bluuebunny/crossref_metadata_2025 huggingface dataset:
Primary polycythaemia: positive diagnosis using the differential response of primitive and mature erythroid progenitors to erythropoietin, interleukin 3 and alpha‐interferon
Authors: Dudley J. M. , Westwood N. , Leonard S. , Eridani S. , Pearson T. C.
Published: March 2008

Forty adult subjects were studied with the aim of establishing positive diagnostic criteria in primary proliferative polycythaemia (polycythaemia vera, PPP). These comprised 14 patients with PPP, eight secondary polycythaemia (SP), five idiopathic erythrocytosis, and 13 normal subjects, classified under standard criteria following comprehensive investigation for causes of SP. Erythroid colony formation from peripheral blood in a serum‐free system was assayed with the addition of recombinant human erythropoietin (Epo), interleukin 3 (IL3), or α‐interferon (α‐IFN). The differential sensitivity of primitive and mature progenitors (BFU‐E) was assessed by counting the number of clusters (‘sub‐colonies’) comprising each erythroid burst. ‘Endogenous’erythroid colonies were found in both PPP (56%) and controls (17%). In Epo containing cultures, the mean number of clusters per burst was lower in PPP than controls, and the percentage of small (≦ 8 clusters) bursts was higher. In PPP primitive BFU‐E demonstrated greater dependence on IL3 than controls, and mature BFU‐E greater inhibition by α‐IFN. These findings suggest an abnormal response to several growth factors, rather than dysfunction of a single growth factor receptor. Regression analysis of these data defined a discriminant of high diagnostic sensitivity and specificity. This discriminant accurately predicted diagnosis in a further nine polycythaemic patients.
Clinical significance of the presence of myeloid associated antigens in acute lymphoblastic leukaemia
Authors: Urbano‐Ispizua Alvaro , Matutes Estela , Villamor Neus , Ribera Josep Maria , Feliu Evarist , Montserrat Emilio , Grańtena Albert , Vives‐Corrons Joan‐Lluis , Rozman Ciril
Published: March 2008

We have analysed the immunological characteristics of blasts from 89 acute lymphoblastic leukaemia (ALL) cases (62 adults and 27 children), by using a panel of antilymphoid and myeloid associated monoclonal antibodies (McAb) and the APAAP method, which detects membrane and cytoplasmic expression of antigens.

The McAb CD19 was the marker most consistently expressed in B lineage ALL, being positive in 100% of cases, compared to CD24 and CD22 expressed in 82% and 79%, respectively. Similarly, for the T lymphoid lineage, the McAb CD3 was the most reliable and specific marker, being expressed in all T‐ALL cases including those with an early thymic phenotype (CD7 +, TdT+).

Lymphoblasts from eight adults (12.9%) and three children (11.1%) expressed one to four myeloid associated antigens recognized by CD13, CD14, CD33 and anti‐myeloperoxidase. There were no substantial clinical and morphological differences between the two ALL groups with or without myeloid associated markers. However, the presence of myeloid associated markers in adult ALL was associated with a significantly lower complete remission (CR) rate (P=0.05) and with a shorter survival (P= 0.001): this variable was independent of advanced age and high WBC. It is concluded that immunophenotypic analysis in ALL should include myeloid markers for its probable, prognostic implications.
Reversal of drug‐resistance by cyclosporin‐A in a patient with acute myelocytic leukaemia
Authors: Sonneveld Pieter , Nooter Kees
Published: March 2008

The emergence of resistant leukaemia in a patient with acute myeloid leukaemia (AML) was evaluated during clinical progression of the disease. At relapse, a decrease of the intracellular accumulation of daunorubicin (DNR) as determined by real time flow cytometry was associated with a relative overexpression of RNA encoding for the multidrug resistance phenotype (MDR1), and by a decreased in vitro sensitivity to DNR of clonogenic AML cells (IC50 0.8‐3.4 μm). Intracellular DNR accumulation and in vitro DNR sensitivity could be completely restored by adding cyclosporin‐A (3 μm) to the cells. At progressive relapse the patient was treated with re‐induction therapy (DNR 30 mg/m2 x 3. cytarabine 200 mg/m2 x 7) to which cyclosporin‐A was added (Cy‐A 4 mg/kg twice daily for 3 d, 2.5 mg/kg twice daily for 2 d). which resulted in elimination of the MDR1 positive AML cells with restoration of the original DNR accumulation and in vitro sensitivity. After 12 weeks the resistant clone reappeared in the blood and bone marrow.
A past mutation at Isoleucine397 is now a common cause of moderate/mild haemophilia B
Authors: Bottema Cynthia D. K. , Koeberl Dwight D. , Ketterling Rhett P. , Bowie E. J. W. , Taylor Sherryl A. M. , Lillicrap David , Shapiro Amy , Gilchrist Gerald , Sommer Steve S.
Published: March 2008

Of the factor IX sequence changes that we have identified in 65 consecutive males with haemophilia B, 11 (17%) are the same mutation. This mutation is a T→C transition at base 31311 which substitutes threonine for isoleucine397 (ile397) in the factor IX molecule. The 11 patients are of Western European descent and have the same haplotype: Hinf1 (‐), Xmn1 (‐), Taq1 (‐), BamH1 (+), Malmö allele = thr148. The frequency of this haplotype was estimated and the probability of the same mutation occurring independently 11 times in this haplotype was miniscule. We conclude that these patients have a common ancestor despite the lack of overlapping pedigrees.

The clinical symptoms of the disease were consistently moderate/mild in these 11 patients, whereas factor IX coagulation values obtained from the medical records varied more than sixfold between individuals. However, when plasma from five individuals was assayed by the same laboratory concurrently, the values varied less than twofold. Thus, in routine practice, clinical severity may correlate better with the presence of a given mutation than the factor IX coagulant activity.

The high frequency of the mutation at ile397 indicates that carrier testing in families of Northern European descent with moderate/mild haemophilia B can be expedited by first determining whether this particular mutation is present. We demonstrate here that the technique of polymerase chain reaction (PCR) amplification of specific alleles (PASA) can be used to rapidly perform carrier testing in families with the ile397 mutation.
Platelet concentrates irradiated with ultraviolet light retain satisfactory in vitro storage characteristics and in vivo survival
Authors: Pamphilon D. H. , Potter M. , Cutts M. , Meenaghan M. , Rogers W. , Slade R. R. , Saunders J. , Tandy N. P. , Fraser I. D.
Published: March 2008

We have previously reported that platelet concentrates (PC) may be irradiated with ultraviolet light (UVL) in a cryostorage pack such that mixed lymphocyte reactions (MLR) are abolished whilst satisfactory platelet function is retained during subsequent storage using Fenwal PL‐1240 containers. We have now studied both platelet structure and function after irradiation in DuPont Stericell bags which are both UV‐permeable and biocompatible. The irradiation dosage was 3000 Joules/m2 of UVL at a mean wavelength of 310 nm; a dose previously shown to abolish MLR. No detriment to platelet function was observed when compared to control as measured by aggregation responses to adenosine diphosphate (ADP), collagen and ristocetin, hypotonic shock response and pH during 5 d of storage. Lactate levels were significantly higher (P<0.01) and glucose levels lower (P<0.01) in UV‐treated PC, although in the majority the lactate level did not exceed 20 mmol/l. Betathromboglobulin and platelet factor 4 levels were higher during storage in the UV group, the latter reaching significance (P<0.05). When whole platelets and platelet membranes were stained with Coomassie blue or Periodic Acid‐Schiff s reagent after electrophoresis in polyacrylamide gels no obvious alterations to major membrane constituents were observed on days 1 and 5 of storage. Paired in vivo autologous studies in healthy volunteers using 111Indium‐labelling were performed at the end of 5 d of storage. The UV‐treated platelets gave satisfactory and equivalent results for recovery, half life and survival when compared to controls. We conclude that PC irradiated with UVL and stored for 5 d in DuPont Stericell containers appear suitable for transfusion and may prove to be non‐immunogenic. Further in vivo studies of haemostatic efficacy and recipient alloimmunization are now warranted.
Frequencies of Band 3 variants of human red cell membranes in some different populations
Authors: Ranney Helen M. , Rosenberg Gwen H. , Morrison Martin , Mueller Thomas J.
Published: March 2008

A variant of Band 3, the major protein of the erythrocyte membrane, was observed by Mueller and Morrison in 1977 in 6–7% of healthy blood donors on sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS–PAGE) of erythrocyte membranes treated with pronase. Pronase treated red cells containing this first recognized variant [here designated ‘Band 3‐Memphis (m)’]§ had two bands of about 63000 and 60000 Mr while pronase treated normal cells had only the lighter Mr band. The present study includes data on the frequency of variants resembling Band 3‐Memphis in patients of different ethnic groups and on random donors obtained earlier in Memphis. These variants were detected by the original method of Mueller and Morrison and were not associated with recognized clinical or haematological abnormalities. Significantly higher gene frequencies for the variants of the (m) type were observed in American Indians, African Americans and Filipinos than in Caucasians: putative heterozygotes and homozygotes were identified among each of these groups. The frequency of silent Band 3 polymorphisms in different populations should be considered in the interpretation of clinical findings associated with the presence of Band 3 variants.
Immunohisto‐cytochemical correlation of DAP IV‐CD 26 reactivity with immunblogic markers of lymphocyte activation in human lymphoid tissues
Authors: Cozzi Marzia , Gloghini Annunziata , Volpe Rachele , Carbone Antonino
Published: March 2008

Summary.The topographic distribution of the dipeptidylami‐nopeptidase IV (DAP IV‐CD 26) and II (DAP II) positive T‐cell population in six reactive lymph nodes and seven follicular B‐cell non‐Hodgkin's lymphomas (NHL) was analysed with regard to the distribution of activated T‐cells, as visualized by a panel of monoclonal antibodies including Tac‐CD 25, HLA‐DR, OKT 9‐CD 71, ICAM‐1‐CD 54, LFA‐1‐CD 11a. For comparative studies serial frozen sections of the lymph nodes were tested by enzyme histochemistry and immunohistochemistry. In addition, cell suspensions obtained from 10 B‐NHL and interleukin‐2 (IL‐2) activated T‐cells were investigated by a combined cytochemical and immunological method for simultaneous visualization of DAP IV‐CD 26 cytoplasmic activity and surface immunostaining for markers of lymphocyte activation. Both in reactive and lymphomatous lymph nodes the topographic distribution of DAP IV‐CD 26+and DAP II+lymphocytes was rather similar to that of Tac‐CD 25+lymphocytes. On the contrary, the DAP IV‐CD 26 and DAP II distribution pattern substantially differed from that of the other immunologic markers. In a cell suspension of IL‐2 activated T‐cells, more than 80% of the cells with a blastic morphology were DAP IV‐CD 26+: DAP IV‐CD 26+cells coexpressing Tac‐CD 25, OKT 9‐CD 71, HLA‐DR positivity, relative to the total number of DAP IV‐CD 26 positive cells, were 90.5%, 70.5% and 87% respectively. Only small (not activated) lymphocytes expressed a focal cytoplasmic DAP II positivity. In cell suspensions from 10 cases of B‐NHL the mean percentage of DAP IV‐CD 26+Tac‐CD 25+cells was 75.8. Only a small number of DAP IV‐CD 26+cells coexpressed HLA‐DR, the mean percentage being 9.6. The results support the view that DAP IV‐CD 26 may be considered as a marker of lymphocyte activation; this marker seems to be restricted to T lymphocytes that reside in the T dependent areas of reactive lymph nodes and to non malignant T‐cells surrounding neoplastic follicles of follicular NHL.
In vivo priming of human normal neutrophils by granulocyte‐macrophage colony stimulating factor: effect on the production of platelet activating factor
Authors: Aglietta Massimo , Monzeglio Clara , Apra Franco , Mossetti Carlo , Stern Angelika C. , Giribaldi Giuliana , Bussolino Federico
Published: March 2008

Summary. The effect of granulocyte‐macrophage colony stimulating factor (GM‐CSF) (recombinant, mammalian, glycosylated, Sandoz, Schering Plough; 4 μg/kg every 12 h for 3 d, s.c.) on platelet activating factor (PAF, 1‐O‐alkyl‐2‐acetyl‐sn glycero‐3 phosphorylcholine) production from neutrophils was studied in five cancer patients with normal haemopoiesis. Peripheral blood counts, PAF production and lyso‐PAF: acetyl transferase (EC 2.3.1.67) (AT) activity in neutrophils were evaluated before treatment, during treatment and 3 d after treatment had been discontinued. GM‐CSF induced a three‐fold increase in the number of circulating neutrophils. Neutrophils obtained during treatment produced about twice as much PAF than before treatment in response to a variety of stimuli (N‐formyl‐methionyl‐leucyl‐phenylalanine, tumour necrosis factor‐alpha, phagocytosis of baker's yeast spores opsonized with C3b). This increased PAF synthesis and release is concomitant with a 2‐3‐fold increase in AT activity. Moreover, lower concentrations of stimuli are sufficient to induce PAF synthesis from neutrophils obtained during GM‐CSF treatment. Three days after treatment had been discontinued, stimulus induced PAF production had returned to baseline levels. Since GM‐CSF induces a marked shift to the left in the Arneth score, the increased PAF release might have been due to the presence of younger granulo‐cytes. This was, however, ruled out by experiments showing that normal neutrophils primed in vitro with GM‐CSF produce more PAF when challenged with the same stimuli. The potential relevance of this effect of GM‐CSF treatment lies on the crucial role of PAF in inflammatory reactions and its intervention in some immune reactions, including delayed hypersensitivity, and in endotoxic shock. Lastly, increased PAF production from neutrophils may explain some toxicities observed during treatment with high doses of GM‐CSF.
MDR1 transcript levels as an indication of resistant disease in acute myelogenous leukaemia
Authors: Sato H. , Preisler H. , Day R. , Raza A. , Larson R. , Browman G. , Goldberg J. , Vogler R. , Grunwald H. , Gottlieb A. , Bennett J. , Gottesman M. , Pastan I.
Published: March 2008

Summary. The expression of the MDR1 gene was studied by Northern blot analysis in leukaemic cell specimens obtained from 74 patients with acute myelogenous leukaemia (AML). No relationship was found between MDR1 RNA levels and FAB type of leukaemia or patient age. Transcript levels tended to be highest in the leukaemic cells of patients with a history of toxic exposure or preleukaemia compared with ‘standard risk’patients at diagnosis but the differences were not significant (P= 0.07). Patients whose leukaemic cells contained high MDR1 transcript levels were difficult to induce into remission and, if remission was induced, the remissions were short. Hence high levels of MDR1 expression may explain, at least in part, the ineffectiveness of anthracycline antibiotic containing treatment regimens in some patients with AML.
Morphologic, immunologic and cytogenetic studies in erythroleukaemia: evidence for multilineage involvement and identification of two distinct cytogenetic‐clihicopathological types
Authors: Cuneo A. , Van Orshoven A. , Michaux J. L. , Boogaerts M. , Louwagie A. , Doyen Ch. , Cin P. Dal , Fagioli F. , Castoldi G. , Van den Berghe H.
Published: March 2008

Summary. Clinical features, as well as morphology, immunophenotype and cytogenetics were retrospectively studied in 20 patients with an original diagnosis of erythroleukaemia (EL) reclassified according to the FAB criteria. Fifteen patients had de novo EL, five patients had therapy‐related EL. Myelodysplasia preceded the onset of EL in eight cases and myelodysplastic features involving multiple haemopoietic lineages were observed at leukaemia presentation in all cases. Immunologic findings confirmed multilineage involvement, showing sub‐populations of cells expressing platelet‐associated markers in more than 50% of cases tested and the presence of a myelomonocytic component, besides glycophorin A‐positive cells. Cytogenetically, major karyotype aberrations (MAKA), defined by the presence of three or more aberrant events in the same clone, were observed in 14 cases, minor karyotype aberrations (MIKA) were observed in four cases and normal karyotype in two cases. No differences in the cytological‐cytogenetic picture of our patients with de novo EL and with therapy‐related EL were found suggesting that aetiological factors and/or pathogenetic mechanisms common to EL and secondary leukaemia may exist. All patients with MAKA had leftward shift of erythropoiesis with proerythroblasts and basophilic erythroblasts usually representing more than 50% of all erythroid cells. In patients with MIKA or normal karyotype, maturation of erythroid cells, though morphologically abnormal, was quantitatively preserved and early erythroblasts never exceeded 25% of erythroid cells. Clinically, the haemoglobin level at presentation, as well as in the proportion of patients achieving complete remission after chemotherapy, appeared to be lower in the maturation arrest‐MAKA group as compared to the preserved maturation‐MIKA/normal karyotype group. Median survival was shorter in the former group (3.5 months) than in the latter (median 13 months). Morphologic‐immunologic‐cytogenetic studies thus allow for the identification of two distinct cytogenetic‐clinicopathological types of EL.
Characterization of a new IgGλ myeloma plasma cell line (EJM): a further tool in the investigation of the biology of multiple myeloma
Authors: Hamilton M. S. , Ball J. , Bromidge E. , Lowe J. , Franklin I. M.
Published: March 2008

Summary. A new IgGλ myeloma plasma cell line known as EJM was established from a peritoneal effusion from a patient with extramedullary myeloma. The EJM cells have a plasmablastic morphology with abundant rough endoplasmic reticulum and grow in liquid culture with a doubling time of 72 h and a labelling index of 36%. In addition to cytoplasmic IgGλ, the cells are positive for CD9, 20, 32, 38, 44, 54, 71, 78, MHC Class II DR. DP and DQ. Studies on the control of the cell line proliferation by cytokines have demonstrated stimulation with interleukin 6. In contrast interferon alpha produces marked inhibition of proliferation in doses of > 100 units/ml. The culture conditions and the importance of accessory cells and cytokines in supporting myeloma plasma cell growth in vitro are discussed.
Long‐term culture of aplastic anaemia bone marrow
Authors: Gibson F. M. , Gordon‐Smith E. C.
Published: March 2008

Summary. Long‐term bone marrow cultures (LTBMC) were established with marrow from 11 patients with aplastic anaemia (AA). Bone marrow from five patients, with low numbers of committed progenitor cells, exhibited an increase in committed progenitor cell production to normal levels in the first week of LTBMC. None of 44 haematologically normal marrow cultures showed this increase. Mature and committed progenitor cell production in all cultures from aplastic anaemia bone marrow, declined faster than in normal cultures. This study indicates that short‐term culture for committed progenitor cells is an underestimate of the proliferative capacity of bone marrow from some patients with AA. LTBMC may provide a useful system for further studies into the mechanisms responsible for this increased growth in some patients with AA.
The distribution of CD45R, CD29 and CD45RO (UCHL1) antigens in mature CD4 positive T‐cell leukaemias
Authors: Worner Inga , Matutes Estela , Beverley Peter C. L. , Catovsky Daniel
Published: March 2008

We have studied the expression of antigens characterizing functional T‐cell subsets in 32 CD4 + mature T‐cell leukaemias. In this analysis we used two monoclonal antibodies (McAb) of the CD4 5R group (2H4 and GRT22) which have been shown to identify the ‘naive/virgin’ T‐cell population that functions as ‘suppressor‐inducer’ cells in vitro, and two McAb, CD29 (4B4) and CD45RO (UCHL1), which characterize non‐identical ‘memory’ cells that proliferate in response to soluble recall antigens and provide help in antigen‐specific IgG synthesis. Four groups of CD4 + cases were identified according to this reactivity: (a) 15 CD45R +, CD29 +; (b) 13 CD45R ‐, CD29 +; (c) three CD45R ‐, CD29 ‐; and (d) one case only CD45R +, CD29 ‐. The high incidence of coexpression of CD45R and CD29 (47% of cases) is a new finding which contrasts with the mutual exclusion of these antigens on normal CD4 + T‐lymphocytes. There was no correlation between subset phenotypes and pathological disease entities. None of the six cases of adult T‐cell leukaemia/lymphoma (ATLL), which is known as a disorder of activated ‘suppressor‐inducer’ cells, had the ‘expected’ CD45R +, CD29 ‐ phenotype. Reactivity with UCHL1 showed a good correlation with CD29 in the CD45R ‐ CD29 + cases which included three with ATLL. These results may help in the further characterization of T‐cell malignancies according to functional subgroups and may clarify further the role of T‐differentiation antigens in health and disease.
Expansion by folinic acid of the peripheral blood progenitor pool after chemotherapy: its use in autografting in acute leukaemia
Authors: Laporte J. P. , Douay L. , Allieri A. , Lopez M. , Montbrun M. C. Dupuy , Mary J. Y. , Giarratana M. C. , Isnard F. , Najman A. , Gorin N. C.
Published: March 2008

We have tested folinic acid (FA) for ability to increase peripheral blood stem cells (PBSC) after chemotherapeutic aplasia in acute leukaemia. Five adult patients (four AML, one ALL) entered the study, each patient underwent two series of three leukapheresis, the first following induction chemotherapy and the second following the first course of consolidation. The first leukapheresis of each series was done when the white blood cell count reached 109/1 with subsequent leukapheresis every other day. Folinic acid (Lederle Laboratories, France) was administered at a dose of 50 mg (i.v.) per day, 15 days from initiation of chemotherapy and continuing through the third leukapheresis of the series (days 25–30). PBSC were collected on a Haemonetics V50 cell separator.

In these five cases we observed an increased yield of both colony‐forming units, granulocyte macrophage (CFU‐GM) and burst forming units‐erythroid (BFU‐E) expressed per ml of cytapheresis product: CFU‐GM × 18, BFU‐E × 3 and if expressed per 104/kg of body weight: CFU‐GM × 30, BFU‐E×3 (CFU‐GM P<0·05, BFU‐E<0·01). Long‐term blood culture (LTSC) from FA stimulated leukapheresis, in an attempt to quantitate the most primitive stem cells, demonstrated that this expansion of the PBSC was sustained in time. We found by means of LTSC that FA did not stimulate CFU‐L from patients with AML (two cases tested).

Finally two AML patients were grafted with FA‐PBSC after Cytotoxan and total body irradiation (TBI). Haematopoietic reconstitution was rapid complete and sustained in time in both patients.

This indication for folinic acid should be further studied with or as an alternative to haematopoietic growth factors.
Alpha interferon therapy for haematological malignancies: correlation between in vivo induction of the 2′,5′ oligoadenylate system and clinical response
Authors: Mel W. C. P. de , Hoffbrand A. V. , Giles F. J. , Goldstone A. H. , Mehta A. B. , Ganeshaguru K.
Published: March 2008

2′,5′ oligoadenylate (2‐5A) synthetase mRNA and enzyme levels have been studied in peripheral blood mononuclear cells from 12 patients with chronic granulocytic leukaemia (CGL), 14 with essential thrombocythaemia (ET) and nine with chronic lymphocytic leukaemia (CLL), undergoing therapy with alpha interferon (αIFN). 2‐5A synthetase mRNA was assayed by hybridization using the dot–blot technique and the enzyme activity was measured biochemically. A statistically significant difference was observed (P<0·05) between the degree of in vivo induction of mRNA by IFNα in the total patients between the good and intermediate responders and the poor responders. There was a similar pattern in each of the CGL, ET and CLL groups although this only reached statistical significance in the CGL group. In vitro induction of either mRNA or of enzyme activity, however, did not show a difference between the responders and poor responders in any of the patient groups. Our findings are consistent with the concept that 2‐5A has an important role in the anti‐tumour activity of αIFN and suggest that measurement of in vivo induction of 2‐5A synthetase mRNA may be useful in predicting clinical response. In vitro studies, on the other hand, do not provide a reliable predictor of clinical response.
Hypercoagulability during l‐asparaginase treatment: the effect of antithrombin III supplementation in vivo
Authors: Gugliotta Luigi , D'Angelo Armando , Belmonte Monica Mattioli , Viganò‐D'Angelo Silvana , Colombo Giuseppina , Catani Lucia , Gianni Lorenzo , Lauria Francesco , Tura Sante
Published: March 2008

To evaluate the occurrence of hypercoagulability during treatment with l‐asparaginase (l‐ase), thrombin‐antithrombin complex (TAT) and d‐dimer levels in plasma were serially measured in 15 consecutive adult patients with acute lymphoblastic leukaemia or lymphoblastic lymphoma who had recently completed a chemotherapy cycle with cytosine arabinoside and methotrexate. The first eight patients (group A) received i.v. l‐ase alone (20000 U/m2 on alternate days over 10 d); the last seven patients (group B) received, in addition to l‐ase, bolus injection of antithrombin concentrate (2000 U) on alternate days for a total of six administrations, beginning with the second l‐ase infusion. Increased levels of TAT (P<0·05) and d‐dimer (P<0·01) were observed prior to l‐ase, possibly related to inflammation and cytolysis secondary to previous chemotherapy. In patients treated with l‐ase alone, further elevation of TAT (P<0·05) and persistence of increased d‐dimer were observed, associated with marked reduction of the anticoagulant activities of protein C, protein S and antithrombin III. At variance, in patients receiving antithrombin III supplementation there was no increase of TAT and a normalization of d‐dimer levels occurred during l‐ase treatment. In these patients, mean plasma antithrombin III activity was maintained at levels higher than 70% of normal throughout the treatment. The rate of decline of fibrinogen, factor IX, protein C and protein S was unaffected by antithrombin III supplementation, indicating that hypercoagulability has little if any relevance for the reduction of coagulation factors and inhibitors induced by l‐ase treatment. The usefulness of antithrombin III concentrates in preventing thromboembolic complications in patients submitted to l‐ase treatment remains to be determined.
Anaemia and marrow blood flow in the rat
Authors: Schoutens A. , Verhas M. , Dourov N. , Verschaeren A. , Mone M. , Heilporn A.
Published: March 2008

An increase in bone marrow blood flow has been previously described in anaemic rabbits and dogs. We examined the effect of haemorrhage and haemolysis in female Sprague‐Dawley rats, with the hypothesis that high blood flow was related to hyperplasia of bone marrow tissue and that the increase would affect bone as well. Blood flow was measured in tibia and femur by the microspheres trapping method. Chronic anaemia was accompanied at day 32 by a marked increase (factor of 1·7–1·9) in blood flow to bone marrow and to bone. On the other hand, no increase in blood flow was observed in chronic compensated anaemia, showing that anaemia per se and not stimulation of erythropoiesis was responsible for the increase. We then explored the effect on blood flow of two factors associated with anaemia. Erythropoietin 4 U/d for 4 d failed to induce any increase in bone and marrow blood flow. Systemic hypoxia (76 mmHg for 3 d) reduced the fraction of cardiac output conveyed to bone and marrow by 40–50%, but this decrease was more than compensated by an increase in cardiac output. We conclude that neither erythropoietin nor low tissue pO2 is the direct cause for the increase in bone and marrow blood flow observed in anaemia.
Unrestricted growth of Plasmodium falciparum in microcytic erythrocytes in iron deficiency and thalassaemia
Authors: Luzzi G. A. , Torii M. , Aikawa M. , Pasvol G.
Published: March 2008

The mechanism(s) underlying the apparent resistance to malaria in certain inherited red cell disorders and iron deficiency anaemia remain poorly understood. The possibility that microcytic erythrocytes might inhibit parasite development, by physical restriction or reduced supply of nutrients, has been considered for many years, and never formally investigated. We sought to determine whether in vitro growth studies of P. falciparum could provide evidence to suggest that small red cell size contributes to malaria resistance in those red cell disorders in which microcytosis is a characteristic feature.

Invasion and development of P. falciparum in iron deficient red cells (mean values for mean cell volume [MCV] 66 fl, mean cell haemoglobin [MCH] 19 pg) and in the red cells of two gene deletion forms of α‐thalassaemia (mean MCV 71 fl, MCH 22 pg) were normal, assessed both morphologically, and by 3H‐hypoxanthine incorporation. Although parasite appearances were normal in all cell types, morphological abnormalities were noted in iron deficient and thalassaemic cells parasitized by mature stages of P. falciparum, notably cellular ballooning and extreme hypochromia of the red cell cytoplasm. Using electron microscopy, the red cell cytoplasm in parasitized thalassaemic cells showed reduced electron density and abnormal reticulation. Normal invasion rates were observed following schizogony in microcytic cells of both types.

Our findings indicate that whilst minor morphological abnormalities may be detected in parasitized iron deficiency and thalassaemic erythrocytes, development of P. falciparum in these conditions is not limited by small erythrocyte size.
Haemoglobin α2β223Val → Ile produced in Escherichia coli facilitates Hb S polymerization
Authors: Pagnier J. , Baudin‐Chich V. , Lacaze N. , Bohn B. , Poyart C.
Published: March 2008

The doubly substituted variant Hb S‐Antilles (β6 Glu → Val, β3 Val → Ile) produces sickling in heterozygous carriers. The Csat value for pure deoxyHb S‐Antilles is nearly half that of deoxyHb S. Dilute solutions of pure Hb S‐Antilles have a lower oxygen affinity than those of Hb A or Hb S. The mutant Hb α2β223 Val → Ile was synthesized in E. coli. It exhibits a decreased oxygen affinity compared to Hb A and does not polymerize in 1·8 m phosphate buffer. Mixtures of equal amounts of Hb S +Hb β23 Val → Ile have a decreased Csat value compared to mixtures of Hb S + Hb A. The β23 Val in Hb S contributes to the axial contact joining molecules in each single filament. Substituting Ile for Val at this site increases the strength of this contact through hydrophobic interactions, allowing increased stability of the lateral contact between filaments in pair, which is the specific unit structure of polymers in deoxyHb S.
Expression of integrins in human bone marrow
Authors: Soligo Davide , Schiró Raffaella , Luksch Roberto , Manara Giancarlo , Quirici Nadia , Parravicini Carlo , Dellliers Giorgio Lambertenghi
Published: March 2008

Expression of integrins, a superfamily of glycoprotein α/β heterodimers which integrate the cytoskeleton with the extracellular matrix and/or mediate cell‐cell adhesive interactions, was examined on normal and leukaemic bone marrow cells by immunohistochemistry and immunotransmission electron microscopy (immuno‐TEM). Among the β1/VLA molecules studied, VLA‐2 and 6 were expressed on megakaryocytes and platelets, while VLA‐4 was present on 40% of haemopoietic cells, including monocytes, erythroblasts and immature cells; this molecule was typically localized at sites of intercellular contact, as seen by immuno‐TEM, suggesting it may be involved in interactions among haemopoietic cells during differentiation. In human longterm bone marrow cultures (LTBMC), VLA‐1 and 3 were present respectively on 35% and 40% of the adherent cells which included flbroblasts and endothelial cells, as shown by double‐labelling experiments; VLA‐2 was expressed only on a subpopulation of flbroblasts. β2/LeuCAM molecules were absent from platelets, megakaryocytes and HLA‐DR+/myelo‐peroxidase− early myeloid precursors, and appeared progressively during maturation in both lymphoid and myeloid cells. Expression of β3/cytoadhesin molecules was restricted to megakaryocytes and platelets and, in the adherent layer of LTBMC, to endothelial cells. The regulated expression and specific localization of integrins in the bone marrow suggest that these molecules may have a role in normal haemopoiesis.
Prognostic factors in stage AO B‐cell chronic lymphocytic leukaemia
Authors: Oscier D. G. , Stevens J. , Hamblin T. J. , Pickering R. M. , Fitchett M.
Published: March 2008

85 patients presenting to a single centre with stage AO B‐cell chronic lymphocytic leukaemia (B‐CLL) have been studied. The duration of follow‐up has ranged from a minimum of 1 year to a maximum of 18 years with a mean of 6 years.

14 patients have had progressive disease and 23 patients have died, of whom nine had CLL‐related deaths. We assessed the prognostic significance of the following parameters: age at presentation, sex, haemoglobin concentration, initial lymphocyte count, surface membrane phenotype. serum immunoglobulin levels at presentation and karyotype.

None of these factors were predictive of survival, but there was a correlation between initial lymphocyte count, surface immunoglobulin MDλ phenotype, and complex karyotypic abnormalities and disease progression.

Two patients with a complex karyotype have been followed for more than 5 years without evidence of progression.
Studies of natural anticoagulant proteins and anticardiolipin antibodies in patients with the lupus anticoagulant
Authors: Lo Samuel C. L. , Salem Hatem H. , Howard Margaret A. , Oldmeadow Michael J. , Firkin Barry G.
Published: March 2008

Components of the natural anticoagulant system (NAS) and anticardiolipin antibodies were examined in 21 patients with lupus anticoagulant (LA), 13 of whom had past histories of thrombotic episodes. No relationship could be shown between the antigenic levels of protein C and S (PC, PS) and a history of thrombosis. Inhibition of the anticoagulant activity of activated protein C (APC) was observed using plasma from 20/21 patients when phospholipid vesicles were used as the surface for the coagulation reaction. This effect was not affected by the addition of PS. When platelet membranes were employed only 2/21 patients demonstrated inhibition of APC. Under the latter condition, PS functional activity was inhibited in 7/21 patients, six of whom had a past history of thrombosis.

Reduced antithrombin III or heparin cofactor II levels were observed in a total of 4/21 patients and may have contributed to the development of thrombosis in three of these patients. Antibodies specifically directed against these proteins were not detected suggesting the possibility of an associated constitutional deficiency. Anticardiolipin antibodies, though elevated in 17/21 patients, did not serve as a useful marker for an increased risk of thrombosis, and the level did not correlate with inhibition of the activity of APC or PS.

We conclude that the mechanism of thrombosis in patients with LA is multi‐factorial. A subset of patients in whom LA specifically inhibits PS function may represent patients who are at significant risk from thrombosis.
Demonstration of the heterogeneity of epitopes of the platelet‐specific alloantigen, Baka
Authors: Take Hironori , Tomiyama Yoshiaki , Shibata Yoichi , Furubayashi Takayasu , Honda Shigenori , Mizutani Hajime , Nishiura Tetsuo , Tsubakio Tadahiro , Kurata Yoshiyuki , Yonezawa Takeshi , Tarui Seiichiro
Published: March 2008

It is well known that the platelet‐specific alloantigen, Baka is carried on glycoprotein (GP) IIb, but little is known about the biochemical characteristics of its epitopes. To clarify the characteristics of the epitopes, we examined the interaction of four anti‐Baka sera (Yam, Lin, Kl and MO) with their epitopes, either with or without modifications by sodium dodecyl sulphate (SDS) and/or neuraminidase. By immunoprecipitation, all four antisera bound to the intact GP IIb/IIIa complex from a Baka‐positive subject. In contrast, immunoblotting demonstrated that Yam, Lin and Kl bound to SDS‐denatured GP IIb, while MO did not. When blotted GP IIb was treated with neuraminidase, Yam and Lin did not bind to desialylated GP IIb, while Kl still did. When the purified GP IIb/IIIa complex or washed platelets were treated first with neuraminidase followed by immunoblotting, the molecular weight of GP IIb decreased from 145 kD to 138 kD; Yam did not bind to desialylated GP IIb, but Kl did. Furthermore, to eliminate the effect of SDS, we examined the interaction of Yam and Lin with neuraminidase‐treated platelets using flow cytometry. The results were the same as those obtained using immunoblotting. Our results thus demonstrate that the expression of the Baka epitopes is not uniform and that sialic acid contributes to the expression of some actual allogenic epitopes.
Abnormal tryptic peptide from the spectrin α‐chain resulting from α‐ or β‐chain mutations: two genetically distinct forms of the Sp αI/74 variant
Authors: Lecomte M. C. , Gautero H. , Garbarz M. , Boivin P. , Dhermy D.
Published: March 2008

Limited tryptic digestion of native spectrin (Sp) has revealed several variants in hereditary pyropoikilocytosis (HPP) and in a subset of patients with hereditary elliptocytosis (HE). In most cases, tryptic peptide corresponding to the αI (N‐terminal) 80 kD domain is wholly or partially replaced by smaller fragments. These variants are provisionally designated according to the molecular weight of the most prominent new peptide. Partial amino acid sequences of the abnormal peptides and DNA analysis of the α‐spectrin gene have shown that most variants result from substitution or insertion of an amino acid in the αI‐domain. However, similar investigations did not detect any such abnormality in the spectrin αI‐domain of an HE black kindred with one of the spectrin variants called Sp αI/74. In this kindred, restriction fragment length polymorphism studies and transmission of the genetic polymorphism relative to the αII‐domain excluded the involvement of the α‐chain in the pathological process. To ascertain whether the abnormal αI 74 kD peptide might be caused by a β‐chain mutation, we reconstituted hybrid dimers combining normal and HE Sp‐chains. The tryptic peptide patterns of spectrin hybrid dimers containing HE α‐chain and control β‐chain showed a normal 80 kD tryptic product. In contrast, the hybrid dimer containing normal α‐chain and HE β‐chain gave rise to increased 74 kD peptide at the expense of the 80 kD, demonstrating that the mutation in this family resides in the β‐chain. The same method was used to show that in two other unrelated white kindreds, the elevated 74 kD peptide arised from a Sp α‐chain defect. Thus an alteration in tryptic susceptibility within the N‐terminal domain of the spectrin α‐chain can be directed by a mutation in the β‐chain. The hybridization technique affords a definitive means of distinguishing between α‐ and β‐chain mutants.
Interleukin 5 activity in sera from patients with eosinophilia
Authors: Enokihara Hideo , Kajitani Hiroshi , Nagashima Shigeki , Tsunogake Satoshi , Takano Naoto , Saito Kenji , Furusawa Shimpei , Shishido Hideo , Hitoshi Yasumichi , Takatsu Kiyoshi
Published: March 2008

Summary. Sera from 10 patients with eosinophilia contained eosinophil colony stimulating factor (Eo‐CSF) activity. Using anti murine (m) interleukin‐5 (IL‐5) antibody, we demonstrated that this activity was mainly derived from IL‐5. Administration of prednisolone to patients decreased both Eo‐CSF activity in sera and the number of eosinophils in blood. These results extend our recent study demonstrating that T cells from eosinophilic patients produce IL‐5 with IL‐2 stimulation and may support the speculation that IL‐5 is an important factor which induces eosinophilia.
Chromosome and bcr rearrangement in chronic myelogenous leukaemia and their correlation with clinical states and prognosis of the disease
Authors: Tien H. F. , Wang C. H. , Chen Y. C. , Shen M. C. , Wu H. S. , Lee F. Y. , Chuang S. M. , Liu C. H.
Published: March 2008

Summary. Among 77 unselected patients with chronic myelogenous leukaemia (CML), 70 had Philadelphia chromosome (Ph1) in blood cells. Extra chromosomal abnormalities were noted in 4%, 55% and 78% of Ph1‐positive patients in chronic phase, accelerated phase and acute blast crisis, respectively. Rearrangement of the bcr was detected in 46 of 47 Ph1‐positive patients studied and also in three of five Ph1‐negative ones. The locations of the breakpoints were mapped to one of four zones of the bcr in 45 patients. The median duration from diagnosis of CML to onset of acute blast crisis was not significantly different between the two groups of patients with breakpoints in the 5’portion (34 months), and in the 3’portion (39 months) of the bcr. In addition, the locations of the breakpoints within the bcr did not change as the disease progressed in the six patients who had DNA analysed both in the chronic phase and subsequently in transformation. In one of them, an additional aberrant band which was not present in the beginning of the acute phase was detected in blood cells taken 2 months later. It is suggested from the studies that transformation of CML may not be related to alterations within the bcr.
Increased serum levels of soluble IL‐2 receptor, CD30 and CD8 molecules, and gamma‐interferon in angioimmunoblastic lymphadenopathy: possible pathogenetic role of immunoactivation mechanisms
Authors: Pizzolo G. , Stein H. , Josimovic‐Alasevic O. , Vinante F. , Zanotti R. , Chilosi M. , Feller A. C. , Diamantstein T.
Published: March 2008

Summary. Abnormal immunoreaction associated with increased cell activation phenomena might play a role in the pathogenetic events leading to the development of angioimmunoblastic lymphadenopathy (AILD). In the present study we investigated the serum levels of some soluble molecules related to cell activation in 24 patients with AILD at presentation. In particular, we measured by immunoenzymatic or immunoradiometric techniques the levels of the Tac peptide (sIL‐2R), soluble CD30 (sCD30) and CD8 (sCD8) antigens, and gamma‐IFN (gIFN). The results show that all the above molecules are increased as compared to normal controls, with a different pattern of increase for the different molecules. The sIL‐2R levels were very high in all cases with no overlap between AILD and control samples (mean 6315±3374 U/ml, controls 271±112 U/ml, P<0.001). Very high values of the sCD30 antigen (722±895 U/ml) were detected in all cases but five, as opposed to the lack of detectable levels in controls. A significant increase of sCD8 (978±646 U/ml, controls 334±95 U/ml, P<0.01) and gIFN (329±236 U/ml, controls 97±43 U/ml, P<0.01) was also observed with some overlap between AILD samples and controls.

The above findings further support the view that a condition of abnormally enhanced cell activation is likely to play a central role in the pathogenetic events leading to the composite clinicopathological picture of AILD.
Type II oestrogen binding sites in acute lymphoid and myeloid leukaemias: growth inhibitory effect of oestrogen and flavonoids
Authors: Larocca L. M. , Piantelli M. , Leone G. , Sica S. , Teofili L. , Panici P. Benedetti , Scambia G. , Mancuso S. , Capelli A. , Ranelletti F. O.
Published: March 2008

Summary. The presence of oestrogen receptors (ER) and type II oestrogen binding sites (type IIEBS) have been investigated by a whole cell assay in seven cases of acute lymphoid leukaemia (ALL) and 16 cases of acute myeloid leukaemia (AML). ER were detected in 6/7 ALL patients with values ranging between 133 and 2268 sites/cell and in 12/16 AML patients with values ranging between 274 and 4197 sites/cell. The apparent dissociation constant (KD) for ER was 0.6±0.3 nM (mean + SD of 20 cases). All blasts from ALL and AML patients expressed type II EBS at variable levels ranging between 3109 and 239 450 sites/cell. The mean (KD) value for these sites was 18.3±5.6 nM (mean±SD of 23 cases). Specificity experiments demonstrated that type II EBS are oestrogen specific relative to the class of steroid hormones. In addition, the flavonol quercetin was able to compete for [3H]17 beta‐oestradiol (E2) binding to type II EBS, the relative binding affinity (RBA) of quercetin being greater than that of diethylstilboestrol (DES). DES and quercetin exerted a dose‐dependent inhibition of ALL and AML blast proliferation in the range of concentrations between 10‐8 and 10‐5 M. The RBA of DES and quercetin for type II EBS correlated well with their potency as cell growth inhibitors. Moreover, the flavonols rutin and hesperidin which compete slightly for [3H]E2 binding to type II EBS, were scarcely effective in inhibiting leukaemic cell proliferation. The inhibitory effect of DES and quercetin was not due to a non‐specific cytotoxic action since after aid culture period, cell viability did not vary between control and treated cells, being greater than 80%. Our results suggest that high oestrogen concentrations and the flavonol quercetin may inhibit leukaemic blast proliferation through a common mechanism involving a binding interaction with type II EBS.
Long‐term prognostic value of serum β2microglobulin in myelomatosis
Authors: Cuzick J. , De Stavola B. L. , Cooper E. H. , Chapman C. , MacLennan I. C. M.
Published: March 2008

Summary.The prognostic value of serum β2microglobulin (sβ2m) measured at entry, at plateau and at 3‐monthly follow‐up times has been assessed for patients in the Medical Research Council's 4th and 5th Myelomatosis Trials. Analysis of 1014 patients confirmed the value of presentation sβ2m of predicting survival in the short term but showed that predictive value was lost for subsequent survival in those patients who had survived for at least 2 years. However, measurements of sβ2m taken during follow‐up were predictive of subsequent survival. The predictive power of these follow‐up measurements was very similar to that for the presenting measurement, and again they were only of value in predicting survival in the next 2 years.
Estimation of PI‐bound proteins on blood cells from PNH patients by quantitative flow cytometry
Authors: Plesner T. , Hansen N. E. , Carlsen K.
Published: March 2008

Summary. The phosphatidylinositol (PI) bound proteins (acetylcholin‐esterase (ACE), decay accelerating factor (DAF), leucocyte function antigen type 3 (LFA‐3) and Fc‐receptor type III (FcRIII)) were estimated by flow cytometry on blood cells from four patients with paroxysmal nocturnal haemoglobinuria (PNH), nine patients with ‘non‐PNH’haemolytic anaemia, four patients with aplastic anaemia and a reference group of 15 healthy individuals to assess the applicability of flow cytometric measurements in the clinical mapping of the PNH defect. Estimation of DAF on granulocytes or monocytes offered the highest diagnostic sensitivity and specificity and may constitute an easy screening method for the PNH defect. One PNH patient had a negative Ham's test at the time of study and normal or near normal levels of PI‐bound proteins on erythrocytes, but reduced expression of DAF and FcRIII on granulocytes and DAF on monocytes. The analytical and biological coefficient of variation for flow cytometric estimation of PI‐bound proteins was in the range of 4.8‐13% and 12‐24%, respectively. Blood samples should be analysed without delay, since storage produced spuriously high results. The results were expressed as molecules per cell after calibration with commercially available standards and validated by comparison with previously reported results obtained by other methods. It is proposed that this way of reporting flow cytometric results should be generally adopted to facilitate comparison of results between laboratories.