bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular charge heterogeneity of human serum erythropoietin
Authors: Wide L. , Bengtsson C.
Published: March 2008

Summary. The charge heterogeneity of human serum erythropoietin (S‐Epo) was studied in 89 serum specimens from 78 subjects by zone electrophoresis in 0.17% agarose suspension at pH 8.6. The electrophoretic elution profiles of S‐Epo were determined with a radioimmunoassay for Epo. The number of Epo peaks indicated that at least 20–30 different forms of Epo were present in a single serum specimen. The median charge of Epo, estimated as its median electrophoretic mobility, was determined for each serum specimen. This median charge was measured in 10 healthy adults, 46 patients with anaemia, six patients with secondary polycythaemia, one patient with polycythaemia Vera treated by phlebotomy, and six healthy newborn infants (cord sera). Forty‐four of the patients with anaemia had a median charge of S‐Epo within the reference range for healthy adults, while all the patients with polycythaemia and the newborn infants had less negatively charged forms of S‐Epo. In nine patients in whom the S‐Epo level had a circadian rhythm, the forms of S‐Epo in the evening were less negative than those in the morning. The median charge of recombinant preparations was much less negative than that of S‐Epo in healthy individuals, while that of the 2nd International Reference Preparation was more negative than in any of the 78 subjects analysed. A significant change to less negatively charged S‐Epo forms was observed 24 h after a subcutaneous injection of recombinant Epo in one patient, who before the injection had a normal median charge and concentration of S‐Epo.

In conclusion, Epo exhibits a considerable charge heterogeneity in individual serum specimens, the forms of S‐Epo in the morning may differ from those in the evening, those in adults differ from those in newborn infants, and those in patients with anaemia differ from those in polycythaemia. The results also suggest that the methods used in this study may be useful for detecting the presence of injected recombinant Epo in the blood in persons with a normal endogenous Epo production.
Serum erythropoietin changes in autologous and allogeneic bone marrow transplant patients
Authors: Ireland R. M. , Atkinson K. , Concannon A. , Dodds A. , Downs K. , Biggs J. C.
Published: March 2008

Summary. Sequential changes in serum erythropoietin (sEPO) levels were measured by radioimmunoassay in six patients receiving autologous rescue (AR) and 11 patients receiving an allogeneic bone marrow transplant (BMT) for malignant disease.

Longitudinal studies showed an inverse relationship between sEPO and haemoglobin levels in the autologous rescue and allogeneic transplant patients throughout the 130 d post‐transplant study period. Early post‐conditioning EPO responses were normal for the haemoglobin level in both groups, but after day 14 post‐transplant, erythropoietin production in response to anaemia became impaired in one autologous rescue patient and eight of the 11 allogeneic transplant patients.

There was no clear association between late impairment of sEPO production and conditioning therapy, infection, graft‐versus‐host disease, immunosuppressive therapy or serum creatinine.

Blood transfusion requirements were similar for both groups in the first month after transplantation, but from days 31 to 90 post‐transplant, BMT patients required an average of 5.5 units per patient compared with 1 unit per patient for the autologous group.

Marrow transplant procedures do not affect early EPO responses but may diminish late responses. The potential value of exogenous rHuEPO in hastening engraftment and decreasing transfusion requirements, particularly for those patients who appear to have impaired EPO responses, remains to be shown by clinical trials.
Expression of the human carbonic anhydrase I gene is activated late in fetal erythroid development and regulated by stage‐specific trans‐acting factors
Authors: Brady Hugh J. M. , Edwards Mina , Linch David C. , Knott Lesley , Barlow Jonathan H. , Butterworth Peter H. W.
Published: March 2008

Summary. Using flow cytometric analysis of red cells from different stages of ontogeny with anti‐CAI antibody, it was shown that the human carbonic anhydrase I (HCAI) gene product appears in a developmental stage‐specific manner. Virtually no CAI protein was detectable in fetal red cells prior to birth. However, at about the time of normal delivery (40 weeks gestation) CAI production is switched on. The proportion of cells containing CAI reaches adult levels during the second half of the first year of life. Northern analysis suggests that the appearance of CAI protein results directly from the activation of the gene and the production of new mRNA. A transient heterokaryon system was set up by fusing the erythroleukaemic cell lines MEL C88 (a mouse cell line in which CAI is expressed) and K562 SAI (a human cell line with an embryonic/fetal phenotype, not expressing CAI). SP6 RNAase mapping of RNA from the fused cells showed activation of the human CAI gene. This shows the developmental stage‐specific expression of HCAI to be regulated by trans‐acting factors.
Differential effects of recombinant human colony stimulating factor (rh G‐CSF) on stem cells in marrow, spleen and peripheral blood in mice
Authors: Bungart B. , Loeffler M. , Goris H. , Dontje B. , Diehl V. , Nijhof W.
Published: March 2008

Summary Previously it has been hypothesized that the granulopoietic and erythropoietic lineages may compete for differentiating stem cells. According to this hypothesis one would expect that a stimulation of granulopoiesis by G‐CSF administration would lead to a reduction of the stem cell pool and be followed by a decline of erythropoietic progenitor numbers. In addition one would expect an enhanced response of granulopoiesis if G‐CSF administration were combined with suppression of erythropoiesis by red cell transfusion.

To evaluate whether this hypothesis holds true C57bl mice were injected subcutaneously for 6 d with 3. 75 μg rh G‐CSF/ mouse/d (150 μg G‐CSF/kg body weight/d). Marrow CFU‐S numbers showed an increase to 160% on day 2, followed by a decrease to 50% of control on day 6. Splenic and peripheral blood CFU‐S increased 20‐fold and 10‐fold, respectively. Marrow CFU‐E declined to 40% of the control value. Splenic CFU‐E increased 10‐fold. The increase in marrow CFU‐GM numbers ranged between 140% and 180%. CFU‐GM obtained from the spleen and the peripheral blood increased 60‐fold and 15‐fold. respectively. Regarding the CFU‐S and CFU‐GM a similar pattern of response was found in an experiment where rh G‐CSF administration was combined with an additional red cell transfusion. These data do not provide convincing evidence for an exhaustion of haemopoietic stem cells during treatment with G‐CSF. They rather suggest that an important side effect of G‐CSF treatment is a release of CFU‐S and progenitors from the marrow to the peripheral blood and a reseeding in the spleen.
Family studies in von Willebrand's disease by analysis of restriction fragment length polymorphisms and an intragenic variable number tandem repeat (VNTR) sequence
Authors: Standen G. R. , Bignell P. , Bowen D. J. , Peake I. R. , Bloom A. L.
Published: March 2008

Summary We have previously identified a microsatellite variable number tandem repeat region of the nucleotide sequence ATCT within intron 40 of the von Willebrand factor (vWF) gene. By polymerase chain reaction (PCR) amplification of this region, eight major alleles have been demonstrated in the South Wales population, with an overall heterozygosity rate of 75%. Direct sequencing has shown that the alleles correspond to lengths of between six and 14 ATCT repeats. In the present study we describe the use of this variable repeat sequence and previously reported restriction fragment length polymorphisms (RFLP) to study inheritance patterns in families with type I, IIA and severe type III von Willebrand's disease (vWD). The results confirm that analysis of this precisely localized intragenic locus provides a highly informative marker for gene tracking studies in the major forms of vWD.
Long‐term trial with the oral iron chelator 1,2‐dimethyl‐3‐hydroxypyrid‐4‐one (L1) I. IRON CHELATION AND METABOLIC STUDIES
Authors: Kontoghiorghes G. J. , Bartlett A. N. , Hoffbrand A. V. , Goddard J. G. , Sheppard L. , And J. Barr , Nortey P.
Published: March 2008

Summary A long‐term clinical trial of 1‐15 months has been carried out with the oral iron chelator 1.2‐dimethyl‐3‐hydroxypyrid‐4‐one (L1) in 13 transfusion‐dependent ironloaded patients. Urinary iron excretion was greatest in patients with thalassaemia major and was related to the number of previous transfusions but not to the serum ferritin level. Substantial increases of urinary iron were observed in all the patients when the frequency of the daily dose was doubled and in response to 2 ± 3 g L1 daily 11 of 12 patients tested excreted > 2 5 mg iron daily, the mean daily intake of iron from transfusion. Serum ferritin levels have fluctuated but overall have remained unchanged. Pharmacological studies in five patients have indicated rapid absorption probably from the stomach and variable plasma half life of 77 ± 35 min (SD). Glucuronation was identified as a major route of L1 metabolism. Short‐term intensive chelation studies using repeated administration of L1 resulted in further increases of urinary iron excretion by comparison to a single dose. In one case 325 mg of iron were excreted in the urine following the administration of 16g (5 ± 2g + 2 ± 3g) within 24 h. Iron excretion studies were carried out in six transfusional iron‐loaded patients who were maintained on a low iron diet before and during chelation. No significant increases of faecal iron excretion were observed with L1 using daily doses of up to 3.3 g and 4 ± 2 g. The high level of compliance during treatment with L1 and the levels of urine iron excretion that can be achieved increase the prospects for oral chelation in transfusional iron‐loaded patients.
LF61: a new monoclonal antibody directed against a trimeric molecule (150 kDa, 125 kDa, 105 kDa) associated with hairy cell leukaemia
Authors: Flenghi L. , Spinozzi F. , Stein H. , Krushwitz M. , And S. Pileri , Falini B.
Published: March 2008

Summary The newly produced monoclonal antibody (mAb) LF61 detects a molecule restricted to hairy cell leukaemia (HCL) among B‐cell non‐Hodgkin's lymphomas. In particutar, the percentage of LF61+HCL cells in different cases ranges from 10% to 100%. In normal lympho‐haemopoietic tissues LF61 reacts with only about 2% of T‐cells, mostly of the CD8 subset in the peripheral blood. extrafollicular areas of the tonsil, red pulp of the spleen and thymic medulla. Expression of the LF61 molecule is observed following stimulation of peripheral blood lymphocytes with phyto‐haemagglutinin (PHA) or pokeweed mitogen (PWM), suggesting that it represents an activation antigen. Due to its restricted reactivity with a small subset of normal CD8 + T‐cells, LF61 in combination with a CD22 mAb is highly suitable for monitoring residual disease in interferon or deoxycoformicin‐treated HCL patients. Polyacrylamide gel gradient electrophoresis shows that LF61 precipitates a 150 kDa. 125 kDa, 105 kDa trimeric molecule from the surface of HCL cells. Immunohistological and immunobiochemical results show that this molecule is the same as the one recognized by the still unclustered anti‐HCL mAb B‐ly7.
Interleukin‐1 is one factor which regulates autocrine production of GM‐CSF by the blast cells of acute myeloblastic leukaemia
Authors: Bradbury D. , Rogers S. , Kozlowski R. , Bowen G. , Reilly I. A. G. , Russell N. H.
Published: March 2008

Summary The role of interleukin‐1 (IL‐1) as a regulator of the autonomous growth of the blast cells of acute myeloid leukaemia (AML) has been studied on samples isolated from 15 patients. In nine out of 10 patients with evidence of partial autonomous blast cell growth. IL‐1 further stimulated cell growth in both suspension culture and in a clonogenic assay. 11,‐1 also stimulated cell growth in two out of three cases with no evidence of autonomous growth whereas no additional stimulation was observed in two cases with totally autonomous growth. In blast cells stimulated by IL‐1, synthesis of granulocyte‐macrophage colony stimulating factor (GM‐CSF) was increased and the proliferative response to IL‐1 was inhibited by an antibody to GM‐CSF. In all samples with evidence of autonomous growth, blast cell conditioned medium (BCCM) contained IL‐1 activity (range 0.7–74.2 units/ml) and a polyclonal antibody to IL‐1 markedly inhibited autonomous growth in four samples. BCCM from three of these four samples contained GM‐CSF, the synthesis of which was suppressed when BCCM was prepared in the presence of anti‐IL‐1. Our data suggests that endogenous IL‐1 is an important factor in the regulation of the production of GM‐CSF and hence of autonomous growth of AML blasts, but that other mechanisms regulating GM‐CSF production may exist.
CD4 and CD8 subpopulation changes during high dose intravenous immunoglobulin treatment
Authors: Macey M. G. , Newland A. C.
Published: March 2008

Summary High doses of immunoglobulin, when given intravenously (IVIgG), influence lymphocyte subset numbers and function. T‐lymphocytes may be subdivided into two functionally different groups. helper/inducer (CD4+) and suppressor/cytotoxic (CD8+). Considerable functional as well as phenotypic heterogeneity exists within the two major subsets. CD4+ cells have been further subdivided into helper/inducer and suppressor/inducer sets by the differential binding of two monoclonal antibodies 4B4 (CDw29) and 2H4 (CD45R). Similarly, the CD8 + subset may be subdivided into suppressor and cytotoxic populations by the differential binding of monoclonal antibodies which identify the C3bi receptor (CD11).

During IVIgG treatment of patients with autoimmune thrombocytopenic purpura (ATP) the change in CD4/CD8, due to an absolute increase in CD8 + cells, has been shown to correlate with the response to treatment as determined by platelet increase. However, the total CD4+ and CD8 + numbers may not reflect changes in their constituent subpopulations. To examine this possibility the CD4 and CD8 subpopulations were analysed in 15 ATP patients, during IVIgG treatment, using a double fluorescence technique. In 10 of these patients the in vitro response to pokeweed mitogen (PWM) and Staphylococcus aureus Cowan I (STA Cowan I) was determined. There was no correlation between the change in CD8 + subpopulations and response to treatment but there was a correlation between the CD4 + change and platelet increment. In addition there was a correlation between the 4B4/2H4 change and the in vitro response to PWM but no correlation with the response to STA Cowan I. These findings suggest that during IVIgG treatment the CD4 + 4B4 + helper/inducer population is influenced resulting in reduced T‐dependent B‐cell activation.
Rh immunization by the partial D antigen of category DVa
Authors: Mayne K. , Bowell P. , Woodward T. , Sibley C. , Lomas C. , Tippett P.
Published: March 2008

Summary Reported here is the first example of a partial D antigen stimulating the production of anti‐D: stimulation was of fetal origin. During her second pregnancy, anti‐D developed in the serum of a D‐negative mother who had received Rh immunoglobulin after the birth of her first D‐positive child. Her second baby had moderate neonatal jaundice and was successfully treated by phototherapy. Subsequently the red cells of the father and of the first child were shown to carry a partial D antigen of category DVa type. Six available batches of Rh immunoglobulin reacted with DVa cells.
Enhanced complement‐susceptibility and dysfunction of lymphocytes in paroxysmal nocturnal haemoglobinuria (PNH)
Authors: Tomiyama Junji , And Haruhiko Ninomiya , Abe Tsukasa
Published: March 2008

Summary We investigated the complement‐susceptibility of paroxysmal nocturnal haemoglobinuria (PNH) lymphocytes in relation to their dysfunction. When assessed by complement‐mediated lysis induced by monoclonal antibodies (CD5 or CD20) and rabbit complement, the complement‐susceptibility of lymphocytes from patients with PNH, both CD5+ (Tcells) and CD20+ (B cells). was greater than that from controls (P<0.001). This susceptibility was further enhanced, both in normal controls (P<0.01) and in patients with PNH (P<0.001). when the activity of decay‐accelprating factor (DAF) on the lymphocytes was blocked with anti‐DAF monoclonal antibody. DAF amounts in mono‐nuclear cells (MNC) from patients with PNH, measured by an enzyme‐linked immunosorbent assay (ELISA), were lower than those of the controls (P<0.001). The expressions of DAF on T cells and on B cells from patients with PNH were significantly decreased (P<0.05 in T cells: P<0.01 in B cells). MNC from patients with PNH responded less to phytohaemagglutinin (PHA) and concanavalin A (Con A) than MNC from the controls (P<0.001). In contrast. the responses of PNH MNC to poke weed mitogen (PWM) or lipopolysaccharide (LPS) were not impaired. MNC from a normal donor preincubated with anti‐DAF became less responsive to PHA or Con A.

We conclude that PNH lymphocytes show enhanced complement‐susceptibility and that they are involved in their own expression of DAF as well as in other types of peripheral blood cells. The results of the responses to various lectins suggest the dysfunction of T cells in PNH.
Teicoplanin compared to flucloxacillin for antibiotic treatment of neutropenic patients
Authors: Smith Carole L. , Milliken S. , Powles R. , Costa F. Da , Gore M. , Benjamin S. , Talbot D. , Ellis L. , And J. Large , Jameson B.
Published: March 2008

Summary Ninety‐eight neutropenic patients were randomized to receive piperacillin and gentamicin in combination with either teicoplanin or flucloxacillin. Sixty‐scven of these patients, most of whom had myeloma. were given this combination as prophylaxis 5 d after high dose chemotherapy. 3 5 receiving flucloxacillin and 32 receiving teicoplanin. Of 31 patients with leukaemia who were febrile and neutropenic following induction chemotherapy or bone marrow transplantation. 18 received flucloxacillin and 13 received teicoplanin. For those given flucloxacillin. the mean number of days to change of antibiotics was 7.8 in the prophylaxis group and 5.1 in the treatment group. In the teicoplanin arm, the mean number of days to change antibiotics was 6.8 in the prophylaxis group and 6.1 in the treatment group. Two patients in the flucloxacillin arm developed drug rashes. Four patients developed rigors after teicoplanin administration and one asthmatic became wheezy. One patient had a progressive rise in creatinine, but overall the patients having teicoplanin did not have any appreciable increase of renal toxicity compared to the flucloxacillin arm. Blood cultures were positive prior to commencement in the treatment group in nine patients, and during treatment in six patients. Organisms grown were Gram‐positive in 14 patients. Teicoplanin appears to be as effective as flucloxacillin when each is used in combination with piperacillin and gentamicin in the treatment of neutropenic patients, with similar rates of toxicity.
A clinical trial on efficacy and safety of teicoplanin in combination with beta‐lactams and aminoglycosides in the treatment of severe sepsis of patients undergoing allogeneic/autologous bone marrow transplantation
Authors: Lang E. , And J. Schmid , Fauser A. A.
Published: March 2008

Summary Early institution of empiric therapy with a broad‐spectrum antibiotic has markedly reduced the morbidity and mortality from infections complicating severe or prolonged cytopenia in patients receiving either an allogeneic or autologous hone marrow transplant. Ceftazidime in combination with an aminoglycoside, i.e. netilmicin, has been established as a combination schedule offering low or even avoiding therapy‐related toxicity.

We evaluated teicoplanin for suspected Gram‐positive infections after inadequate response to initial beta‐lactam and aminoglycoside combination therapy. All 11 patients so far included in this study received either an allogeneic (five patients) or an autologous (six patients) bone marrow transplant for acute myeloid leukaemia (AML). non‐Hodgkin lymphoma (NHL. high grade) or other malignant diseases. All patients developing a primary septicaemia of unknown origin (10 patients) or a catheter related septicaemia (one patient) were treated with 400 mg teicoplanin, administered i.v. once daily in combination with a cephalosporin and an aminoglycoside (ceftazidime 2 g. i.v., t.i.d., netilmicin 400 mg once daily). All 11 patients responded to therapy. 10 patients were clinically cured, one patient improved under therapy. The therapeutic regimen was well tolerated and adverse drug reactions did not occur. We have not observed any delayed take or prolonged neutropenia or thrombocytopenia with this therapeutic regimen when compared to other bone marrow transplant patients who did not receive this antimicrobial therapy.

Our preliminary results suggest that teicoplanin is a potentially effective and well‐tolerated antimicrobial agent in bone marrow transplant patients with infections not responding primarily to beta‐lactams and aminoglycosides.
Prophylactic teicoplanin during insertion of Hickman catheters
Authors: Lim S. H. , Smith M. P. , Machin S. J. , Goldstone A. H.
Published: March 2008

Summary With the extensive use of Hickman catheters in patients requiring cytotoxic chemotherapy there is now a resurgence of Gram‐positive septicaemia among these patients during the neutropenic periods. We are currently running a prospective randomized study of prophylactic teicoplanin during insertion of Hickman catheters to determine if it reduces the incidence of Gram‐positive septicaemia. A total of 44 patients have completed the study (23 in the teicoplanin group and 21 in the control group). The diagnoses were: acute myelogenous leukaemia (13). non‐Hodg‐kin'slymphoma (16). Hodgkin's disease (11), chronic granulocytic leukaemia (3) and acute lymphoblastic leukaemia (1). The number of days between insertion of Hickman catheters and the development of neutropenia was: teicoplanin group: mean 14.7, median 11, range 0–53; control group: mean 11.8, median 10. range 0–37.

In the treated patients, there were four episodes of line‐associated Gram‐positive septicaemia and a total of four organisms were isolated. In the control group, 10 organisms were isolated from a total of nine episodes of line‐associated Gram‐positive septicaemia. The organisms were: coagulase‐negative staphylococci (8). Streptococcus B, (1), Strep. faecalis (1), Strep. mitis (1). alpha streptococcus (1). diphtheriods (1) and Staphylococcus aureus (1). All organisms were sensitive to teicoplanin. No adverse reaction was observed in any patient.

Prophylactic teicoplanin during insertion of Hickman catheters may therefore reduce the incidence of line‐associated Gram‐positive septicaemia in neutropenic patients.
A randomized prospective study of ceftazidime and ciprofloxacin with or without teicoplanin as an empiric antibiotic regimen for febrile neutropenic patients
Authors: Lim S. H. , Smith M. P. , Goldstone A. H. , Machin S. J.
Published: March 2008

Summary Febrile neutropenic patients were randomized to receive ceftazidime and ciprofloxacin with the addition of teicoplanin in cases with clinical suspicion of a Hickman line‐associated infection. At 48 h both clinical and bacteriological analyses were made. Patients were categorized as: success, in cases of improvement or stability in the clinical condition: failure, if there was a deterioration of the clinical condition; and non‐evaluable, in cases of protocol violation or death due to other causes.

A total of 86 patients have completed the study so far: 43 patients were randomized to the ceftazidime and 43 to the ciprofloxacin group. Teicoplanin was added to the mono‐therapy in 12 patients in the ceftazidime group and 15 in the ciprofloxacin group. The diagnoses were: acute myelogenous leukaemia (25), non‐Hodgkin's lymphoma (34), Hodgkin's disease (17); acute lymphoblastic leukaemia (9) and chronic granulocytic leukaemia (1).

Forty‐eight hour clinical assessment showed a response rate in the ceftazidime group of: success 18/31 (58%). failure 13/31 (42%): in the ceftazidime and teicoplanin group: success 8/12 (67%), failure 4/12 (33%): in the ciprofloxacin group: success 23/28 (82%). failure 5/28 (18%): and in the ciprofloxacin and teicoplanin group: success 11/15 (73%), failure 4/15 (27%).

Blood cultures were positive in 48/86 (56%) cases, with more than one organism isolated in seven of these 48 cases. Organisms isolated were: coagulase‐negative staphylococci (20), Escherichia coli (13), Pseudomonas aeruginosa (4). Staphylococcus aureus (3), diphtheroids (3), Klebsiella sp. (3), Enterobacter cloacue (2), Streptococcus faecalis (2). Ent. adenocarboxylata (1). Clostridium septicum (1). Strept. B (1). alpha streptococcus (1) and P. fluorescens (1). In‐vitro testing showed that all Gram‐positive organisms were sensitive to teicoplanin, and Gram‐negative organisms to ciprofloxacin or ceftazidime. There were seven cases of superimposed infections with eight organisms isolated, all cases occurring in patients receiving ciprofloxacin alone: coagulase‐negative staphylococci (4). Strep. sangius (1). Strep. viridans (1), diphtheroids (1) and P. maltophilia (1). Both ciprofloxacin and ceftazidime are comparable in efficacy as empirical mono‐therapy for febrile neutropenic patients. There is, however, a significant increase in the incidence of superimposed infection in patients receiving only ciprofloxacin. In view of the very high incidence of Gram‐positive septicaemia, we suggest that teicoplanin should be added as part of the initial empiric antibiotic regimen in all febrile neutropenic patients.
In vitro growth pattern and differentiation predict for progression of myelodysplastic syndromes to acute nonlymphocytic leukaemia
Authors: Raymakers R. , Witte T. De , Joziasse J. , Van Der Lely N. , Boezeman J. , Haanen C.
Published: March 2008

Summary. In 153 consecutive patients with myelodysplastic syndrome (MDS) the prognostic value of FAB‐classification, cytogenetics, Bournemouth score, a history of previous radio‐or chemotherapy and in vitro bone marrow growth were retrospectively analysed, for both acute nonlymphocytic leukaemia (ANLL) development and survival. Thirty‐eight of the 153 patients (25%) showed progression to ANLL, 63 (41%) died during the myelodysplastic phase due to infection or bleeding and three (2%) received allogeneic bone marrow transplantation (BMT). Univariate analysis showed that the FAB‐classification, in vitro growth pattern and differentiation, and cytogenetics had a predictive value for ANLL development and survival. The Bournemouth score was predictive only for survival. Most predictive for the development of ANNL were in vitro growth pattern and maturation. Patients with normal in vitro growth progressed to ANLL in 6% of the cases, in patients with hypoplastic or leukaemic growth 32.5% developed ANLL (P <0·0001). The ANLL incidence in patients with normally differentiated in vitro colonies was 14.5%, compared with a 52% incidence in cases showing no in vitro cell maturation (P=0·001). The combination of growth pattern and differentiation revealed an ANLL incidence of 4.2% in cases of normal growth and differentiation, and 60.4% if the in vitro growth and/or differentiation was abnormal (P=0·006). In vitro maturation was the only parameter predictive for ANLL development in multivariate analysis.

From our data it is concluded that the predictive value of in vitro bone marrow culturing in patients with MDS can be increased by including in vitro maturation as a distinct parameter. The in vitro prognostic data can be important in selecting MDS patients for intensive chemotherapy or BMT.
Serum‐free liquid marrow culture in patients with acute lymphoblastic leukaemia: a potential application to purge marrow for autologous transplantation
Authors: Da Wan Ming , Douay Luc , Barbu Véronique , Fabrega Sylvie , Allieri Maria‐Alessandra , Drouet Xavier , Deloux Jacqueline , Giarratana Marie‐Catherine , Oszahin Hulya , Van Den Akker Jacqueline , Vanhaeke Danielle , Gorin Norbert‐Claude
Published: March 2008

Summary. We have previously established a serum‐free (SF) culture medium, which supports normal haemopoietic progenitor cell growth for at least 4 weeks as does conventional serum dependent (SD) medium. In the present study, we investigated the efficacy of such a defined SF liquid medium which sustained in vitro residual normal haemopoietic proliferation of marrow derived from ALL patients and which was detrimental for the leukaemic population. Evidence for a potential selective effect of SF culture was obtained by a leukaemic progenitor cell assay (ALL‐CFU) and the detection of the bcr/abl translocation by polymerase chain reaction (PCR). In 13 experiments including 12 patients, morphological blast cells and ALL‐CFU were dramatically reduced within 3 weeks of incubation in both SF and SD cultures. Likewise, in 5/5 experiments in SD and 2/5 experiments in SF conditions, leukaemic cells expressing the bcr/abl fusion gene disappeared within 3–4 weeks. In contrast, the absolute numbers of supernatant cells harvested weekly from SF and SD cultures were similar. No difference in CFU‐GM production was detected for the two culture systems. Erythropoiesis in SF medium exhibited a slower decline than that found in SD. These results indicate that liquid marrow culture may selectively deplete leukaemic lymphoblastic cells and enable repopulation by residual normal haemopoietic cells. This technique may be useful to purge leukaemic cells for clinical autologous bone marrow transplantation in patients with ALL.
Interaction of two GPIIb/IIIa monoclonal antibodies with platelet Fc receptor (FcγRII)
Authors: Rubinstein Eric , Kouns William C. , Jennings Lisa K. , Boucheix Claude , Carroll Roger C.
Published: March 2008

Summary We have previously used the IV‐3 monoclonal antibody specific for FcγRII to demonstrate that platelet activation by CD9 monoclonal antibodies such as ALB‐6 is mediated by the FcγRII. Here, we show that platelet activation following addition of a monoclonal antibody directed against GPIIb/IIIa, P256 is completely blocked by IV‐3, as monitored by serotonin release, calcium and pH modifications. However, aggregation was only partially inhibited. D3GP3 is another monoclonal antibody directed against GPIIIa which has been shown to induce platelet aggregation by exposure of the fibrinogen binding site. The present study demonstrates that this phenomenon is not accompanied by calcium flux or pH modification, nor is it blocked by pretreatment of platelet by IV‐3. Despite its apparent independence from the FcγRII activation pathway, D3GP3, but not its Fab fragment, was able to inhibit ALB‐6 induced activation, including serotonin release, calcium flux and pH modifications. Binding studies demonstrated that D3GP3 (20 μg/ml. 0·13 μM) does not block ALB‐6 binding to CD9 antigen but completely blocks IV‐3 binding to the Fc receptor for concentrations of IV‐3 ranging from 0 to 15 nM. Together, these results suggest an interaction between GPIIb/IIIa, FcγRII and GPIIb/IIIa monoclonal antibodies which in some cases can result in activation of platelets through FcγRII.
Polymerase chain reaction (PCR) mutagenesis enabling rapid non‐radioactive detection of common β‐thalassaemia mutations in Mediterraneans
Authors: Lindeman R. , Hu S. P. , Volpato F. , Trent R. J.
Published: March 2008

Summary The IVS‐1–110 (G→A) and IVS‐1–1 (G→A) mutations occur in approximately 33% and 9% respectively of β‐thalassaemia alleles in Mediterraneans (Kazazian & Boehm, 1988). They are generally detected in polymerase chain reaction (PCR)‐amplified material by allele‐specific oligonucleotide (ASO) hybridization patterns. In this study, artificial base substitutions in amplified material have been created to distinguish normal from mutant alleles on the basis of restriction enzyme digestion patterns. Invariant target sites provide an internal control for restriction enzyme activity. Mutagenesis was achieved by 3’base mismatches in primers selected to anneal immediately adjacent to target sites. Digestion of PCR products from normal and thalassaemic alleles with the restriction enzymes MboI (IVS‐1–110) and Hinfl (IVS‐1–1) produced different fragments on electrophoresis. The above strategy was validated by allele‐specific oligonucleotide probing. Identification of the three commonest mutations in this population (IVS‐1–110, codon 39 and IVS‐1–1), which account for approximately 69% of thalassaemic alleles (Kazazian & Boehm, 1988), was subsequently undertaken in seven chorion villus biopsies.
AUTOTRANSPLANTS IN ACUTE LEUKAEMIA
Authors: Gale Robert Peter , Horowitz Mary M. , Butturini Anna
Published: March 2008

Summary The data we review indicate that in adults with ALL in first remission intensive chemotherapy and radiation given before a transplant is more effective in eradicating leukaemia than current chemotherapy. This is not so in adults with AML in first remission where more intensive therapy does not reduce the likelihood of relapse. Furthermore, leukaemia relapse because of re‐infused leukaemia cells is an important issue in autotransplants for ALL. Whether re‐infusing leukaemia cells would be important in AML were more effective pretransplant therapy developed is unknown. Presently, there appears to be little sense to test in vitro approaches to remove leukaemia cells in autotransplants for AML because efficacy cannot be evaluated. (A randomized trial is an exception but would not be expected to show a difference.)

Another conclusion is that attempts to induce GVHD in autotransplant recipients, such as by using cyclosporine post‐transplant (Jones et al, 1989), are more likely to succeed in AML than ALL since the impact of GVHD is substantially greater (Horowitz et al, 1990). However, the GVHD‐related antileukaemia effect in AML is associated with chronic GVHD whereas cyclosporine treatment of autotransplant recipients results in acute GVHD. Also, attempts to separate clinical and antileukaemia effects of GVHD were unsuccessful (Sullivan et al, 1989). Another caution is that in AML we detected an immune antileukaemia effect distinct from GVHD (termed GVL) only after HLA‐identical sibling transplants. Since GVL was absent in twins it is unlikely to operate after autotransplants.

In summary, there is sense in studying autotransplants in adults with acute leukaemia in first remission. However, there are currently no convincing data that autotransplants are superior to current therapy. More intensive treatment seems effective in ALL; the focus should be on increasing the antileukaemia efficacy of pretransplant therapy and on attempts to remove leukaemia cells from the graft. In AML, there is no evidence that more intensive therapy is more effective. This problem needs resolution before evaluating attempts to remove leukaemia cells from the graft. The best place to test new pretransplant regimens is in twins and recipients of HLA‐identical sibling transplants without GVHD. The data and ideas we review and discuss should be useful in planning clinical trials.
Circulating progenitor cells in myelofibrosis: the effect of recombinant α2b interferon in vivo and in vitro
Authors: Craig Jenny I. O. , Anthony Robert S. , Parker Alistair C.
Published: March 2008

Summary We have studied circulating progenitor cells in patients with myelofibrosis, the effects of recombinant α2b interferon (IFN) treatment on these cells in vivo and in vitro and the patients’clinical response to IFN treatment. A 75‐fold increase in circulating granulocyte macrophage colony forming units (CFU‐GM) and a 25‐fold increase in multi‐lineage colonies (CFU‐GEMM) were seen in the patients compared with controls. Patients who had undergone splenectomy had circulating progenitors within the normal range. IFN treatment of two patients resulted in clinical improvement and reduction in spleen size, but was complicated by a fall in platelet count and persistent malaise. Whilst on treatment the circulating progenitor cells increased up to 5‐fold. In contrast, the addition of IFN in vitro to the CFU‐GM and CFU‐GEMM culture plates resulted in a dose‐dependent inhibition of colony growth which was unaffected by the removal of T‐cells and monocytes. Thus we confirm that circulating progenitors are raised in patients with myelofibrosis, IFN may be of benefit clinically, reducing spleen size but may increase levels of these cells in vivo. This is in contrast to the inhibitory effect of IFN in vitro on CFU‐GM and CFU‐GEMM growth.
Selective purging by human interleukin‐2 activated lymphocytes of bone marrows contaminated with a lymphoma line or autologous leukaemic cells
Authors: Gambacorti‐Passerini Carlo , Rivoltini Licia , Fizzotti Marco , Rodolfo Monica , Sensi Maria Luisa , Castelli Chiara , Orazi Attilio , Polli Nicoletta , Bregni Marco , Siena Salvatore , Borin Lorenza , Pogliani Enrico , Corneo Gian Marco , Parmiani Giorgio
Published: March 2008

Summary The ability of recombinant interleukin 2 (rIL2) activated lymphocytes (LAK) to purge BM samples contaminated by tumour cells was evaluated. Human BM mononuclear cells were contaminated with 10% of the lymphoma line CA46 and then cultured in liquid medium containing 1000 U/ml of rIL2 and/or LAK autologous to the used BM. At the end of coculture the growth of residual tumour cells and of CFU‐GM were evaluated by clonogenic assay. No tumour cell growth was observed in 5/5 independent experiments after 18 h of coculture with LAK. No significant inhibition of CFU‐GM growth was also noted. Subsequently, the effect of LAK on BM obtained from four leukaemic patients and contaminated with 20–50% of their own AML and ALL cells was studied using MAb as a tool for identifying leukaemic cells. LAK eliminated 24–78% of contaminating cryopre‐served uncultured autologous leukaemic cells. In five cases the BM was contaminated by a low (2%) amount of ALL cells. In these patients the monoclonal heavy chain rearrangement typical of ALL was no longer visible after coculture with LAK. Evidence for selective tumour cytotoxicity by LAK was confirmed by using autologous BM cells as hot and cold targets in a 51Cr release assay. Finally, successful haematologic reconstitution of lethally irradiated BALB/c mice was obtained using syngeneic BM cocultured with LAK. These results support the investigational use of rIL2 and LAK in the treatment of human leukaemia.
Treatment of anti‐recombinant interferon‐alpha 2 antibody positive CML patients with natural interferon‐alpha
Authors: Wussow P. v. , Jakschies D. , Freund M. , Hehlmann R. , Brockhaus F. , Hochkeppel H. , Horisberger M. , Deicher H.
Published: March 2008

Summary Of 38 patients with a Philadelphia‐chromosome positive chronic myeloid leukaemia treated with recombinant interferon alpha (rIFN‐α) 2a or 2b and monitored for emergence of IFN‐antibodies in their sera 11 patients developed rIFN‐α2 binding and 10 rIFN‐α2 neutralizing antibodies. rIFN‐α neutralizing antibody positive patients experienced significantly (P<0·025) more clinical relapses (6/10) than IFN‐antibody negative patients (6/28) during continuous IFN‐therapy. Furthermore, IFN‐antibody‐positive patients with titre above 400 INU/ml were more likely to relapse under rIFN‐α‐therapy than IFN‐antibody‐negative patients with titre below 400 INU/ml (P<0·05).

Seven rIFN‐antibody‐positive patients experiencing a clinical relapse or a primary non‐responsiveness were treated with two‐ to three‐fold increased doses of rIFN‐α2. Only one of these seven patients developed a partial haematological remission upon intensification of the rIFN‐α2 therapy. Consecutively, the six patients failing high dose rIFN‐α treatment were switched to a natural IFN‐α preparation (3 × 9 × 106 I.U. weekly s.c.). Under such treatment two of the six patients achieved a long‐lasting complete, one a partial haematological remission.

In high‐titred IFN‐antibody positive patients significantly altered serum‐IFN‐titre and minimal IFN‐inducible Mxhomologue concentrations were measured; in contrast, nIFN‐α induced normal IFN‐titre and dose‐equivalent Mxhomologue amounts in these patients. The data prove that high‐titred rIFN‐α neutralizing antibodies abrogate the biological action of rIFN‐α, but not of nIFN‐αin vivo and explains why nIFN‐α can be effective in the anti rIFN‐α2 positive patients.
Proteolysis of factor VIII heavy chain polypeptides in plasma and concentrates
Authors: Kemball‐Cook G. , Edwards S. J. , Barrowcliffe T. W.
Published: March 2008

Summary Factor VIII heavy chain (FVIII HC) polypeptides have been studied in both normal plasma and FVIII concentrates on exposure to three coagulation proteases.

FVIII samples were incubated with labelled affinity‐purified anti‐FVIII Fab’fragments, immunocomplexes formed were visualized by autoradiography after sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE), and apparent relative molecular masses (Mr) of each band assigned. FVIII HC polypeptides were detected in all types of samples, including plasma, without further purification.

Normal plasma contained a range of polypeptides with the largest dominant band at a net apparent Mr of 250–300 kD, and the smallest at 80–90 kD: the bands visualized correspond to the 90–210 kD HC species seen on conventional analysis of purified FVIII. No bands were produced from samples of haemophilic plasma.

Treatment of plasma or FVIII concentrate with low concentrations (1 IU/ml) of thrombin removed the 250–300 kD and other intermediate bands, intensified then removed the 80–90 kD polypeptide and produced a band at 40–50 kD. Thrombin‐associated rise and fall in FVIII clotting activity by one‐stage assay correlated with intensity of the 80–90 kD polypeptide. A polypeptide of Mr 40–50 kD was also produced after incubation with activated factor X: activated factor VII plus thromboplastin had no effect on HC structure.

FVIII polypeptides were visualized in prothrombin complex concentrates, with a more degraded profile seen in a deliberately ‘activated’product.
A new dual‐label technique for platelet survival studies with the use of 111indium and 114mindium tropolonate
Authors: De Vries R. A. , De Bruin M. , Oldenburg S. J. , Zwiers A. , Marx J. J. M. , Poortman J. , Hart H. Ch. , Van De Wiel A.
Published: March 2008

Summary A dual‐label technique to study the survival of two different populations of platelets within one individual was developed using 111indium and 114mindium. The validity of the technique was demonstrated in seven individuals with an expected equal survival time of two platelet populations and in two persons with an expected difference in platelet survival time.

Since the energy spectra of the two indium isotopes are very close, a well‐type germanium semiconductor detector was applied. By adaptation of the counting time the effective dose equivalent of the dual‐label procedure could be restricted to 1·6 mSv.

The dual‐label technique provides an instrument for studying the survival of two different populations of platelets simultaneously within one individual.
Red blood cell alloimmunization in transfused patients on AZT
Authors: Calverley D. , Haley L. , Ballem P.
Published: March 2008

Summary Significant numbers of patients receiving azidothymidine (AZT) develop anaemia requiring an adjustment of AZT dosage. Fear of red blood cell (RBC) alioimmunization may act as a deterrent to exposing a patient to long‐term transfusion therapy which is the alternative to AZT dosage reduction or discontinuation. This retrospective study was done to assess the incidence of RBC alloimmunization in transfused patients on AZT. Records of 72 patients receiving long‐term AZT were analysed to assess the incidence of alloimmunization. Other study parameters included duration of transfusion therapy, total number of pRBC units transfused/patient, alloantibody specificity, rates of autoantibody detection and their specificity. For comparison, a parallel analysis was carried out on 188 male patients transfused during the same period of time for chronic renal failure (CRF). Only one of 72 AZT patients (1·4%) developed an alloantibody compared with seven of the 118 CRF patients (5·9%) (P=NS). The incidence of autoantibodies and positive direct antiglobulin testing in the AZT and CRF populations was found to be similar (9·7%v. 7·6% respectively: P=NS). The two populations were similar with respect to mean number of pRBC units transfused (AZT: 10·5 v. CRF: 11·5 units: P=NS): however, there was a significant difference in the mean duration of transfusion therapy (AZT: 4·5 months v. CRF 19·4 months; P<0001). We conclude there is an insignificant incidence of alloimmunization in the multiply‐transfused AZT population. Furthermore, in this study the incidence of RBC autoantibodies in AZT patients appears to be low and no different from that of patients with chronic renal failure.
High‐dose cytostatic agents in allogeneic bone marrow transplantation: comparison of the engraftment promoting potential
Authors: Gassmann W. , Uharek L. , Erbersdobler A. , Glass B. , Wottge H.‐U. , Schmitz N. , Löffler H. , Mueller‐Ruchholtz W.
Published: March 2008

Summary. We investigated the potential of various cytostatic agents for preventing graft rejection following allogeneic bone marrow transplantation. LEW rats received a lethal dose (35 mg/kg) of busulfan followed by injection of 1 x 108 F1(CAP x LEW) marrow cells, which are unable to induce a graft‐versus‐host reaction in LEW recipients. Rejection of the marrow graft was assessed by monitoring haematocrit and granulocyte counts. Due to its weak immunosuppressive activity, busulfan by itself is unable to allow engraftment of allogeneic marrow. Therefore, agents administered in addition to busulfan can be tested for their capacity to prevent marrow graft rejection. 120 mg/kg of cyclophosphamide, 20 mg/kg of ACNU and 240 mg/kg of ifosfamide completely prevented rejection of the allogeneic marrow. Maximum doses of BCNU applicable in conjunction with busulfan reduced the rejection rate to 12% (30 mg/kg) and 17% (40 mg/kg), whereas the antitumour agents thiotepa, melphalan, and carboplatin exhibited a very limited engraftment‐promoting potential in this experimental setting. Thus, BCNU (carmustine), ACNU (nimustine), and ifosfamide might be suitable candidates for conditioning of allogeneic bone marrow graft recipients.
Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and granulocyte colony‐stimulating factor (G‐CSF) in serum during induction treatment of acute leukaemia
Authors: Sallerfors Bengt , Olofsson Tor
Published: March 2008

Summary. Granulocyte‐macrophage colony stimulating factor (GM‐CSF) and granulocyte colony‐stimulating factor (G‐CSF) are increasingly used to stimulate granulopoiesis in neutropenic patients but in most cases without any knowledge of the endogenous CSF‐levels. With the purpose to define serum levels of GM‐CSF and G‐CSF during induction chemotherapy and haematological reconstitution in patients with acute leukaemia we have used enzyme‐linked immuno‐sorbent assay (ELISA) techniques to measure these growth factors in 18 patients with acute myeloid leukaemia (AML) and eight patients with acute lymphoblastic or undifferentiated leukaemia (ALL/AUL). G‐CSF above 0·05 ng/ml was detected in 54% of the analysed AML samples, median 0·29 (range 0·05–2·80) ng/ml; and in 40% of analysed ALL/AUL samples, median 0·09 (range 0·05–3·00) ng/ml. In patients with AML there was a clear correlation between an elevated serum concentration of G‐CSF and documented infections. On the other hand, 15/18 of the patients with acute myeloid leukaemia and 8/8 patients with ALL/AUL had non‐detectable levels of GM‐CSF (<0·10 ng/ml). Two patients had measurable levels of GM‐CSF in all samples, median 0·71 (range 0·26–1·18) ng/ml and in these patients the levels successively decreased during and after chemotherapy and did not increase in response to infections. In normals detectable levels of GM‐CSF were found in 2/35 individuals and G‐CSF in 0/10 individuals.