l. Patients with WBC >100 × 109/l, however, almost always relapse, and the intensified chemotherapy might not be tolerated well by patients over 70 years of age.
Vitamin D treatment in myelodysplastic syndromes
Authors: Mellibovsky  , Díez  , Pérez‐Vila  , Serrano  , Nacher  , AubÍa  , Supervía  , Recker Robert R.
Published: March 2003

Myelodysplastic syndromes (MDS) are a group of clonal disturbances with defective cellular differentiation. Vitamin D3 (VD) analogues can act on the differentiation and maturity of different cell lines. We studied the effects of VD on a series of patients with MDS in an open‐design trial. Nineteen patients, 12 men and seven women, with MDS were included. Patients were 74.8 ± 5.6 years (mean ± SD), seven had refractory anaemia with ringed sideroblasts, five had refractory anaemia, one had refractory anaemia with excess of blasts and six had chronic myelomonocytic leukaemia. All the patients were in a low to intermediate risk group. Mean follow‐up period was 26.21 months, range 9–75. Responders were defined as follows: granulocyte or platelet count increase by 50%, or haemoglobin increase of 1.5 g/dl or transfusion needs decrease by 50%. The first five patients received 266 μg of calcifediol three times a week and the other 14 received calcitriol (0.25–0.75 μg/d). Response was observed in 11 patients. In the calcifediol‐treated group, one case responded, three were non‐responders, and one showed progression. In the calcitriol group, 10 were responders (two with major response), and four were non‐responders. No correlation was observed between baseline levels of vitamin D metabolites and the presence of response. No hypercalcaemia was observed. Treatment with vitamin D3 metabolites could induce a long‐standing response of the haematological disturbance in some low‐intermediate risk MDS patients without inducing hypercalcaemia.
A case of myelodysplastic syndrome with an intronic point mutation of the p53 tumour suppressor gene at the splice donor site
Authors: Kikukawa Masayuki , Aoki Naoto , Mori Mayumi
Published: March 2003

We analysed genomic DNA and mRNA of the p53 gene in a case of myelodysplastic syndrome (MDS) with monosomy of chromosome 17. DNA analysis revealed a mutation at the splice donor site (GT to GC) of intron 5. mRNA analysis revealed the presence of abnormal splicing with 46 nucleotide deletion in exon 5, producing a downstream frame shift and a predicted truncated protein which lacked normal function. The p53 gene mutation at the splice donor site contributes to the inactivation of the p53 gene function and may play an important role in the pathogenesis, progression and therapeutic responsiveness of MDS.
Impaired redistribution of aminophospholipids with distinctive cell shape change during Ca2+‐induced activation of platelets from a patient with Scott syndrome
Authors: Bettache Nadir , Gaffet Patrick , Allegre Nicolas , Maurin Lucien , Toti Florence , Freyssinet Jean Marie , Bienvenüe Alain
Published: March 2003

We have investigated phospholipid redistribution, membrane vesicle shedding, shape change, and granule release following A23187 activation of platelets from a patient with Scott syndrome, characterized by impaired transmembrane migration of phosphatidylserine (PS) accompanied by haemorrhagic complications, and two of her children. Electron spin resonance spectroscopy measurement of phospholipids redistribution showed that the internalization of PS was unaffected by the disorder but, after activation, PS exposure was significantly reduced in platelets from the homozygous‐type patient. Vesicle shedding was also reduced in these platelets. However, the slow redistribution of phosphatidylcholine was similar to that observed in normal platelets. When treated with calpeptin, platelets from the homozygous‐type patient, unlike normal or heterozygous Scott syndrome platelets, showed a smoothly rounded shape without filopods after activation. Following A23187 activation of normal platelets, filopod formation was consecutive to the re‐exposition of aminophospholipids on the outer leaflet of the plasma membrane, and the existence of a floppase (outward aminoPLs translocase) has been suggested. In homozygous Scott syndrome platelets the deficiency in PS re‐exposition, the absence of filopod formation, and low vesicle shedding are correlated with each other, and argue in favour of a disruption of the proposed floppase activity.
Deficiency of glycosylphosphatidyl inositol‐anchored proteins in patients with aplastic anaemia does not affect response to immunosuppressive therapy
Authors: Lord Corinne De , Tooze Jennifer A. , Saso Radovan , Marsh Judith C. W. , Gordon‐Smith Edward C.
Published: March 2003

Deficient expression of glycosylphosphatidyl inositol (GPI)‐anchored proteins in aplastic anaemia (AA) patients has previously been reported to be associated with a poor response to immunosuppressive (IS) therapy. Here we report the response to IS therapy of 111 patients with AA and correlate this with GPI‐anchored protein expression on peripheral blood cells by flow cytometry. A GPI‐anchored protein deficient population was identified in 15% (17/111) of patients with AA who had a negative Ham's test and no laboratory evidence of haemolysis. Patients were treated with antilymphocyte globulin and/or cyclosporin A, or oxymetholone. Bone marrow transplantation was performed in 12 patients, seven of whom had not responded to IS therapy. In patients tested for GPI‐anchored protein expression prior to IS therapy there was no difference in response rate to IS therapy between AA patients with a GPI‐anchored protein deficiency and those with normal GPI‐anchored protein expression (50% response rate versus 75%, respectively). Survival in these two groups was similar at 90% with follow‐up over 140 months from diagnosis. Eight of the 17 AA patients who developed a GPI‐anchored protein‐deficient population later went on to develop a positive Ham's test. From this study we demonstrate a lower incidence of GPI‐anchored protein deficiency in AA patients compared with previous reports. In addition we have shown that the presence of a GPI‐anchored protein‐deficient cell population in patients with AA who have a negative Ham's test is not a poor prognostic factor in terms of response and survival after IS therapy.
Dendritic cells cultured from mononuclear cells and CD34 cells in myeloma do not harbour human herpesvirus 8
Authors: Cull  , Timms  , Haynes  , Russell  , Irving  , Ball  , Thomson
Published: March 2003

Dendritic cells (DC) are antigen‐presenting cells with the potential to be a powerful adjuvant in the immunotherapy of haematological malignancy, including myeloma. Recently, human herpesvirus 8 (HHV‐8) infection of dendritic cells in the long‐term bone marrow stromal cultures of patients with myeloma has been reported. This finding is of great potential importance regarding oncogenesis in myeloma in addition to having significant implications for the use of DC in the immunotherapy of this disease. Therefore DC generated from mobilized blood mononuclear cells (MO‐DC) and purified CD34+ cells (CD34‐DC) of myeloma patients were examined for the presence of HHV‐8 using a sensitive PCR technique. HHV‐8 was not demonstrated in MO‐DC or CD34‐DC and we conclude that these cells remain a suitable vehicle for investigation in the immunotherapy of myeloma.
Detection of minimal residual disease in B‐lineage acute lymphoblastic leukaemia by quantitative flow cytometry
Authors: Farahat Nahla , Morilla Alison , Owusu‐Ankomah Kwasi , Morilla Ricardo , Pinkerton C. Ross , Treleaven Jennie G. , Matutes Estella , Powles Ray L. , Catovsky Daniel
Published: March 2003

The clinical significance of detecting minimal residual disease (MRD) in B‐lineage acute lymphoblastic leukaemia (ALL) was evaluated by quantitative flow cytometry using a combination of TdT with CD10 and CD19. 53 patients with B‐cell precursor ALL were followed during and after completion of treatment (median follow‐up 23 months). Nine patients relapsed and MRD had been detected in six of them, 5–15 weeks before relapse despite morphological complete remission. 43 patients remain in clinical remission and in none of these was MRD detected. Disease‐free survival based on the detection of MRD by flow cytometry showed a statistically significant difference between both groups (P < 0.0001). The absence of MRD correlates with a low relapse rate, whereas the presence of MRD predicted early relapse. This study has shown that flow cytometry can improve the morphologic assessment of bone marrow (BM) remission status in B‐lineage ALL. The finding of < 5% blasts in BM aspirates did not correlate with ‘true’ remission in a proportion of cases as residual leukaemic blasts were detected by flow cytometry in nine samples from six patients. On the other hand, the presence of > 5% blasts assessed by morphology was not necessarily a feature of relapse in five patients as these cells were shown to have a phenotype identical to normal TdT‐negative B‐cell precursors. Quantitative flow cytometry was more informative than conventional morphology to assess remission status and showed a strong correlation with clinical outcome. This methodology is useful to define MRD in the majority of patients with B‐lineage ALL and should be tested in prospective clinical trials.
DNA extraction from archival Giemsa‐stained bone‐marrow slides: comparison of six rapid methods
Authors: Vince Adriana , Poljak Mario , Seme Katja
Published: March 2003

The ability of six rapid DNA extraction procedures to provide DNA for the polymerase chain reaction from archival Giemsa‐stained bone marrow slides was tested on 120 samples. Boiling in distilled water, freeze–thaw method, boiling in 10% Chelex‐100 resin solution, proteinase K/Tween 20/NP‐40 method coupled with simplified phenol/chloroform/isoamyl alcohol protocol or salting‐out procedure using saturated NaCl and modification of commercial QIAamp procedure (Qiagen, Chatsworth, Calif.) gave DNA extraction efficiencies of 50%, 70%, 85%, 95%, 100% and 100%, respectively. Our results demonstrate that rough DNA extraction methods have decreased efficiencies compared to complete DNA extraction protocols and that the latter are required to ensure highly reproducible results from archival Giemsa‐stained bone marrow slides.
Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood
Authors: Schipper  , Brand  , Reniers  , Melief  , Willemze  , Fibbe
Published: March 2003

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34+ progenitor cells in cord blood. Cells were cultured in both liquid and semi‐solid cultures containing 50 ng/ml TPO. The CD34+/CD45RA− and CD34+/CD38− subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU‐meg assay. Progenitor cells derived from the CD34+/CD45RA− and CD34+/CD38− subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19‐fold expansion of the total CD34+ cell fraction, whereas in the CD34+/CD45RA− and CD34+/CD38− subfractions the mean expansion was 23‐ and 50‐fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.
Differential activity of P‐glycoprotein in normal blood lymphocyte subsets
Authors: Ludescher Christof , Pall Georg , Irschick Eveline U. , Gastl Günther
Published: March 2003

To better understand the phenomenon of P‐glycoprotein (P‐170) expression we investigated lymphocyte subpopulations for P‐170 function in healthy volunteers. Studies were based on three‐colour flow cytometry including the fluorescent probe rhodamine 123 (Rh123), which is transported by P‐170. Marked Rh123 efflux was detected in CD8+ T lymphocytes with CD8+/CD45RA+ T cells (naive cells) showing significantly higher P‐170 activity as compared with CD8+/CD45RA− cells (P < 0.04). Vice versa, CD8+/CD45RO+ T cells (memory cells) demonstrated less P‐170 activity than CD8+/CD45RO− cells (P < 0.04). P‐170 function was less prominent in CD4+ T cells, however, Rh123 efflux was higher in the CD4+/CD45RA+ and CD4+/CD45RO− subpopulations (P < 0.025) corresponding to the CD8+ results. Dye efflux differed significantly between activated and non‐activated CD8+ and CD4+ as well as CD8+/CD11b+ and CD8+/CD11b− T lymphocytes. Since CD16+ natural killer cells (NK) expressed the highest level of P‐170, the NK cytotoxicity against 51Cr‐labelled K562 target cells was assayed in the presence or absence of P‐170 inhibitors. NK related cytotoxicity was significantly reduced in the presence of R‐verapamil and dexnigaldipine‐HCP in a dose‐dependent manner.  The differential expression of P‐170 activity in naive and memory T cells together with the reduced NK related cytotoxicity in the presence of MDR‐modulators suggest a physiological role of P‐170 in immunological functions of these lymphocyte subsets. Consequently, the addition of MDR modulators to conventional chemotherapy as a strategy to overcome drug resistance should consider possible adverse immunosuppressive effects.
Elevated numbers of reticulated platelets in hyperthyroidism: direct evidence for an increase of thrombopoiesis
Authors: Stiegler Gabriele , Stohlawetz Petra , Brugger Stefan , Jilma Bernd , Vierhapper Heinrich , Höcker Paul , Panzer Simon
Published: March 2003

We studied thrombopoietic activity in hyperthyroidism by determination of reticulated platelet counts. At the time of hyperthyroidism 14/15 patients had higher reticulated platelets than after achievement of euthyroidism (P < 0.001). There was no difference in peripheral platelet counts and mean platelet volumes at the time of hyperthyroidism when compared to euthyroidism. Three patients had pan‐ and auto‐reactive platelet antibodies during hyperthyroidism. These antibodies were directed against GPIIb/IIIa in two patients and against GPIb/IX in one patient. Our findings provide direct evidence that hyperthyroidism is associated with increased platelet production, as reflected by an increase in reticulated platelets.
Highly‐efficient gene transfer with retroviral vectors into human T lymphocytes on fibronectin
Authors: Fehse Boris , Schade Ulrika M , LI Zhixiong , Uhde Almut , Koch Stefan , Goller Bernhard , RÜger RÜdiger , Fehse Natalia , StochschlÄder Marcus , Zander Axel R
Published: March 2003

Genetically modified lymphocytes have been successfully used for correction of ADA deficiency in children and in controlling graft‐versus‐host disease (GvHD) after allogeneic bone marrow transplantation. Low transduction efficiencies are, however, limiting for gene therapeutic strategies based on lymphocytes. In this study we compared protocols for highly efficient gene transfer into human T cells using retroviral vector‐containing supernatant. We showed that infection of both human primary T cells and CD4+ Jurkat cells is most efficient on the matrix component fibronectin. Transduction was carried out with a retroviral vector encoding both the human intracytoplasmatically truncated low‐affinity nerve growth factor receptor (ΔLNGFR) as a gene transfer marker and the Herpes simplex virus thymidine kinase for negative selection. Based on ΔLNGFR expression genetically modified cells were enriched to near purity by magnetic cell sorting (MACS). Enriched cells could be shown to be highly sensitive to ganciclovir.
Paroxysmal nocturnal haemoglobinuria clones in patients with myelodysplastic syndromes
Authors: Iwanaga Masako , Furukawa Koichi , Amenomori Tatsuhiko , Mori Hiroyuki , Nakamura Hideo , Fuchigami Kengo , Kamihira Shimeru , Nakakuma Hideki , Tomonaga Masao
Published: March 2003

Among acquired stem cell disorders, pathological links between myelodysplastic syndromes (MDS) and aplastic anaemia (AA), and paroxysmal nocturnal haemoglobinuria (PNH) and AA, have been often described, whereas the relationship between MDS and PNH is still unclear. We analysed blood cells of patients with MDS to determine the incidence of the PNH clone, and analysed the PIG‐A gene to find mutations characteristic of the PNH clone in MDS. In four (10%) of 40 patients with MDS, flow cytometry showed affected erythrocytes and granulocytes negative for decay‐accelerating factor (DAF) and CD59. The population of affected erythrocytes was smaller in MDS patients with PNH clone (MDS/PNH) than in patients with de novo PNH, and haemolysis was milder in the MDS/PNH patients. PIG‐A mutations were found in granulocytes of all patients with MDS/PNH. In type and site, the PIG‐A mutations were heterogenous, similar to that observed in de novo PNH; i.e. no mutation specific to MDS/PNH was identified. Of note, three of four patients with MDS/PNH each had two PNH clones with different PIG‐A mutations, suggesting that PIG‐A is mutable in patients with MDS/PNH. In a MDS/PNH patient with trisomy 8, FISH detected a distinct karyotype in a portion of granulocytes with PNH phenotype, indicating that PNH and MDS partly shared affected cells. Thus, MDS predisposes to PNH by creating conditions favourable to the genesis of PNH clone. Considering the increasing prevalence and incidence of MDS, these disorders could be useful for investigating the mechanism by which PIG‐A mutation is induced.
Prenatal diagnosis of Glanzmann thrombasthenia using the polymorphic markers BRCA1 and THRA1 on chromosome 17
Authors: French Deborah L. , CP;;er Barry S. , Usher Saly , Berkowitz Richard , Eng Christine , Seligsohn Uri , Peretz Hava
Published: March 2003

Glanzmann thrombasthenia is an autosomal recessive bleeding disorder caused by mutations in the genes encoding platelet GPIIb or GPIIIa. Both genes map to chromosome 17q21 and polymorphisms within this chromosomal region have been identified. In the current study, prenatal diagnosis was performed for a family that already had one affected child, patient 1, who had a compound heterozygous mutation in GPIIb. At the time of prenatal diagnosis, the maternal GPIIb mutation had been identified but the paternal GPIIb mutation was unknown. By sequence analysis, the fetus was identified as a carrier of the mother's mutation. To determine the probability of the fetus inheriting the father's mutation, haplotype analysis of DNA samples from the fetus, mother, father and affected child were performed using polymorphic markers on chromosome 17q12‐q21. These markers included polymorphisms within the thyroid hormone receptor α1 gene (THRA1), the breast cancer gene (BRCA1), GPIIb, GPIIIa, and an anonymous marker D17S579. Heterozygosity within the THRA1, BRCA1 and GPIIIa polymorphic markers predicted that the fetus carried the father's normal allele. Based on genetic linkage studies, no recombination was identified with any of the informative markers, and from the map distance between GPIIb and BRCA1 the accuracy of diagnosis was predicted to be >98%. The father's mutation was subsequently identified and direct sequence analysis of fetal DNA confirmed that the fetus did not inherit the fathers' mutant allele.
IL‐10 inhibits LPS‐induced human monocyte tissue factor expression in whole blood
Authors: Lindmark  , Tenno  , Chen  , Siegbahn
Published: March 2003

Interleukin 4 (IL‐4), IL‐10 and IL‐13 are all known to modulate several proinflammatory functions in human monocytes. They have also previously been shown to down‐regulate lipopolysaccharide (LPS)‐induced tissue factor (TF) expression in isolated cultured monocytes. In this study we investigated the effect of these three cytokines on the induction of monocytic TF in a whole blood environment at three levels: mRNA quantitation, surface antigen expression and procoagulant activity. We showed that IL‐10 attenuated LPS‐induced monocyte TF expression and activity in whole blood in a concentration‐dependent manner, both when added to the blood prior to LPS and, although to a lesser extent, when added up to 1 h subsequent to LPS challenge. Maximum inhibition occurred at 5 ng/ml of IL‐10 when the cytokine was added before LPS. IL‐4 and IL‐13, however, did not exhibit any inhibitory effect in the whole blood environment, contrary to the reported findings in cell culture experiments. Our results confirm the potential of IL‐10 as an anti‐inflammatory, TF‐preventing drug, whereas the effects of IL‐4 and IL‐13 on monocytes in whole blood seem more complex, and require further investigation.
Aberrant TSG101 transcripts in acute myeloid leukaemia
Authors: Lin Pai‐Mei , Liu Ta‐Chih , Chang Jan‐Gowth , Chen Tyen‐Po , Lin Sheng‐Fung
Published: March 2003

Recently, a tumour susceptibility gene, TSG101, has been identified at chromosome 11p15. A large intragenic deletion of this gene has been demonstrated in primary breast tumours. To evaluate the role of the TSG101 gene in leukaemia, bone marrow and/or peripheral blood from 68 acute myeloid leukaemia patients, five haemopoietic cell lines (HL60, U937, Raji, KG‐1, K562) and 30 normal controls were analysed by reverse transcription of the TSG101 mRNA, followed by PCR amplification and sequencing of the products. The results showed aberrant TSG101 transcripts in 24/68 (35%) acute myeloid leukaemia (AML) patients, all of the cell lines (100%) and 3/30 (10%) normal controls. Our study indicated that the abnormal transcripts may have resulted from aberrant RNA splicing as evidenced by these aberrant transcripts. Also, normal full‐length transcripts were present in all specimens examined. The aberrant transcript occurred more frequently in the AML and cell lines. However, because aberrant transcripts of TSG101 were also found in the normal controls, the role of TSG101 as a tumour suppressor gene should be evaluated carefully.
Absence of Bruton's tyrosine kinase (Btk) mutations in patients with acute myeloid leukaemia
Authors: Ritis Konstantinos , Speletas Matthaios , Tsironidou Victoria , Pardali Evaggelia , Kanariou Maria , Moschese Viviana , Orlandi Paola , Skordala Mariana , Rossi Paolo , Kartalis Georgios , Bourikas Georgios , Sideras Paschalis
Published: March 2003

Bruton's tyrosine kinase (Btk) is a non‐receptor protein tyrosine kinase (PTK) that is expressed in all haemopoietic lineages except mature T cells and plasma cells. Despite the broad range of expression, mutations that inactivate this molecule affect primarily the development of the B‐cell lineage. As a PTK, Btk could potentially be involved directly or indirectly in the processes that relate to the malignant transformation of all the cell lineages where this molecule is expressed. Previous studies have failed to demonstrate mutations in patients with B‐cell origin acute lymphoblastic leukaemia (ALL).

We have utilized a recently developed method that enables the rapid and convenient detection of mutations at the cDNA level, namely, the non‐isotopic RNase cleavage assay (NIRCA) to analyse Btk sequences from 27 patients with different types of acute myeloid leukaemia (AML). The only alteration that we observed was a polymorphism at position 2031. This polymorphism has already been seen in previous studies. Furthermore, using the same methodology, we identified the Btk mutations in six XLA (X‐linked agammaglobulinaemia) patients. Our results, although they do not exclude the involvement of Btk mutations in the development or progression of some type of AML, nevertheless suggest that such mutations do not constitute a major co‐factor in the development of myeloid malignancies.
Small GTP‐binding proteins in human endothelial cells
Authors: De Leeuw Hubert P. J. C. , Koster Pauline M. , Calafat Jero , Janssen Hans , Van Zonneveld Anton‐Jan , Van Mourik Jan A. , Voorberg Jan
Published: March 2003

Small GTP‐binding proteins of the Ras superfamily control an extensive number of intracellular events by alternating between GDP‐ and GTP‐bound conformation. The presence of members of this protein family was examined in human umbilical vein endothelial cells employing RT‐PCR. Sequence analysis of 215 cDNA clones revealed the presence of a total of 28 different partial cDNAs encoding small GTP‐binding proteins. Two sequences corresponded to novel isoforms of Rab2 and Rab9. In addition, human analogues of Rab4b, Rab7, Rab9, Rab14 and Rab15 were identified. Besides Rab proteins, members of other subfamilies were detected as well. As a first step towards elucidation of the function of the different small GTP‐binding proteins identified we have isolated full length cDNA corresponding to Rab30 from a human endothelial cell cDNA library. In order to assess the subcellular localization of Rab30, we expressed epitope‐tagged Rab30 cDNA in monkey kidney COS‐1 cells. Immunoelectron‐microscopy of transfected COS‐1 cells indicated that Rab30 is associated with Golgi stacks.
Anti‐myeloma activity of pamidronate in vivo
Authors: Dhodapkar Madhav V. , Singh Joginder , Mehta Jayesh , Fassas Athanasios , Desikan  , Perlman Martin , Munshi Nikhil C. , Barlogie Bart
Published: March 2003

Two patients with progressive myeloma were treated with pamidronate disodium every 2‐4 weeks. Pamidronate therapy resulted in a significant reduction of marrow plasmacytosis and plasma cell labelling index (PCLI), together with durable (20 months) stabilization of immunoglobulin (Ig) levels and an increase in bone mineral density in the first patient and >50% reduction in Ig levels and bone marrow plasmacytosis in the second. This, to our knowledge, is the first report of an anti‐myeloma effect of bisphosphonates in humans and provides evidence that a therapeutic intervention largely directed at the myeloma microenvironment may alter the natural history of the disease.
Enhanced human cell engraftment in mice deficient in RAG2 and the common cytokine receptor γ chain
Authors: Goldman Jacki P , Blundell Michael P , Lopes Lucien , Kinnon Christine , DI Santo James P , Thrasher Adrian J
Published: March 2003

Xenotransplantation of human cells into immunodeficient mice has been used to develop models of human haemopoiesis and lymphoid cell function. However, the utility of existing mouse strains can be limited by shortened life‐spans, spontaneous production of functional lymphocytes with ageing, and residual innate immunity leading to variable levels of engraftment. Mice with a deletion of the common cytokine receptor γ chain (γc) gene have reduced numbers of peripheral T and B lymphocytes, and absent natural killer cell (NK) activity. A genetic cross with a recombinase activating gene 2 (RAG2)‐deficient strain produced mice doubly homozygous for the γc and RAG2 null alleles (γc−/RAG2−). These mice have a stable phenotype characterized by the absence of all T lymphocyte, B lymphocyte and NK cell function. Injection of human B‐lymphoblastoid cells resulted in earlier fatal metastatic lymphoproliferative disease than in NOD/LtSz‐scid controls. This was particularly evident in animals injected intravenously, possibly because of residual NK activity in NOD/LtSz‐scid mice. Levels of engraftment with peripheral‐blood‐derived human lymphocytes were also increased and associated with higher CD4/CD8 ratios. These findings demonstrate that this new strain of immunodeficient mice has significant advantages over existing strains for engraftment of human cells, and may be useful for study of adoptive immunotherapy and novel therapies for GvHD and HIV infection.
Molecular characterization of the PK‐LR gene in pyruvate kinase deficient Spanish patients
Authors: Zarza  , Alvarez  , Pujades  , Nomdedeu  , Carrera  , Estella  , Remacha  , SÁnchez  , Morey  , Cortes  , PÉrez Lungmus  , Bureo  , Vives Corrons
Published: March 2003

The PK‐LR gene has been studied in 12 unrelated patients with red cell pyruvate kinase deficiency and hereditary nonspherocytic haemolytic anaemia (CNSHA). The entire codifying region of the R‐type PK gene and the flanking intronic regions were analysed by single‐stranded conformation polymorphism (SSCP) followed by direct sequencing of abnormal DNA. 10 different mutations were identified in 22/24 alleles at risk. Eight of these were missense mutations that caused the following single amino acid changes: G514C (172Glu‐Gln), G1010A (337Arg‐Gln), G1015C (339Asp‐Gln), T1070C (357Ile‐Thr), C1223T (408Thr‐Ile), G1291A (431Ala‐Thr), C1456T (486Arg‐Trp) and G1595A (532Arg‐Gln). Two were nonsense mutations: G721T (241Glu‐Stop) and C1675T (559Arg‐Stop). 7/22 alleles demonstrated the same C1456→ T mutation. The study of the polymorphic site at nucleotide (nt) 1705 performed in all cases disclosed a 1705 C/C mutation in 10 and a 1705 A/C mutation in three.

This is the first report on the presence of several different L‐type PK gene mutations within Spanish population. Furthermore, from the PK gene mutations found, six were unique and not previously described (1015C, 1070C, 1223T, 1291A, 1595A and 1675T) and one (C1456T) seems to be predominant in Spain. Interestingly, no case with the 1529A mutation commonly found in Northern European populations was present here.
Longitudinal observation and outcome of nonfamilial childhood haemophagocytic syndrome receiving etoposide‐containing regimens
Authors: Chen Jiann‐Shiuh , Lin Kai‐Hsin , Lin Dong‐Tsamn , Chen Rong‐Long , Jou Shiann‐Tarng , Su Ih‐Jen
Published: March 2003

The long‐term outcome of 22 children treated with etoposide‐containing regimens for haemophagocytic syndrome (HS) were longitudinally studied; none of them had a family history of the disease. All patients received etoposide‐containing (150 mg/m2/d) regimens, combined, in 16 cases, with intravenous immunoglobulin (IVIG) and prednisolone. Complete remission (CR) was achieved in 12 patients, partial remission in seven, and early mortality occurred in three. Of the 12 CR patients, only four remain alive and disease‐free, with a median follow‐up of 47.4 months; one CR patient died due to infection and the remaining seven had relapsed diseases. Three patients with a partial response or with relapsed disease progressed to T‐cell lymphoma, characterized, in the two cases tested, by clonal chromosomal abnormalities. Epstein‐Barr virus (EBV) infection was implicated in disease pathogenesis in 15/22 patients. The overall survival was 45.5%, 40.9% and 40.9% at 1, 3 and 5 years, respectively, and disease‐free survival for CR patients at these same times was 45.5%, 36.4% and 36.4%. The etoposide‐containing regimen would appear to be an effective initial therapeutic option for childhood HS. However, in view of the frequency of partial remissions and relapsed disease, a more intensive chemotherapy or bone marrow transplantation should be applied. The progression to EBV‐containing T‐cell lymphoma in three patients is consistent with the previous observation that EBV‐associated HS is a potentially malignant disease.
Near‐haploid common acute lymphoblastic leukaemia of childhood with a second hyperdiploid line: a DNA ploidy and fluorescence in‐situ hybridization study
Authors: Ma S. K. , Chan G. C. F. , Wan T. S. K. , Lam C. K. , Ha S. Y. , Lau Y. L. , Chan L. C.
Published: March 2003

Near‐haploidy is a rare cytogenetic finding in childhood acute lymphoblastic leukaemia (ALL) and is associated with a poor prognosis. A second hyperdiploid line, occurring presumably by endoreduplication of the near‐haploid stemline, is often observed. We present a case of common ALL in relapse characterized morphologically by a dual population of small and large lymphoblasts. Cytogenetic analysis supplemented with fluorescence in‐situ hybridization (FISH) studies localized near‐haploidy and hyperdiploidy to the small and large blast population respectively. DNA ploidy determination confirmed two abnormal clones with near‐haploidy as the predominant one. A novel t(9;12)(q11;q13) was present in the near‐haploid clone and was duplicated in the hyperdiploid clone. This finding identified cells bearing near‐haploidy to be the clonogenic population following malignant transformation and confirmed endoreduplication as the mechanism for the presence of associated hyperdiploidy.
Bolus administration of cladribine in the treatment of Waldenström macroglobulinaemia
Authors: Liu Eva S. , Burian Carol , Miller William E. , Saven Alan
Published: March 2003

This phase II clinical trial evaluated bolus cladribine as a single agent in Waldenström macroglobulinaemia (WM).

Cladribine was administered to 20 patients at a dose of 0.12 mg/kg/d by 2 h intravenous infusion for 5 consecutive days at monthly intervals for three courses. Partially responding patients were continued on therapy until maximal response and/or prohibitive toxicity, to a maximum of eight courses. Complete responders were treated with one additional course of cladribine.

After a median of three courses of cladribine, all 20 patients were evaluable; one achieved a complete response (CR) (5%) and 10 achieved a partial response (PR) (50%). The median duration of response follow‐up was 28 months (range 1–37 months). Four of 7 (57%) untreated and 7/13 (54%) previously treated patients responded. The major toxicity encountered was myelosuppression with 60% of patients demonstrating grade 3 or 4 neutropenia. Non‐haematological toxicities included two patients with herpes zoster and two patients with non‐melanoma skin cancers. At a median follow‐up duration of 20 months, 17 patients remain alive and three have died.

We confirm that bolus cladribine is an effective and safe method of drug delivery in WM patients. Recommendations regarding the equivalence of the continuous infusion and bolus methods in untreated patients requires further study. Bolus cladribine is more convenient and less costly than infusional cladribine since it obviates the need for central catheters and infusional devices.
Effects of four species of interferon‐α on cultured erythroid progenitors from congenital dyserythropoietic anaemia type I
Authors: Menike  , Wickramasinghe
Published: March 2003

The in vitro effects of interferon (IFN)‐α on erythroid progenitor cells derived from the peripheral blood of five patients with congenital dyserythropoietic anaemia (CDA) type I and seven healthy adults were studied. Ficol‐hypaque‐separated mononuclear cells were cultured for 14 d in StemGEM‐1d medium with 0, 1, 10 and 100 U/ml of recombinant IFN‐α2a, recombinant IFN‐α2b, the genetically‐engineered hybrid molecule IFN‐α1‐8 or the laboratory‐designed molecule IFN‐αconsensus. Erythroid bursts and colonies were counted, picked and processed for electron microscopy. In the experiments employing IFN‐α2a there were no differences in the numbers of erythroid bursts or colonies between four patients with CDA type I and seven healthy adults. All five patients with CDA type I showed the ‘Swiss‐cheese’ ultrastructural abnormality of the heterochromatin in a proportion of the erythroblasts when the progenitor cells were cultured in the absence of added IFN‐α. A statistically significant reduction in the proportion of erythroblasts showing the ‘Swiss‐cheese’ defect was seen when the erythroid progenitors were cultured in the pre‐sence of 0.01–0.5 U/ml IFN‐α2a (five patients), or 0.1 U/ml of IFN‐α2b (two patients). In contrast, no reduction was seen in cultures containing 0.1–100 U/ml of either IFN‐α1‐8 or IFN‐αconsensus or 20 μM hydroxyurea (two patients). The partial correction of the ‘Swiss‐cheese’ abnormality by low concentrations of IFN‐α2ain vitro provides an experimental model with which the mechanisms underlying the haematological response that occurs after the in vivo administration of this species of IFN‐α may be investigated.
Overexpression of lung‐resistance protein and increased P‐glycoprotein function in acute myeloid leukaemia cells predict a poor response to chemotherapy and reduced patient survival
Authors: Borg Anton G. , Burgess Robert , Green Linda M. , Scheper Rik J. , Liu Yin John A.
Published: March 2003

We investigated the role of the drug resistance‐related proteins LRP, MRP and Pgp and the apoptotic suppressor, bcl‐2, in relation to other clinical characteristics, with respect to response and survival in 91 patients with newly diagnosed AML, treated with standard chemotherapy. Multivariate analysis showed that poor response to chemotherapy was associated with increasing age (P = 0.0004), LRP expression (P = 0.0001) and Pgp function (P = 0.015). The significant predictors of both leukaemia‐free survival (LFS) and overall survival (OS) were LRP (LFS, P = 0.01; OS, P = 0.0001), Pgp function (LFS, P = 0.0001; OS, P = 0.0003) and cytogenetic abnormalities (LFS, P = 0.0001; OS, P = 0.0005). Patients with the lowest expression of LRP and Pgp function and favourable karyotype (group I) had an LFS of 30.2 months compared to 8.5 months in the group with the highest expression of LRP and Pgp and poor prognosis karyotype (group III, P = 0.002). OS decreased from 75.4 months in group I to 7.9 months in group III patients (P  < 0.0001). Neither MRP nor bcl‐2 were significantly associated with chemotherapy response and survival. Correlations were found between increasing expression of LRP and older age (P = 0.05) and an unfavourable karyotype (P = 0.005), but these variables were independent of each other in analysis of treatment response and patient survival. Our findings suggest that both LRP and Pgp are clinically relevant drug‐resistance proteins and it may be necessary to modulate both LRP and Pgp functions in order to reverse the multidrug resistance phenotype in AML.
Characterization of the response of human bone marrow endothelial cells to in vitro irradiation
Authors: Gaugler Marie‐Hélène , Squiban Claire , Claraz Marie , Schweitzer Karin , Weksler Babette , Gourmelon Patrick , Van der Meeren Anne
Published: March 2003

Endothelial cell dysfunction is a classic consequence of radiation damage. Bone marrow endothelial cells (BMEC) are a critical component of the stroma in the regulation of haemopoiesis. In animal models, radiation‐induced injury of BMEC has been described and a role for BMEC in haemopoietic regeneration after irradiation has been suggested. However, functions of BMEC involved in the haemopoietic regeneration have not been assessed. Therefore we studied the functional response of human BMEC to irradiation using the transformed human BMEC line (TrHBMEC) irradiated with 2, 5 or 10 Gy. Our results showed a time‐ and a dose‐dependent increase in damage to irradiated TrHBMEC measured by a decreased number of adherent cells which correlated with increased apoptosis and augmented release of soluble ICAM‐1 and von Willebrand factor. 2 Gy irradiated TrHBMEC expressed more ICAM‐1 on their surface than non‐irradiated cells, whereas no change in VCAM‐1, E‐selectin and PECAM‐1 expression was observed. An increased production of G‐CSF, GM‐CSF, IL‐8, IL‐6, IL‐1α, IL‐11, MIP‐1α and SCF and no production of LIF, TNF‐α, TPO and IL‐3 by 2 Gy irradiated TrHBMEC was observed. The haemopoietic supportive function of TrHBMEC was not altered after a 2 Gy exposure. These results suggest that although radiation induces endothelial cell damage, irradiated cells still support the proliferation and the differentiation of CD34+ haemopoietic cells.
Absence of p53 deletions in bone marrow plasma cells of patients with monoclonal gammopathy of undedetermined significance
Authors: Ackermann Jutta , Meidlinger Petra , Zojer Niklas , Gisslinger Heinz , Ludwig Heinz , Huber Heinz , Drach Johannes
Published: March 2003

We have recently shown that presence of a p53 deletion in multiple myleoma is an independent predictor for short survival. We therefore investigated whether or not this chromosomal abnormality can be identified in patients with monoclonal gammopathy of undetermined significance (MGUS). Using a triple staining method combining staining for cytoplasmic immunoglobulins and fluorescence in situ hybridization (FISH) with chromosome 17‐centromere and p53‐gene specific probes, we studied plasma cells from 15 patients with MGUS. In all patients, concordant signal numbers with both probes were obtained (including one patient with trisomy 17), indicating that allelic loss of p53 does not occur in MGUS.
Human factor VIII can be packaged and functionally expressed in an adeno‐associated virus background: applicability to haemophilia A gene therapy
Authors: Gnatenko Dmitri , Saenko Evgueni , Jesty Jolyon , Cao Liang‐Xian , Hearing Patrick , Bahou Wadie
Published: March 2003

Adeno‐associated virus (AAV) is a single‐stranded DNA parvovirus displaying several attractive features applicable to haemophilia A gene therapy, including non‐pathogenicity and potential for long‐term transgene expression from either integrated or episomal forms. We have generated and characterized two B‐domain‐deleted (BDD) fVIII mutants, deleted in residues Phe756 to Ile1679 (fVIIIΔ756–1679) or Thr761 to Asn1639 (fVIIIΔ761–1639). [35S]metabolic labelling experiments and immunoprecipitation demonstrated intact BDD‐fVIII of the predicted size in both lysates and supernatants (Mr ~ 155 kD for fVIIIΔ756–1679 and Mr ~ 160 kD for fVIIIΔ761–1639) after transient transfection into COS‐1 cells. Functional fVIII quantification appeared maximal using fVIIIΔ761–1639, as evaluated by Coatest and clotting assay (98 ± 20 mU/ml/1×106 cells and 118 ± 29 mU/ml/1×106 respectively, collection period 48 h). To bypass potential size limitations of rAAV/fVIII vectors, we expressed fVIIIΔ761–1639 using a minimal human 243 bp cellular small nuclear RNA (pHU1‐1) promoter, and demonstrated fVIII activity ~30% of that seen using CMV promoter. This BDD‐fVIII (rAAV(pHU1‐1) fVIIIΔ761–1639) can be efficiently encapsidated into rAAV (107% of wild type), as demonstrated by replication centre and DNAase sensitivity assays. A concentrated recombinant viral stock resulted in readily detectable factor VIII expression in COS‐1 cells using a maximally‐achievable MOI ~35 (Coatest 15 mU/ml; clotting assay 25 ± 2.0 mU/ml/1×106 cells). These data provide the first evidence that rAAV is an adaptable virus for fVIII delivery, and given the recent progress using this virus for factor IX delivery in vivo, provide a new approach towards definitive treatment of haemophilia A.