bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin 2 interacts with myeloid growth factors in serum‐free long‐term bone marrow culture
Authors: Douay Luc , Giarratana Marie‐Catherine , Mary Jean‐Yves , Gorin Norbert‐Claude
Published: March 2008

Summary. IL2 infusion may benefit patients with haematological malignancies by lowering the disease burden. However, conflicting data have been reported on IL2 effects on myelopoiesis, in vitro as well as in vivo. In the present study we investigated the ability of IL2 to act on committed and primitive bone marrow progenitor cells in defined serum‐free (SF) culture conditions which avoid many technical biases such as interference by exogenous stimulating or inhibiting factors. Low doses of IL2 (0·1–1000 U/ml) were studied without or in combination with recombinant IL3, GM‐CSF and erythropoietin, in SF long‐term marrow culture (LTMC). We report data in favour of an inhibitory activity of IL2 limited to committed progenitors and excluding more primitive haemopoietic stem cells, as shown by an alteration of CFU‐GM proliferation during the first 5 weeks of LTMC., decreasing with time, unaffected BFU‐E and increased nucleated cell production. Beyond week 5, no difference was observed between IL2 supplemented cultures and the SF control cultures. In parallel, IL2 induced the adherence of fibroblastic cells and their progeny. In addition to the inhibitory effect, IL2 appeared to limit the stimulating effect on granulopoiesis and erythropoiesis of myeloid growth factors (GF) such as combination of IL3, GM‐CSF and EPO. Indeed, in SF‐LTMC conditions, IL2 inhibitory effect is effective on CFU‐GM production throughout the 7 weeks of LTMC and on BFU‐E during the first 2 weeks only. These data confirm the interaction of IL2 with other GFs in the complex interplay of the cytokine network.
Decreased retinoblastoma protein expression in acute myeloblastic leukaemia is associated with the autonomous proliferation of clonogenic blasts
Authors: Zhu Yong‐Ming , Bradbury Dawn , Russell Nigel
Published: March 2008

Summary. We have previously shown that blasts from some patients with acute myeloblastic leukaemia (AML) grow autonomously in vitro and that this growth pattern is related to the production of autocrine growth factors including granulocyte‐macrophage colony stimulating factor (GMCSF) and interleukin‐1β. However, another potential mechanism of autonomous growth involves the deletion of growth inhibitory molecules. One such inhibitory protein is the product of the retinoblastoma (Rb) gene which is expressed in normal haemopoietic cells and functions in cell cycle control and as a transcriptional repressor. Deletion of the Rb gene has been reported in various types of malignancy. We have examined the expression of the Rb gene product in blasts from 39 patients with AML by Western analysis. Using an antibody, Rb (Ab‐2), which recognizes the C‐terminal region, 11/39 samples (28%) failed to express Rb protein. The results of the Western blot analysis were confirmed by flow cytometry using a second antibody, Rb (Ab‐1), against residues 300–380 of the Rb protein. Samples from seven of the 11 Rbnegative cases were studied for the expression of Rb mRNA: Rb mRNA was absent in four and three cases were positive. Analysis of the growth characteristics of these cells showed that blasts from 24/39 cases (62%) had partially or totally autonomous growth in a blast cell colony assay. Of these 24 samples with autonomous growth, 10 (41%) were Rb protein negative; in contrast, of the 15 cases with non‐autocrine growth only one was Rb protein negative (7%. P<0·05). Also, in Rb positive blasts, suppression of Rb protein production using an antisense oligonucleotide significantly increased proliferation of clonogenic AML blasts, confirming that the Rb protein acts as a negative regulator of growth in AML blasts. Our data suggest that deletion of Rb protein expression is frequently found in AML and is associated with the acquisition of autocrine growth characteristics, possibly as the consequence of derepression of genes involved in growth control and cytokine production.
Characterization and modulation of drug transport kinetics in K562 cl.6 daunorubicin‐resistant cell line
Authors: Jiang Xu‐Rong , Macey Marion G. , Collins Peter W. , Newland Adrian C.
Published: March 2008

Summary. The effects of cyclosporin A (CSA) and cellular energy depletion on daunorubicin (DAU) transport kinetics were investigated in a human erythroid leukaemia cell line K562 c1.6 selected for resistance to daunorubicin. K562 c1.6/DAU resistant cells displayed high levels of P‐glycoprotein and a high level of multidrug resistance against several antitumour drugs. The resistance factors of K562 c1.6/DAU cells to DAU, doxorubicin, vinblastine and etoposide were 106, 114, 85 and 13 respectively. A 1·6‐fold decrease (P<0·01, n= 8) in DAU accumulation and a 4‐fold increase (P<0·001, n= 8) in DAU efflux were shown in the resistant cells when compared to K562 c1.6 drug‐sensitive parental cells. K562 c1.6/DAU cells were also shown to reach a DAU saturation level (SL) 8‐fold faster (P<0·001, n= 8) than the parental cells. Addition of CSA to the resistant cells led to a dose‐dependent increase in cellular DAU retention, while no such effect was observed in the sensitive cells by the introduction of CSA. Resistance to the antitumour drugs could be reduced to various extents by CSA. The patterns of changes and modulations of DAU transport kinetics, as well as chemosensitivity in K562 c1.6/DAU cells were found to be similar to a vinblastine‐resistant leukaemia cell line CEM/VLB100. However, K562 cl.6/DAU cells were mere resistant to DAU, doxorubicin and etoposide than the CEM/VLB100 cells. An increase in DAU accumulation, intracellular SL and the time to reach 90% saturation level (SL90), and a decrease in DAU efflux in the resistant but not the sensitive cells were found in response to ATP depletion by sodium azide. These effects could be completely reversed by addition of glucose. Our results suggest that the presence of an energy‐dependent effluxing mechanism responsible for the decreased drug accumulation and enhanced drug efflux may make a major contribution to the mechanism of resistance in K562 c16/DAU resistant cells.
ADP causes partial degranulation of platelets in the absence of aggregation
Authors: Janes Sarah L. , Wilson Darren J. , Cox Alison D. , Chronos Nicolas A. F. , Goodall Alison H.
Published: March 2008

Summary. Whole blood flow cytometry has revealed that platelets undergo partial degranulation in response to ADP, in the absence of aggregation, as evidenced by the expression of the P‐selectin and CD63 antigens of the α‐granule and lysosomal membranes respectively. With maximum ADP (10‐5m) fibrinogen bound to 76·1 ± 7·2% of platelets but P‐selectin and CD63 antigen were expressed on 26·9±9·8% and 8·6±3·5% of platelets respectively. Maximum fibrinogen binding, P‐selectin and CD63 expression induced by α‐thrombin were 96·1±1·4%, 92·8±2·3% and 77·6±9·7% respectively. β‐thromboglobulin release from the ADP‐stimulated platelets correlated closely with the expression of P‐selectin and CD63 (r=0·98·0±02 for both antigens). No platelet aggregates were seen by flow cytometry and the absence of aggregation was confirmed by single cell counting. Addition of the GPIIb–IIIa antagonist echistatin. at concentrations that totally blocked fibrinogen binding to ADP‐stimulated platelets, had no effect on the expression of the granule membrane antigens. The partial degranulation of normal platelets was independent of thrombin generation since it was not inhibited by hirudin (5 units/ml). In conclusion, ADP is capable of causing partial degranulation of platelets independently of aggregation, fibrinogen binding or thrombin generation. Thus release of potent procoagulant, vasoactive and mitogenic substances from the platelets could continue in the presence of thrombin inhibitors and GPIIb‐IIIa antagonists.
A comparative study of partial primary structures of the catalytic region of mammalian protein C
Authors: Murakawa Masahiro , Okamura Takashi , Kamura Takumi , Kuroiwa Mika , Harada Mine , Niho Yoshiyuki
Published: March 2008

Summary. Protein C (PROC) is a plasma vitamin K‐dependent zymogen of a serine protease which regulates blood‐clotting cascade through proteolytic inactivation of the non‐enzymatic cofactors of blood coagulation, Va and VIIIa. We characterized the partial nucleotide and amino acid sequences for the catalytic domain of PROC in six mammalian species, rhesus monkey, dog, cat, goat, horse and mouse, and compared these sequences with known ones from humans, the bovine and rat. By using a pair of primers based on the nucleotide sequences from human and bovine PROC cDNA, the PROC gene fragments were enzymatically amplified from their genomic DNAs and were sequenced by the dideoxytermination method. The cloned PROC gDNA encoded a part of the heavy chain of PROC including the lesions of active site residues corresponding to human PROC Asp‐257 and Ser‐360. Comparison of the sequences from these species revealed that there was a high degree of homology at the nucleotide and amino acid levels: from 69% to 96% of the amino acids in the catalytic region were identical among the nine species including humans, the bovine and rat. The locations of five Cys residues as well as the putative carbohydrate attachment sites were evolutionally conserved. All the amino acids recognized in the human abnormal PROC variants were conserved across species, suggesting their functional importance, and a comparison of the conserved residues among PROC from multiple species will provide considerable information in the investigations of PROC functions.
Uncommon clinical presentation of a lymphocytic lymphoma of intermediate differentiation in a patient with systemic sclerosis
Authors: Baldini L. , Guffanti A. , Ferrari A. , Castagnone D. , Mascagni B. , Fracchiolla N. , Colombi M. , Maiolo A. T.
Published: March 2008

Summary. The association of systemic sclerosis (SSc) and non‐Hodgkin lymphoma is a rare event and its pathogenetic mechanism remains to be clarified. We describe a previously unreported association of SSc with a lymphocytic lymphoma of intermediate differentiation (IDL), involving the gastrointestinal tract, gallbladder and one salivary gland. Whereas the morphological, immunological and cytogenetic features were typical of IDL, the extensive extranodal involvement and the association with an autoimmune disorder were suggestive of two other lymphomas of mantle lineage: mucosa‐associated lymphoid tissue (MALT) lymphoma and monocytoid B‐cell lymphoma (MBCL).
Successful reversal of neutropenia in Felty's syndrome with recombinant granulocyte colony stimulating factor
Authors: Choi Man‐Fung Vincent , Mant Michael J. , Turner A. Robert , Akabutu John J. , Aaron Stephen L.
Published: March 2008

Summary. We report two patients with Felty's syndrome and chronic skin ulcers treated successfully with recombinant granulocyte colony stimulating factor (GCSF). In both cases granulocytes returned to the normal range within days of starting treatment, and their cutaneous ulcers improved. In one patient granulocytes were maintained at normal levels with a regimen of GCSF 3 μg/kg twice weekly for 14 months.
Raised urinary calmodulin levels in idiopathic myelofibrosis: possible implications for the aetiology of fibrosis
Authors: Eastham J. M. , Reilly J. T. , Mac Neil S.
Published: March 2008

Summary. Platelet growth factors (e.g. PDGF and TGF‐β) are thought to be pathogenetically important in the stromal reaction characteristic of idiopathic myelofibrosis (IM). We have investigated a possible pathogenetic role for a further platelet mitogen, calmodulin. Platelets are rich in calmodulin, of which 30–40% is releasable with a time course that differs from α‐granule proteins. In IM urinary calmodulin concentrations were 3‐fold those of the normals controls. We suggest that an abnormal release of calmodulin may occur from platelets/megakaryocytes in patients with IM, and that calmodulin should be considered, along with other growth factors, in the pathogenesis of marrow fibrosis.
Recombinant human interleukin‐4 inhibits the production of granulocyte‐macrophage colony stimulating factor by blood mononuclear cells
Authors: Sato Norihiro , Sawada Ken‐ichi , Tarumi Takashi , Koizumi Kazuki , Yasukouchi Taro , Takahashi Tsuneo A. , Sekiguchi Sadayoshi , Koike Takao
Published: March 2008

Summary. The effect of recombinant human (rh) interleukin‐4 (rIL‐4) on human blood BFU‐E was investigated using two populations of cells: platelet‐depleted low‐density mononuclear cells (FH, Pl− cells), as unpurified cells, and highly purified BFU‐E. When FH, Pl− cells were cultured with rherythropoietin (rEp), rIL‐4 inhibited BFU‐E growth in a dose‐dependent manner. However, the addition of rIL‐4 did not affect rh‐interleukin‐3 (rIL‐3) supported BFU‐E growth. Limiting dilution analysis (LDA) of FH, Pl− cells showed that rIL‐4 suppressed endogenous production of burst‐promoting activity (BPA) by accessory cells. Highly purified BFU‐E were used as target cells to measure BPA in the conditioned medium (CM) that was prepared by FH, Pl− cells. When 100 purified BFU‐E were cultured in 0·5 ml clots with 20% (vol/vol) of the CM, the number of BFU‐E colonies was increased by the CM. The increase was significantly reduced by the addition of the CM prepared in the presence of rIL‐4, but anti‐IL‐4 blocked the effect of rIL‐4. The concentration of IL‐3 and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in CM was determined by an enzyme‐linked immunoadsorbent assay (ELISA). The spontaneous production of GM‐CSF but not IL‐3 was detected, and this was significantly decreased in the presence of rIL‐4. Anti‐GM‐CSF but not anti‐IL‐3 inhibited CM supported BFU‐E growth, indicating that the main BPA in the CM is GM‐CSF and that rIL‐4 suppresses the spontaneous production of GM‐CSF by accessory cells. From these studies, we conclude that rIL‐4 has a unique mechanism as a negative regulator on erythropoiesis through the inhibition of BPA production by blood mononuclear accessory cells.
Gene amplification in non‐Hodgkin's lymphoma
Authors: Ben‐Yehuda Dina , Houldsworth Jane , Parsa Nasser Z. , Chaganti R. S. K.
Published: March 2008

Summary. Among 426 consecutively ascertained and karyotypically abnormal non‐Hodgkin's lymphoma (NHL) tumours, cytological evidence for gene amplification in the form of homogeneously staining regions (HSRs) was encountered in nine cases of large cell diffuse lymphoma (LC‐DL). The mean age of patients with HSRs was 62·9 years and four died within a year of diagnosis. To identify candidate gene(s) amplified in these tumours, we performed a Southern blot analysis of tumour DNA using probes for 23 known protooncogenes and the multidrug resistance gene, PGY1, Besides a two‐fold amplification of the BCL2 gene in two cases, no evidence for overt amplification of any of the genes assayed was found. To confirm DNA amplification in these specimens we performed the DNA in‐gel renaturation assay. Evidence for presence of amplified DNA fragments was obtained in four of seven specimens. These results suggest amplification of a novel gene(s). To our knowledge, this is the first formal study of gene amplification in a large consecutively ascertained series of fresh lymphoma biopsies.
Additive in vitro effects of anti‐sickling drugs
Authors: Stuart J. , Mojiminiyi F. B. O. , Stone P. C. W. , Culliford S. J. , Ellory J. C.
Published: March 2008

Summary. To study the effect of anti‐sickling drugs on cellular dehydration induced by entry of Ca, sickle cells were subjected to cyclical oxygenation–deoxygenation for 15 h in Ca‐containing buffer. The consequential loss of cation (K) via the Ca‐dependent K efflux (Gardos) channel caused cell dehydration and loss of deformability. Inhibition of a specific fraction of Ca entry by verapamil had no rheologically protective effect, whereas inhibition of the Gardos channel by clotrimazole or nitrendipine had a marked protective effect. When Gardos channel inhibition (by either clotrimazole or nitrendipine) was combined with stabilization of the oxyconformation of sickle haemoglobin (by the substituted benzaldehyde 12C79), an additive protective rheological effect was achieved with 60–78% reduction in clogging rate of 5 μm diameter pores when compared with no drug. Therapeutic use of anti‐sickling compounds in combination may achieve increased efficacy with lower toxicity.
Long‐term follow‐up of aplastic anaemia
Authors: Narayanan M. N. , Geary C. G. , Freemont A. J. , Kendra J. R.
Published: March 2008

Summary. Of 61 patients with aplastic anaemia (AA) diagnosed in our hospitals, 37 survived more than 2 years; actuarial survival of this latter group was 58%, with a median follow‐up of living patients of 10.2 years. Laboratory and clinical data pertaining to these long‐term survivors was scrutinized to determine the incidence of clonal disorders, which was identified in 43%. Morphological evidence of the myelodysplastic syndrome (MDS) was found in 13 (35%), including four cases of RAEB; four (11%) developed PNH. Of 23 patients studied, four showed karyotypic abnormalities, but these did not always coincide with morphological features of MDS.

Although four patients now have completely normal blood and marrow morphology, and another had normal blood and marrow morphology at the time of death due to unrelated disease, the study confirms the high incidence of cytopenia and morphological abnormality, sufficient to justify a diagnosis of MDS, in patients with a history of AA. No definite survival plateau was identified. However, the natural history of MDS secondary to AA seems to be different to that of MDS arising de novo; the clinical course is relatively indolent, possibly implying a different biology.
Vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe
Authors: Savage David , Gangaidzo Innocent , Lindenbaum John , Kiire Clement , Mukiibi Joshua M. , Moyo Alpha , Gwanzura Christine , Mudenge Boniface , Bennie Anne , Sitima John , Stabler Sally P. , Allen Robert H.
Published: March 2008

Summary. In a study of the pathogenesis and clinical features of megaloblastic anaemia in southern Africa, we evaluated 144 consecutive Zimbabwean patients with megaloblastic haemopoiesis. Vitamin B12 deficiency was diagnosed in 86.1% of patients and was usually due to pernicious anaemia; isolated folate deficiency accounted for only 5/5% of cases. Anaemia was present in 95.8% of patients; the haemoglobin (Hb) was 6 g/dl in 63.9%. Neurological dysfunction was noted in 70.2% of vitamin B12‐deficient patients and was most striking in those with Hb values > 6 g/dl. Serum levels of methylmalonic acid, homocysteine, or both, were increased in 98.5% of patients.

Vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe and, contrary to textbook statements, is often due to pernicious anaemia. Isolated folate deficiency is less common. As reported in industrialized countries 75 years ago, anaemia is almost always present and often severe. Neurological dysfunction due to vitamin B12 deficiency is most prominent in patients with mild to moderate anaemia.
Alleles at D6S265 and D6S105 define a haemochromatosis‐specific genotype
Authors: Worwood M. , Raha‐Chowdhury R. , Dorak M. T. , Darke C. , Bowen D. J. , Burnett A. K.
Published: March 2008

Summary. Hereditary haemochromatosis is an HLA‐linked, recessive disorder with HLA‐A3 a strong marker for the gene. We have identified molecular markers for two serologically indistinguishable subtypes of HLA‐A3 and examined these in 42 patients with haemochromatosis. The common HLA‐A3 subtype HLA‐A* 0301 (highly correlated with allele 1 of D6S265) was a slightly better marker for haemochromatosis (RR = 10.1, Chi2= 30) than the serologically recognized A3 antigen (RR = 9.1; Chi2 27.3). Allele 8 of the more telomeric locus D6S105 was also strongly associated with haemochromatosis (RR = 13.0; Chi2= 21.1) but alleles at this locus were not in strong linkage disequilibrium with HLA‐A alleles in the control subjects. The co‐occurrence of D6S265–1 and D6S105–8 alleles yielded a higher risk (RR = 16.9; Chi2= 44). Homozygosity for the haplotype including these markers was specific for haemochromatosis, i.e. did not occur in 376 healthy subjects but was observed in 21.4% of patients. These results refine the HLA‐A3 association with haemochromatosis, suggest that the haemochromatosis gene is located on the telomeric side of HLA‐A and define a possible haplotype in which the first mutation may have occurred.
Acute lymphoblastic leukaemia following Hodgkin's disease is associated with a good prognosis
Authors: Narayanan M. N. , Morgenstern G. R. , Chang J. C. , Harrison C. J. , Ranson M. , Scarffe J. H.
Published: March 2008

Summary. Acute leukaemia, both myeloid and lymphoblastic, in patients treated for Hodgkin's disease (HD) is thought to have a poor prognosis. We report four adults who developed secondary acute lymphoblastic leukaemia (ALL) following chemoradiotherapy for HD. The chromosomal translocation t(4;11) (q21;q23) was found in two patients who received a chemotherapeutic regimen containing the DNA topoisomerase II inhibitor etoposide. Three of the four patients are alive and in unmaintained first remission at 3, 5 and 9 years from diagnosis of ALL, two following autologous bone marrow transplantation. These results suggest that ALL following HD may have a good prognosis when treated aggressively.
Altered surface marker expression and function of G‐CSF‐induced neutrophils from test subjects and patients under chemotherapy
Authors: Spiekermann Karsten , Emmendoerffer Andreas , Elsner Joern , Raeder Evelin , Lohmann‐Matthes Marie‐Luise , Prahst Arndt , Link Hartmut , Freund Mathias , Welte Karl , Roesler Joachim
Published: March 2008

We have previously reported an altered surface marker expression and chemotaxis of G‐CSF‐induced neutrophils from patients with severe congenital neutropenia. However, effects of G‐CSF and influence of the underlying disease on neutrophils could not be discerned. In this study we have evaluated the effects of G‐CSF on neutrophil phenotype and function in patients under chemotherapy and in healthy test subjects. We found a significantly enhanced expression of FcγRI, CD14 and CD54 and a decrease in the level of FcγRIII during G‐CSF treatment. In addition, motility of G‐CSF‐induced neutrophils was significantly decreased. The effects were seen in patients under cytotoxic chemotherapy and in healthy test subjects. Surface marker alterations and neutrophil motility were affected by G‐CSF administration in a dose‐dependent manner. Kinetic studies on neutrophils from healthy test subjects demonstrated that all effects could be seen after a single administration of 300 μg G‐CSF and began to appear within 4 h. Release of partially immature neutrophils from the bone marrow and indirect activation of these cells by G‐CSF are discussed as possible reasons for the findings presented. They demonstrate that G‐CSF has profound effects on neutrophil phenotype and function in vivo which might have clinical implications.
Intensive induction/consolidation therapy without maintenance in adult acute lymphoblastic leukaemia: a pilot assessment
Authors: Wernli M. , Tichelli A. , Fliedner V. von , Re G. Brun del , Chaputs B. , Fey M. F. , Fopp M. , Gmür J. , Grob J‐P. , Jacky E. , Pedrazzini A. , Schmid L. , Senn H‐J. , Tobler A. , Gratwohl A.
Published: March 2008

. Maintenance chemotherapy for up to 3 years is traditionally given to patients with acute lymphoblastic leukaemia (ALL) achieving complete remission. We questioned the value of such maintenance therapy in adult patients treated with intensive induction/consolidation. In a phase II study (SAKK 33/86) 63 patients between 17 and 72 years of age (median 27 years) with newly diagnosed ALL were treated with three intensive cycles of marrow‐ablative chemotherapy. All subtypes were included. No maintenance phase was added. 53 patients (84%) entered a complete remission (CR) and 21 (33%) continue to be in unmaintained remission for 11‐69 months (median 21 months). The disease‐free survival of patients achieving CR and completing all three cycles is 40% at 3 years, with a 95% confidence interval of ± 19%. These findings are comparable to the results of conventional studies. We conclude that maintenance therapy might not be needed in all adult ALL patients. Its value should be tested in a randomized trial. For patients failing, novel approaches are needed to improve outcome in adult ALL.
A case of myelodysplasia with eosinophilia having a translocation t(5;12)(q31;q13) restricted to myeloid cells but not involving eosinophils
Authors: Jani K. , Kempski H. M. , Reeves B. R.
Published: March 2008

Summary. A chromosomally abnormal clone characterized by a translocation, t(5;12)(q31;q13), was detected in the marrow of a child with myelodysplasia and associated eosinophilia which included a generalized skin infiltration. Combined immunophenotyping and fluorescence in situ hybridization on interphase bone marrow cells showed that the chromosomal rearrangement was restricted to the granulocyte lineage but was not present in the eosinophils. If the chromosome rearrangement is important in the overproduction of eosinophils in this case, the lineage restriction found suggests that its effect must be indirect.
Inactivation of the retinoblastoma gene appears to be very uncommon in myelodysplastic syndromes
Authors: Preudhomme Claude , Vachee Anne , Lepelley Pascale , Vanrumbeke Mickaéul , Zandecki Marc , Quesnel Bruno , Cosson Alain , Fenaux Pierre
Published: March 2008

Summary. Rearrangements of the retinoblastoma (RB) gene have been reported in a few cases of myelodysplastic syndromes (MDS). In addition, low or absent expression of the RB protein is found in 20‐30% of cases of acute myeloid leukaemias (AML), particularly in AML with a monocytic component (M4 or M5). We performed Southern blot analysis of the RB gene in 90 cases of MDS, including 37 cases of chronic myelomonocytic leukaemia (CMML). None of them had progressed to AML at the time of study. In 37/90 patients (including 20 CMML) Northern blot analysis, study of RB protein by immunocytochemistry on bone marrow slides, and detection of point mutations in exons 20‐24 of the RB gene was also made, using single strand conformation polymorphism analysis (SSCP).

No abnormal Southern profile was found in any of the 90 patients. Northern blot and immunocytochemical study of RB protein were normal in the 37 cases studied. SSCP analysis detected a point mutation in 2/37 patients tested. Direct sequencing confirmed the mutation in each case, which involved intron 21 and intron 23, respectively, and was located outside splicing sites of the neighbouring exons.

These findings suggest that abnormalities of the RB gene and its expression must be very rare in MDS, and play a minor role, if any, in the pathophysiology of those disorders, at least before progression to AML.
Prevalence of antithrombin deficiency in the healthy population
Authors: Tait R. C. , Walker Isobel D. , Perry D. J. , Islam S. I. A. M. , Daly M. E. , McCall F. , Conkie J. A. , Carrell R. W.
Published: March 2008

. In a cohort of 9669 blood donors we have identified 16 cases of congenital AT deficiency (1 in 600) by way of family studies and AT gene analysis. Two donors had type I AT deficiency (prevalence 0.21 per 1000; 95% CI = 0.03/1000 to 0.75/1000), their families displaying a symptomatic phenotype. 14 donors had a type II deficiency (prevalence 1.45 per 1000; 95% CI = 0.79/1000 to 2.43/1000): one recurring and three unique mutations. None of these type II deficiencies appeared to confer a high thrombotic risk despite many of the affected individuals having experienced potentially prothrombotic challenges. The high frequency of these relatively asymptomatic variants may reflect a selection bias in the study population. However, their existence should not only add to our understanding of structure‐function relationships of AT but may also influence our management of asymptomatic deficient individuals identified in epidemiological or presurgical screening programmes.
The effect of deferiprone (L1) and desferrioxamine on myelopoiesis using a liquid culture system
Authors: Al‐Refaie F. N. , Wilkes S. , Wonke B. , Hoffbrand A. V.
Published: March 2008

Summary. Agranulocytosis was observed in a 63‐year‐old patient with myelodysplasia 6 weeks after commencing chelation with the oral iron chelator deferiprone (1,2‐dimethyl‐3‐hydroxypyrid‐4‐one, L1) at a daily dose of 79 mg/kg. Using a liquid culture system no difference was observed when L1 toxicity to normal and patient myelopoiesis was compared (IC50: 150 v 172 μM respectively). L1 was found to be less toxic than desferrioxamine (DFX) (IC50: 150 u 9 μM respectively) to normal myelopoiesis. Delayed addition of iron to myeloid cultures containing an inhibitory concentration of L1 or DFX was associated with reversal of chelator‐induced inhibition of myelopoiesis up to 6 h but not after 24 h. Further studies are needed to determine the incidence and elucidate the pathogenesis of agranulocytosis associated with L1 therapy.
Biochemical and molecular characterization of a new sporadic glucose‐6‐phosphate dehydrogenase variant described in Italy: G6PD Modena
Authors: Cappellini Maria Domenica , Sampietro Maurizio , Toniolo Daniela , Carandina Giovanni , Pittalis Salvatore , Montemuros Franco Martinez di , Tavazzi Dario , Fiorelli Gemino
Published: March 2008

Summary. A new glucose‐6‐phosphate dehydrogenase variant detected in an Italian man from the Po delata is described and designated as G6PD Modena. Biochemical characterization of the variant enzyme revealed an activity 21% of normal, a slow electrophoretic mobility, increased Km value for NADP, decreased Km value for G6P and a complete absence of NADPH inhibition, which could account for the apparently nonhaemolytic feature of this variant. The cloning and sequencing of the G6PD Modena allele showed a GC transition at nucleotide 844 in exon VIII causing a Asp His amino acid substitution. On the basis of biochemical characterization, G6PD Modena is classified as a genuine variant but it has the same mutation as G6PD Seattle‐like.
Scedosporium inflatum infection in immunocompromised haematological patients
Authors: Tapia Manuel , Richard Carlos , Baro Julio , Salesa Ricardo , Figols Javier , Zurbano Felipe , Zubizarreta Alberto
Published: March 2008

We report four cases of Scedosporium inflatum (S. inflatum) infection in severely immunocompromised haematological patients. Six well‐documented cases of S. inflatum disseminated infection in haematological patients have been reported: four in Australia and two in Spain. Their clinical and pathological characteristics are heterogenous, particularly in the Australian cases. However, the clinical and pathological profile emerging from our and other Spanish cases is homogenous and very similar to the clinico‐pathological spectrum of other disseminated mycoses, including Aspergillus and S. apiospermum. The optimal treatment of S. inflatum infection is unknown and the outcome in haematological patients is very poor. Eight patients died despite systemic antifungal treatment.
Postnatal changes of CD45 expression in peripheral blood T and B cells
Authors: Lai Raymond , Visser Lydia , Poppema Sibrand
Published: March 2008

SUMMARY. One known postnatal change of CD45 expression is the decline of the CD45RAhigh CD45ROlow T subsets and the reciprocal increase of the CD45RAlow CD45ROhigh T subsets in the peripheral blood. Using a panel of monoclonal antibodies reactive with either protein or carbohydrate epitopes on the variable regions of CD45, we were able to detect more postnatal changes of CD45 expression. These changes are largely caused by modulation of the CD45 glycosylation, including: (I) lesser sialylation of the CD45RA region on T cells, and (2) differential sialylation of the CD45RB region leading to the distinction of CD45RBhigh and CD45RBlow T and B subsets. In addition, the existence of the CD45RAdim CD45ROdim labelled as transitional T cells is only found during the postnatal life. These changes may reflect the maturation of the immune system.
Production of fibronectin and adherence to fibronectin by human myeloma cell lines
Authors: Riet Ivan Van , Greef Catherine De , Favero Herwig Del , Demanet Christian , Camp Ben Van
Published: March 2008

SUMMARY. In the present study we examined the production of fibronectin (FN) in 10 human myeloma cell lines (HMCL). By Northern blot analysis we could detect the presence of FN‐mRNA in most of these lines. A majority of the cell lines (LP‐1, OPM1, SKMM‐2, EJM, JJN3 and ARH‐77) hybridized with two probes recognizing total FN while the mRNA of one cell line (LB84‐1) was shown to hybridize also with a probe recognizing the EDA segment of cellular FN. In one cell line (L363) FN‐mRnA could only be detected after PCR amplification. Using an enzyme‐linked immunosorbent assay, we could also demonstrate that HMCL secrete FN in their culture medium. Seven myeloma cell lines that produce FN showed a significant adherence to soluble FN. By blocking experiments, this adhesion was found to be mediated by the VLA‐4 (α4β1) receptor. The production of fibronectin and the expression of a functional receptor for this protein may represent independent features of myeloma cells but may also be functionally linked. Since fibronectin has recently been identified as a crucial co‐factor of IL6 in the regulation of the terminal B cell differentiation, the endogenous FN production may be part of an autocrineline process mediating the autonomous growth of these cell lines. Alternatively, the FN production may also reflect a mechanism that myeloma cells use to communicate with their natural environment, i.e. the bone marrow stroma. In order to examine whether FN can be regarded as a reliable tumour marker for myeloma, serum FN levels were measured in 24 myeloma patients and were found to be increased when compared to normal controls (mean values 40.7 mg/dl versus 29.2 mg/dl; P < 0.01). It was, however, not possible to relate the FN concentration to serum IL6 and β2m levels or bone marrow plasmacytosis.
Increased expression of myeloid antigen markers in adult acute lymphoblastic leukaemia patients: diagnostic and prognostic implications
Authors: Lauria F. , Raspadori D. , Martinelli G. , Rondelli D. , Ventura M. A. , Farabegoli P. , Tosi P. , Testoni N. , Visani G. , Zaccaria A. , Gamberi B. , Cenacchi A. , Tura S.
Published: March 2008

Summary. By applying direct immunofluorescence with a dual‐staining technique we were able to demonstrate that 20/37 (54%) patients with acute lymphoblastic leukaemia (ALL) expressed both lymphoid and myeloid antigens on the same leukaemic cells. CD13 and CD33 myeloid antigens were detected in 18/20 and in 15/20 cases respectively, and both in 13. Molecular studies confirmed that the four patients with T‐cell phenotype had molecular rearrangement of T‐cell receptor (TcR) β chain, and 26 ALL patients with ‘B‐cell’phenotype showed JH rearrangement. Two ALL patients without (ALL/My‐) and three with myeloid antigens (ALL/My+) also demonstrated bcr/abl rearrangement. Both groups of patients had similar presenting features such as age, sex, Hb level, white blood cells, platelet counts and cytogenetic features. Complete response was achieved in 16/17 (94%) ALL/My‐ patients and in 15/18 (83%) ALL/My+ patients (two deaths occurred during induction) with a mean duration of 17 and 16 months respectively and with similar survival and event‐free survival curves.

Myeloid antigen expression in adult ALL patients may occur more frequently than previously reported. The presence of myeloid antigen does not identify, in our series, a higher‐risk subgroup of patients, although lack of any statistical evidence of prognostic significance needs to be confirmed in a larger case study.
The relative levels of βA and βS mRNAs in Hb S heterozygotes and in patients with Hb S‐β+ ‐thalassaemia or Hb S‐β+ ‐HPFH combinations
Authors: Dimovski A. J. , Efremov D. G. , Gu L‐H. , Huisman T. H. J.
Published: March 2008

SUMMARY. We have used a quantitative reverse transcription/ polymerase chain reaction (RT/PCR) procedure to evaluate the relative amounts of βA and βS mRNA transcripts in eight subjects with a simple Hb S heterozygosity, in six with Hb S‐β+ ‐thalassaemia (thal), and in three individuals with Hb S‐β+ ‐HPFH [hereditary persistence of fetal haemoglobin (Hb)] [two with the Atlanta type and one with the Gγ‐202 (C ± G) substitution]. A balanced synthesis of βA and βS mRNAs was observed in all Hb S heterozygotes, whereas the βA mRNA levels were reduced to ˜ 16% of that of the βS mRNA in the six Hb S‐β+ ‐thal compound heterozygotes, to ˜43% in the two subjects with Hb S‐β+ ‐HPFH (Atlanta type), and to 23.8% in the one individual with Hb S‐β+ ‐HPFH [Gγ‐202 (C ± G) substitution]. The higher Hb A versus Hb S levels observed in all groups of the patients studied, further confirm a post‐translational control mechanism in determining the levels of Hb A and Hb S in the peripheral blood of these individuals. The procedure described here provides an accurate and easy method for studying the relative expression of particular globin genes at the transcriptional level in patients with various haemoglobinopathies.
Myelodysplasia in a patient with pre‐existing paroxysmal nocturnal haemoglobinuria: a clonal disease originating from within a clonal disease
Authors: Longo L. , Bessler M. , Beris P. , Swirsky D. , Luzzatto L.
Published: March 2008

SUMMARY. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemolytic anaemia, clonal in nature, due to somatic mutation. PNH may evolve to aplastic anaemia; more rarely to a myelodysplastic syndrome (MDS) or to acute myeloid leukaemia (AML). We have studied a patient who suffered from PNH and later developed refractory anaemia with ringed sideroblasts (RARS) associated with trisomy 8. By testing peripheral blood cells with appropriate antibodies we have shown that all of the red cells, neutrophils and monocytes, as well as 20% of the lymphocytes, belonged to the PNH clone; in contrast, only 43% of neutrophils and 22% of monocytes belonged to the MDS clone. We infer that the MDS must have arisen from within the PNS clone.
Eosinophilic leukaemia with a t(2;5) (p23;q35) translocation
Authors: Sato Hiroshi , Danbara Mikio , Tamura Masato , Morita Makoto
Published: March 2008

SUMMARY. Although the diagnosis of rare eosinophilic leukaemia is possible based on cytogenetic abnormalities, the chromosomal aberrations reported are diverse. We found a t(2;5) (p23;q35) translocation on bone marrow cells of a patient with chronic eosinophilia who suffered from multiple pustular folliculitis but lacked the clinical symptoms commonly observed in hypereosinophilic syndrome. No morphological abnormalities in an eosinophilic series were apparent and other haemopoietic cells were well preserved. A therapeutic trial with interferon‐α failed after a 2‐month period, and the patient is currently undergoing a combination therapy with interferon and intermittent administrations of hydroxyurea.
John Hageman's factor and deep‐vein thrombosis: Leiden Thrombophilia Study
Authors: Koster Ted , Rosendaal Frits R. , Briet Ernest , Vandenbroucke Jan P.
Published: March 2008

SUMMARY. Because the relationship between factor XII deficiency and venous thrombosis in unclear and study results seem contradictory, we undertook a populationbased case‐control study.

Among 350 unselected patients younger than 70 years, with a first, objectively confirmed, episode of deep‐vein thrombosis and without underlying malignant disease we detected a 6% frequency (21/350 patients) of factor XII deficiency (activity level 57%). Among 350 healthy control subjects, matched for age and sex, the frequency was 5% (18 subjects). Thus there is no increase in prevalence of factor XII deficiency among thrombosis patients and no increase in thrombosis risk for subjects with low factor XII levels (mathced odds ratio 1.2 (95% CI 0.6‐2.4)). In addition, there was no relation between strata of factor XII levels and thrombosis risk. In conclusion, we do not consider factor XII to be a determinant of deep‐vein thrombosis.
Prognostic significance of loss of a chromosome 17p and p53 gene mutations in blast crisis of chronic myelogenous leukaemia
Authors: Nakai Hiroyuki , Misawa Shinichi , Taniwaki Masafumi , Horiike Shigeo , Takashima Teruyuki , Seriu Taku , Nakagawa Hitoshi , Fujii Hiroshi , Shimazaki Chihiro , Maruo Naoyuki , Akaogi Teruaki , Uike Naokuni , Abe Tatsuo , Kashima Kei
Published: March 2008

SUMMARY. In 31 cases of chronic myelogenous leukaemia (CML) we examined the prognostic significance of chromosomal loss of a 17p and p53 mutations at the onset of blast crisis (BC). p53 mutations were closely related to a shortened survival in CMLBC (P < 0.005 by the logrank test), whereas loss of a 17p by itself was not a poor prognostic indicator. The prognostic significance of loss of a 17p, however, emerged when combined with its predominance in the metaphases analysed. This predominance might easily and rapidly be screened by polymerase chain reaction‐based analysis in about half of the cases.
Typing of the granulocyte‐specific NA antigens by restriction fragment length polymorphism analysis
Authors: Stein E.‐L. , Bux J. , Santoso S. , Mueller‐Eckhardt C.
Published: March 2008

SUMMARY. An RNA‐based method has been developed to genotype donors for the granulocyte‐specific alloantigens NA1 and NA2. mRNA was isolated from granulocytes, reversely transcribed into cDNA and amplified using an Fcgamma‐receptor III‐1 sequence‐specific primer in the polymerase chain reaction (PCR). PCR products were analysed by restriction fragment length polymorphism (RFLP) using the restriction endonuclease Taq I, which provided a distinct restriction fragment pattern corresponding to the NA alleles. 17 donors were typed by PCR‐RFLP and the results were in close accordance with those obtained by serological phenotyping by granulocyte immunofluorescence and the antigen capture assay MAIGA.
Response of T‐beta CD8+ lymphocytosis‐associated neutropenia to G‐CSF
Authors: Vickers M. , Stross P. , Millard P. , Barton C.
Published: March 2008

SUMMARY. Only limited data are reported on the response to granulocyte‐colony stimulating factor (G‐CSF) of large granular lymphocytosis‐associated neutropenia. We report features of such a case who developed a suppurating leg ulcer following a dog bite. G‐CSF was used to increase the neutrophil count, allowing the ulcer to be successfully skin grafted.
Transforming growth factor‐β and megakaryocytes in the pathogenesis of idiopathic myelofibrosis
Authors: Martyré M. C. , Romquin N. , Le Bousse‐Kerdiles M. C. , Chevillard S. , Benyahia B. , Dupriez B. , Demory J. L. , Bauters F.
Published: March 2008

Summary. Although the disease is well described, the pathogenesis of bone marrow fibrosis in idiopathic myelofibrosis still remains unclear. We previously reported elevated intraplatelet transforming growth factor‐β (TGF‐β) levels in patients with this myeloproliferative disorder, compared with healthy subjects. Here, in a series of 16 patients, we show that TGF‐β expression is also increased in patients' periperal blood mononuclear cells (PBMC): (i) at the mRNA level analysed by Northern blot hybridization and/or reverse transcription‐polymerase chain reaction (RT‐PCR): (ii) and/or at the secreted peptide level as evaluated in conditioned media from patients' mononuclear cells by a growth inhibition assay on CC164 cells.

By immunostaining with a polyclonal anti‐TGF‐β1 antibody, TGF‐β was localized in morphologically heterogenous cells; these cells were characterized as megakaryocytes by labelling with a gpIIbIIIa monoclonal antibody.

Thus we provide evidence that both TGF‐β and megakaryocytes are linked in the pathogenesis of idiopathic myelofibrosis.
CD45 isoform expression on human haemopoietic cells at different stages of development
Authors: Craig William , Poppema Sibrand , Little Marie‐Terese , Dragowska Wieslawa , Lansdorp Peter M.
Published: March 2008

Summary. Alternate splicing and glycosylation produce multiple CD45 isoforms which are selectively expressed on the surface of cells of the haemopoietic system. The expression of CD45RA, CD45RB and CD45RO on CD34+ and CD34‐ haemopoietic cells from umbilical cord blood, bone marrow and fetal liver were studied by flow cytometry. CD34+ subpopulations defined by CD45 isoform expression were sorted from bone marrow and tested in long‐term culture assays. By combining results of functional studies with phenotypic data and previously published information, the following pattern of CD45 isoform expression on early haemopoietic cells was established. The most primitive CD34+ cells are CD45RO+ CD45RB+ and express low or undetectable levels of CD45RA. Upon erythroid differentiation, CD34+ cells remain CD45RO+ CD45RB+, whereas commitment into the myeloid and lymphoid lineages coincides with down‐regulation of CD45RO and up‐regulation of CD45RA. As a result, the majority of CD34+ cells can be divided into two mutually exclusive populations of cells which express either CD45RO or CD45RA. This notion was confirmed in this study by three‐colour immunofluorescence. The alternative expression of various CD45 isoforms on functionally distinct haemopoietic cells suggests an important role for these molecules in the proliferation and differentiation of haemopoietic cells.
Recovery of T‐lymphocytes for adoptive immunotherapy by lymphapheresis of HIV‐infected patients without alterations of virological, immunological or clinical parameters
Authors: Lunzen Jan VAN , Schmitz Jörn , Dengler Kathrin , Schmidt Lutz , Schmitz Herbert , Dietrich Manfred
Published: March 2008

Summary. We performed repeated continuous flow cytaphereses (CFC) on 13 asymptomatic HIV‐1‐infected patients to study the feasibility of cell separation procedures to recover high yields of peripheral blood T‐lymphocytes for adoptive immunotherapy in HIV‐infected patients and to determine immunological and virological alterations following such procedures.

A mean yield of 6.23 * 109 lymphocytes could be obtained by each cytapheresis, containing 1.82 * 109 CD4+, 3.23 * 109 CD8+ T‐lymphocytes and 8.39 * 106 CD34+ peripheral progenitor cells. The CD4/CD8 ratio (mean 0.53, SD ± 0.15) in the cell samples reflected the distribution of the lymphocyte subsets in vivo. Absolute lymphocyte counts decreased at a mean of 404/mm3 (25%) immediately after CFC but were replaced from the extravascular pool within 1 h. The CD4/CD8 ratios, p24‐antigenaemia. HLA‐DR expression and neopterin levels did not change significantly after cell separation. No alteration of the number of T‐cells with integrated proviral DNA copies (1/103 to 1/106) could be detected in peripheral T‐helper cells by PCR after lymphapheresis.

We conclude that high yields of peripheral T‐lymphocytes can be obtained by continuous flow lymphapheresis for cell mediated immunotherapy, withou deterioration of virological or immunological parameters in HIV‐infected patients. The separated T‐cells are fully replaced from extravascular pools after 1 h.