bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of porcine von Willebrand factor with the platelet glycoproteins Ib and IIb/IIIa complex
Authors: Pareti F. I. , Mazzucato M. , And E. Bottini , Mannucci P. M.
Published: March 2008

Summary Porcine von Willebrand factor (PvWF) induces platelet aggregation which is thought to be responsible for the thrombocytopenia that occurs in haemophilic patients treated with commercial preparations of porcine factor VIII. This study demonstrates that such aggregation can be completely inhibited by a monoclonal antibody against human platelet glycoprotein GPIb and partially inhibited by an antibody directed against platelet GPIIb/IIIa. The interaction of PvWF with GPIb is also demonstrated by the inhibitory effect of purified glycocalycin on aggregation. The binding site of PvWF to GPIb is very close to that of human vWF, since a recombinant peptide blocks the binding of both molecules to GPIb. When platelets are incubated with PvWF, the GPIIb/ IIIa receptor is activated and binds fibrinogen. PvWF also binds to GPIIb/IIIa when platelets are stimulated with thrombin, suggesting that the molecule has the same RGD sequence as other adhesive proteins (human vWF, flbrinogen, fibronectin and vitronectin). These findings identify the dual mechanisms responsible for in vivo platelet aggregation induced by PvWF, i.e. binding to GPIb and activation of the GPIIb/IIIa receptor.
Analysis of African Burkitt's and high‐grade B cell non‐Burkitt's lymphoma for Epstein‐Barr virus genomes using in situ hybridization
Authors: Prevot S. , Hamilton‐Dutoit S. , Audouin J. , Walter P. , Pallesen G. , Diebold J.
Published: March 2008

Summary. We investigated the association of Epstein‐Barr virus (EBV) with African cases of Burkitt's lymphoma (BL) and high grade B cell non‐Burkitt's lymphoma (non‐BL) occurring in areas where BL is endemic. The presence of EBV genomes was analysed in 24 cases using in situ hybridization with a 15S‐labeiled EBV probe applied to paraffin sections. EBV DNA was detected in each of 10 cases of BL in which technically satisfactory results were obtained, the virus being homogenously distributed in all identifiable tumour cells. Two other cases of BL could not be evaluated because of technical problems. In contrast, EBV DNA was not detected in any case of high‐grade non‐BL (10 centroblastic and two immunoblastic lymphomas). These results confirm previous reports of the strong association of EBV with endemic BL, but suggest that the virus is not important in the pathogenesis of other types of African high‐grade B cell lymphoma from regions where BL is endemic.
Central role of tumour necrosis factor, GM‐CSF, and interleukin 1 in the pathogenesis of juvenile chronic myelogenous leukaemia
Authors: Freedman Melvin H. , Cohen Amos , Grunberger Tom , Bunin Nancy , Luddy Ruth E. , Saunders E. Fred , Shahidi Nasrollah , Lau Alan , Estrov Zeev
Published: March 2008

Summary. In previous studies on patients with juvenile chronic myelogenous leukaemia (JCML), we found excessive proliferation of malignant monocyte‐macrophage elements in the absence of exogenous growth factor, and impaired growth of normal haematopoietic progenitors. In the current study, six newly‐diagnosed JCML patients were investigated to characterize the disease further. In co‐cultures, JCML cell culture supernatant as well as patient plasma obtained at diagnosis produced a striking reduction in numbers of control marrow BFU‐E, CFU‐GM, CFU‐Meg and CFU‐GEMM colonies. Monoclonal anti‐tumour necrosis factor alpha neutralizing antibodies (anti‐TNF‐α Ab) abolished these inhibitory properties. In sharp contrast, JCML supernatants exerted a marked growth‐promoting effect on autologous JCML cells cultured in clonogenic assays. Anti‐TNF‐α Ab and anti‐granulocyte‐macrophage colony‐stimulating factor neutralizing antibodies (anti‐GM‐CSF Ab) both reversed the stimulating effect. Recombinant GM‐CSF and recombinant TNF‐α produced a profound increase in JCML colonies when tested individually and anti‐GM‐CSF Ab reversed the TNF‐α effect. Expression studies of TNF‐α and TNF‐α receptor genes of cultured JCML cells demonstrated mRNAs for both. Further, TNF‐α activity was assayed in a wide variety of cell culture supernatants and in normal and patients’plasma, and only the JCML specimens showed increased TNF‐α values. Recombinant interleukin‐1 alpha (IL‐1α) also stimulated JCML colony growth, but polyclonal anti‐IL‐1 neutralizing antibodies did not suppress JCML colony numbers nor did it reverse the effects of TNF‐α or GM‐CSF. The evidence indicated that the JCML monokine which inhibits normal haematopoiesis is TNF‐α and that the endogenously‐produced TNF‐α and GM‐CSF from JCML cells play an important role in the pathogenesis of the disease by acting as autocrine growth factors. IL‐1α also stimulates JCML cell proliferation as an accessory factor and augments the effect of GM‐CSF, TNF‐α or both.
Impact of active and passive exclusions on the results of a clinical trial in multiple myeloma
Authors: Hjorth Martin , Holmberg Erik , Röder Stig , Westin Jan ,   for the Myeloma Group of Western Sweden
Published: March 2008

Summary. During the 3 years 1984–86, 314 cases of multiple myeloma were diagnosed in the Health Care Region of Western Sweden. 180 of these cases were included in a clinical trial; 71 were notified to the trial but excluded; 49 cases were not reported to the trial; 14 were diagnosed post mortem.

The crude incidence rate of myeloma was 6.3 cases per 100000 inhabitants per year, corresponding to an age‐adjusted (world standard population) incidence rate of 2.9 cases per 100000 inhabitants per year.

The excluded and the non‐reported patients had a significantly shorter survival than those included in the clinical trial (median survival 22, 13 and 33 months, respectively). This was partly due to differences in age and proportion of actively treeated patients between the groups, but the same tendency remained also after correction for these factors.

Considering the included patients separately, the effect of tentative application of presumptive exclusion criteria corresponding to major prognostic factors was studied. Prolonged survival was seen when the upper age limit was lowered and when patients with renal failure or low performance status were excluded.

The results illustrate the fact that for multiple myeloma the survival in a trial population is markedly influenced by active and passive exclusion of patient groups with unfavourable prognostic characteristics. When reporting results of clinical trials, discussion of the representativeness of the trial population for the total patient population is recommended in order to facilitate application of the trial results to medical practice and comparisons between trials.
Haematological effects of radioimmunotherapy in cancer patients
Authors: Stein Rhona , Sharkey Robert M. , Goldenberg David M.
Published: March 2008

Summary. The haematologic effects of radioimmunotherapy (RAIT) in cancer patients have been studied in order to better understand the aetiology of RAIT‐associated myelosuppresion. Evaluations were performed on patients treated with 131I‐anti‐carcinoembryonic antigen (CEA) and 131I‐LL2, a monoclonal antibody (MAb) reactive with non‐Hodgkin's B‐cell lymphoma. Both groups of patients experienced decreases in WBC and platelets. Nadirs were observed 42–51 d post first injection in the lymphoma patients, and 49–66 d post first injection (30–43 d post high‐dose therapeutic injection) in the carcinoma patients. Within the WBC population, B cells were the most radiosensitive. The evaluations of the binding of these MAbs to peripheral blood cells and the effect of RAIT on lymphocyte subpopulations indicated a drop of 26–92% in the percentage of B lymphocytes within 1 week following treatment with both 131I‐LL2 and 131I‐anti‐CEA MAbs even though only LL2 binds to B cells. The percentage of T lymphocytes was not affected by the 131I‐antibody treatments.

These observations suggest that the marked drop in circulating B lymphocytes and platelets after the administration of radioiodinated antibodies is a nonspecific radiation effect, and not necessarily related to the binding of MAb to normal B cells. Thus, among WBCs, B cells are uniquely radiosensitive, and will be unusually affected by antibody‐directed internal radiation.
Bone marrow transplantation for severe sickle cell anaemia
Authors: Ferster Alina , Valck Christine De , Azzi Nadira , Fondu Pierre , Toppet Michèle , Sariban Eric
Published: March 2008

Summary. Five children with sickle cell anaemia underwent bone marrow tranplantation (BMT) for severe clinical disease. The conditioning regimen for BMT was in busulfan plus cyclophosphamide. The allograft contained more than 5.108 nucleated cells per kg recipient. Prophylaxis of GVHD consisted of methotrexate and cyclosporin A. Therapy was well tolerated. Duration of neutropenia (<0.5 × 109/1) was short (14–25 d). Platelet recovery (>50 × 109/1) occurred between day 12 and 45. The patients have been folowed up for 8–28 months. No major infections occurred and long‐term BMT‐related toxicity was limited to mild, chronic GVHD in one patient. Mean haemoglobin levels remained above 10 g/dl. Haemoglobin electrophoresis showed AS patterns in all grafted patients—all marrow donors having sickle cell trait. From our preliminary data, we conclude that BMT or sickle cell anaemia is curative, well tolerated and should be proposed for suitable patients.
Molecular basis of chronic non‐spherocytic haemolytic anaemia: a new G6PD variant (393 Arg→His) with abnormal KmG6P and marked in vivo instability
Authors: Filosa S. , Calabrò V. , Vallone D. , Poggi V. , Mason P. , Pagnini D. , Alfinito F. , Rotoli B. , Martini G. , Luzzatto L. , Battistuzzi G.
Published: March 2008

Summary. More than 80 genetic variants of glucose‐6‐phosphate dehydrogenase (G6PD) are associated with chronic non‐spherocytic haemolytic anaemia (CNSHA). In order to help clarify the molecular basis of this association, we have carried out a detailed biochemical and genetic characterization of two G6PD deficient brothers affected by CNSHA. The G6PD from the two patients has altered electrophoretic mobility, abnormally elevated Michaelis constant (Km) for G6P, and extreme instability in vivo and in vitro. By comparison with published information we found that this is a new G6PD variant which we have designated G6PD Portici. The entire coding region of the gene has been sequenced, and a single point mutation, a G→A transition, was found at position 1178 in exon X, causing a substitution of histidine for arginine at residue 393 in the polypeptide chain. By polymerase chain reaction (PCR) amplification followed by diagnostic restriction enzyme analysis and allele‐specific oligonucleotide hybridization we have demonstrated the inheritance of this mutation in the patient's family. Our results support the notion of a causative link between this mutation in the G6PD gene and CNSHA. Our data, in combination with previous data in the literature, suggest that the three‐dimensional structure of G6PD is such as to cause interaction in the binding of its two substrates, G6P and NADP.
The neurologic aspects of transcobalamin II deficiency
Authors: Hall Charles A.
Published: March 2008

Summary. Thirty‐four symptomatic cases of inherited transcobalamin II (TCII) deficiency were analysed in order to determine the frequency and nature of neurologic manifestations. In no instance was there definite evidence of a neurologic disorder at the time of presentation as a young infant. One child of 21/2 years transiently lost deep tendon reflexes at a time of suboptimal treatment. A syndrome of mental retardation and other neurologic manifestations was observed in three cases, all with the following in common: (1) an extended duration of illness of 2–17 years; (2) inadequate or no treatment with Cbl; (3) treatment with folic or folinic acid. TCII deficiency rarely if ever presents with neurologic manifestations. However, neurologic disorders can be produced subsequently by improper treatment.
Antigenic analysis of human haemopoietic progenitor cells expressing the growth factor receptor c‐kit
Authors: Strobl Herbert , Takimoto Masafumi , Majdic Otto , Höcker Paul , Knapp Walter
Published: March 2008

Summary. The cell surface molecule encoded by the proto‐oncogene c‐kit has recently been identified as the receptor for a growth factor variously termed stem cell factor (SCF), mast cell growth factor or steel factor. Using the c‐kit antibody 17F11 we analysed, in triple staining experiments, the surface molecule profile and scatter characteristics of c‐kit+CD34+ human haemopoietic progenitor cells. In 10 normal bone marrow samples we found 19–51% of CD34+ bone marrow progenitor cells to coexpress c‐kit. These c‐kit+CD34+ bone marrow cells turned out to represent a phenotypically heterogeneous population. A considerable proportion coexpressed CD33 (52±23%), and/or CD71 (62 ±26) antigens, marker molecules previously shown to be expressed by committed in vitro colony forming cells but not by their precursors. In line with a relatively differentiated phenotype c‐kit+CD34+ cells also gave rise to on average higher forward and right‐angle light scattering signals. The proportions of CD38 and/or HLA‐D expressing cells were similar in the c‐kit+ and in the c‐kit− subsets of CD34+ progenitor cells. Coexpression of CD19 was found to be less frequent in the c‐kit+ (4 ± 5%) as compared to the c‐kit− (17 ± 14%) fraction of CD34+ cells. CD7+CD34+ bone marrow cells were hardly detectable and their numbers too low to allow further subdivision in c‐kit+ and c‐kit− subsets.
Primary myelodysplastic syndrome in children: the clinical experience in 33 cases
Authors: Tuncer M. A. , Pagliuca A. , Hicsonmez G. , Yetgin S. , Ozsoylu S. , Mufti G. J.
Published: March 2008

Summary. We describe the clinicomorphological features in 33 cases of primary myelodysplastic syndrome classified according to the FAB classification which presented to a single centre over a 12 year period. Presenting features were typically related to pancytopenia although hepatosplenomegaly and granulocytic sarcomas were far more prevalent than in the adult population.

Morphological assessment of the peripheral blood and the bone marrow showed seven patients had refractory anaemia (RA), 13 patients had RA with excess of blasts (RAEB), nine patients had RAEB in transformation (RAEB‐t) and four patients had chronic myelomonocytic leukaemia (CMML). The overall mean survival was short (9.9 months) in all the subgroups and the leukaemic transformation rate was high. None of the patients scored 0–1 according to the Bournemouth Scoring System; four patients scored 2 whereas 29 patients scored 3 or 4. We conclude that unlike adults, the myelodysplastic syndromes in children run an aggressive clinical course, irrespective of the FAB subtype, and the pathogenesis of these diseases in paediatric practice warrants scientific scrutiny. Intensive chemotherapy such as the one used in de novo— AML lead to complete remission in some children and these early results suggest that this should be the treatment of choice in paediatric MDS.
Sustained correction of the bleeding time in an afibrinogenaemic patient after infusion of fresh frozen plasma
Authors: Cattaneo Marco , Bettega Donato , Lombardi Rossana , Lecchi Anna , Mannucci Pier Mannuccio
Published: March 2008

Summary. We have evaluated the duration of the effect of replacement therapy with two different doses of fibrinogen on the prolonged bleeding time of an afibrinogenaemic patient and the relationship between changes in bleeding time and plasma and platelet fibrinogen concentrations. The infusion of 40 mg/kg fibrinogen, as fresh frozen plasma (FFP). corrected the prolonged bleeding time of the patient from longer that 30 min to 8 min. The bleeding time was still normal 9 d after infusion, at a time when the plasma and platelet fibrinogen levels were low (0.13 g/l and 27 μg/109 platelets; normal ranges 1.6–4.0 and 60–190). Two months later, the infusion of a smaller dose of fibrinogen (4 mg/kg) also corrected the bleeding time, which remained normal until the second day after infusion, despite the fact that plasma and platelet fibrinogen were very low (0.02 g/l and 3.4 μg/109 platelets). The bleeding time returned to the prolonged baseline values only by day 6 post‐infusion, when plasma and platelet fibrinogen levels were 4 × 10–4 g/l and 1.4 μg/109 platelets. Therefore, sustained correction of the prolonged bleeding time may be obtained in afibrinogenaemic patients with a single infusion of fibrinogen at lower doses than usually recommended.
The low molecular weight heparin Enoxaparin inhibits the consumption of factor VII and prothrombin activation in vivo associated with elective knee replacement surgery
Authors: Orosu Frederick A. , Leclerc Jacques , Delorme Fernand , Craven Sharon , Shafai Shamiran , Frewin Lori , Blajchman Morris A.
Published: March 2008

Summary. Patients over 40 years of age who undergo elective orthopaedic surgery have a relatively high risk for developing post‐surgical deep vein thrombosis (DVT). Prophylactic use of heparin or low molecular weight heparins can reduce the incidence of post‐operative DVT by up to 80%. It is not known whether prophylaxis is achieved by inhibition of prothrombin activation or catalysis of thrombin inhibition in vivo. We determined the changes in concentrations of factor VII zymogen and thrombin‐antithrombin III (the latter as an index of prothrombin activation) in the plasmas of 129 patients randomized to receive two daily subcutaneous injections of placebo or 30 mg of Enoxaparin after elective knee surgery. Enoxaparin reduced the frequency of post‐surgical DVT by 70%. The concentration of factor VII zymogen had decreased by ∼50% within 24 h after the knee surgery, followed by a gradual increase to near pre‐surgical values. Additionally, post‐Enoxaparin plasmas had statistically significant higher concentrations of factor VII zymogen than post‐placebo plasmas. Post‐Enoxaparin plasmas had significantly lower concentrations of endogenous thrombin‐antithrombin III than comparable post‐placebo plasmas. Finally, post‐Enoxaparin plasmas inactivated exogenous factor Xa and thrombin more effectively than comparable post‐placebo plasmas. As Enoxaparin moderated the generation of endogenous thrombin‐antithrombin III after elective knee surgery, inhibition of prothrombin activation in vivo by Enoxaparin may be important for its prophylactic antithrombotic effect.
Prophylactically equivalent doses of Enoxaparin and unfractionated heparin inhibit in vivo coagulation to the same extent
Authors: Ofosu F. A. , Levine M. , Craven S. , Dewar L. , Shafai S. , Blajchman M. A.
Published: March 2008

Summary. This study compared how Enoxaparin and unfractionated (UF) heparin influenced in vivo coagulation in patients randomized to receive, by twice daily subcutaneous injections, either 30 mg of Enoxaparin or 7500 I.U. of UF heparin after elective hip surgery. These two regimens were equally effective in reducing the incidence of post‐operative deep vein thrombosis DVT. We compared the concentrations of endogenous thrombin‐antithrombin III in pre‐ and post‐surgical plasmas to determine how each prophylactic regimen influenced prothrombinase activity in vivo, and found the same concentrations of endogenous thrombin‐antithrombin III in post‐heparin and post‐Enoxaparin plasmas. However, significantly higher concentrations of endogenous thrombin‐antithrombin III were found in pre‐ and post‐surgical plasmas of patients who developed post‐operative DVT than the levels found in comparable plasmas of patients who remained DVT‐negative, regardless of the drug received for prophylaxis. Human factor Xa was added to an equal volume of each patient's plasmas and the amount of added enzyme inactivated by antithrombin III measured using an enzyme‐linked immunosorbent assay for factor Xa‐anti‐thrombin III. Post‐heparin and post‐Enoxaparin plasmas inactivated ∼4 times more factor Xa than the pre‐surgical plasmas, regardless of the clinical outcome. Thus, before and after surgery, a higher than normal in vivo prothrombinase activity may be a significant risk factor for developing postoperative DVT.
Transfer of specific immunity from donor to recipient of an allogeneic bone marrow graft: evidence for donor origin of the antibody producing cells
Authors: Labadie J. , van Tol M. J. D. , Dijkstra N. H. , van der Kaaden M. , van der Zijde C. M. Jol , de Lange G. G. , Zwaan F. E. , Vossen J. M.
Published: March 2008

Summary. A rapid recovery of specific humoral immunity in the recipient of an allogeneic bone marrow transplantation (BMT) can be observed after immunization of the donor before graft sampling. This has been attributed to transfer of specific immunity from donor to recipient. However, to maintain the concept of transfer the origin of the antibody producing cells in the recipient after BMT must be demonstrated. To this end, donor‐recipient pairs with differences in Gm‐allotypes were selected and immunized before BMT with the neo‐antigen Helix pomatia haemocyanin (HPH) according to three immunization protocols. Additionally, the recipients were immunized at day 42 after BMT. Serum samples were weekly obtained from the recipients in the first 100 d after BMT. The origin of HPH‐specific antibody producing cells was assessed by two approaches: (1) determination of the Gmallotypes of anti‐HPH antibodies within a distinct IgG subclass, (2) analysis of anti‐HPH antibody spectrotypes by isoelectric focusing combined with immunoblotting. The results obtained with these two approaches show concordance in most instances and led to the conclusion that the antibody producing cells are of donor origin.
Regulation of iron absorption in iron loaded subjects with end stage renal disease: effects of treatment with recombinant human erythropoietin and reduction of iron stores
Authors: Hughes R. T. , Smith T. , Hesp R. , Hulme B. , Dukes D. C. , Bending M. B. , Pearson J. , Raja K. B. , Cotes P. Mary , Pippard M. J.
Published: March 2008

Summary. The effects on iron absorption of variation in erythroid activity, haemoglobin and iron stores were studied in six anaemic dialysis‐dependent subjects in whom iron stores were increased from previous red cell transfusions. Gastrointestinal mucosal uptake and whole body retention of oral iron were measured at the beginning of the study, after starting treatment with recombinant erythropoietin (but before significant increase in haemoglobin), after partial correction of anaemia, after further reduction of iron stores by repeated phlebotomy, and when erythropoiesis decreased from the discontinuation of treatment with erythropoietin. Between successive measurements, valid comparisons were made in five subjects.

Correction of anaemia decreased whole body retention of iron through decreased mucosal uptake (P=0.032). Further reduction in iron stores through repeated phlebotomy whilst the increase in haemoglobin was maintained by treatment with erythropoietin, tended to increase whole body retention of iron through an increase in mucosal transfer (P= 0.010). With initial enhancement of erythropoiesis in anaemic iron‐loaded subjects there was no change in any measured component of iron absorption. However, after correction of anaemia and reduction of iron stores, a decrease in erythropoiesis was associated with decreased whole body iron retention in all subjects through decreased mucosal transfer (P= 0.028). The data suggest that anaemia upregulates mucosal iron uptake, and that erythroid activity upregulates mucosal transfer but that this latter effect may be counterbalanced by iron overload which downregulates mucosal transfer.
Transferrin uptake by bone marrow macrophages is independent of the degree of iron saturation
Authors: Rama R. , Sanchez J.
Published: March 2008

Summary. The uptake of transferrin by macrophages was studied in relation to the degree of iron saturation. Rat bone marrow derived macrophages were incubated with transferrin labelled with 59Fe and 3H. At 37°C the amount of 59Fe incorporated by macrophages was dependent on the time of incubation. 3H labelled transferrin was found degraded in the supernatants of the cell culture (material not precipitated by trichloroacetic acid) in a time dependent fashion. Taking into account the specific activity of 59Fe‐3H labelled transferrin, we found that 95% of the transferrin uptake was degraded. This suggests that most of the uptake of transferrin was not mediated by a receptor‐dependent mechanism, but by a phase fluid endocytosis. 3H‐labelled apotransferrin appears in the supernatant of the cell culture at the same rate as 59Fe‐3H labelled diferric transferrin, showing an identical uptake for the two types of transferrin. Uptake of apo‐ or diferric transferrin by macrophages was identical in relation to time of incubation and the amount of transferrin used. These studies suggest that most of the transferrin uptake by bone marrow macrophages (non‐activated or non‐elicited cells) is mediated by a non‐receptor mechanism that is independent of the degree of transferrin saturation.
Long‐term assessment of efficacy and safety of L1, an oral iron chelator, in transfusion dependent thalassaemia: Indian trial
Authors: Agarwal M. B. , Gupte S. S. , Viswanathan C. , Vasandani D. , Ramanathan J. , Desai Neena , Puniyani R. R. , Chhablani A. T.
Published: March 2008

Summary. From August 1989 to May 1991. 52 patients with transfusion dependent thalassaemia major received L1 (1.2‐dimethyl‐3‐hydroxypyrid‐4‐one), the oral iron chelator, for a period of 3–21 months (mean±SD: 14.2±6.8). Mean (±SD) urinary iron excretion varied from 6.2±4.6 mg/d on 25 mg/kg/d of L1 to 42.3±37.1 mg/d on 100 mg/kg/d of L1. Mean (±SD) drop in S ferritin was 1465±990 μg/l after 5.0±0.8 months to 3641.2 ±2299.3 μg/l after 20.1 ±0.9 months of therapy. There was no evidence of neutropenia, thrombocytopenia, ear or eye toxicity. L1‐related arthralgia, which was reversible on dose reduction or stoppage, was seen in 20 patients (38.5%), while minor gastrointestinal (GI) tract symptoms occurred in seven (3.5%) cases.

We conclude that although L1 is an effective iron chelator, further studies are required to understand the mechanism of L1 related arthralgia and also to find a safer but effective dose on which incidence of L1 related arthralgia is minimal.
Towards a new predictor of AIDS progression through the quantisation of HIV‐1 DNA copies by PCR in HIV‐infected individuals
Authors: Lefrère Jean‐Jacques , Mariotti Martine , Wattel Eric , Lefrère Francois , Inchauspe Geneviève , Costagliola Dominique , Prince Alfred
Published: March 2008

Summary. The proviral copy (PVC) number in peripheral blood mononuclear cells (PBMCs) of human immunodeficiency virus (HIV)‐infected individuals was measured by a quantitative polymerase chain reaction (PCR) assay to determine over time the relation between the viral load and the evolution towards the disease in HIV‐infected people: 67 anti‐HIV‐1 positive individuals (60 stage II/III, 7 stage IV) were studied. The mean PVC number per 1.5 × 105 PBMCs in stage II/III individuals (14.4 ± 14.2) and in stage IV individuals (32.2 ± 22.9) was significantly different (P <0.02). PVC number was inversely correlated to the CD4 lymphocyte count (P <0.01). In the logistic regression, the PVC number was a better marker of evolution towards the disease than the CD4 lymphocyte count. The mean proportion of HIV‐infected PBMCs in stage IV individuals and in stage II/III individuals was 1/4606 and 1/10714, respectively.
A distinct large granular lymphocyte (LGL)/NK‐associated (NKa) abnormality characterized by membrane CD4 and CD8 coexpression
Authors: Richards Stephen J. , Sivakumaran M. , Parapia L. A. , Balfour Ian , Norfolk Derek R. , Kaeda Jaspal , Scott Colin Stephen
Published: March 2008

Summary. In a study of 870 individual patients with either lymphocytosis (excluding known lymphoproliferative disease), increased proportions of blood lymphocytes with granular morphology (LGL), or neutropenia, 14 cases were found with abnormally increased CD3+CD4+CD8+ components. Eleven of these were further investigated and 10 shown in follow‐up studies to be persistent in nature. Morphological assessments revealed increased LGL in 9/11 cases, and in seven of these > 50% lymphocytes had discernable cytoplasmic granulation. Immunophenotypic studies indicated that CD8 expression by CD4+ lymphocytes in these patients was of low density (CD8dim+), and that both the CD4+CD8− and CD4+CD8dim+ fractions in each patient was characterized by a CD11b+ CD16−CD56+CD57+ composite NK‐associated (NKa) phenotype (in contrast to normal CD4+ CD8− blood lymphocytes and CD4+CD8+ thymocytes which were consistently CD11b−CD16−CD56−CD57−). TCR genotypic studies revealed rearranged components (β plus 7. or β alone) in 5/11 cases, but there were no obvious relationships between TCR configuration (including rearranged band densities) and immunophenotypes, absolute lymphocyte or neutrophil numbers, the proportions of blood LGL, or the proportions of CD4+ cells coexpressing CD8. The occurrence of identical NKa phenotypic profiles in both germline and rearranged TCR cases does, however, suggest the possiblity of an evolutionary process from a non‐clonal expansion to a clonal state. Serum studies, including soluble CD4. CD8 and IL2‐R concentrations and autoantibody investigations, of representative germline and rearranged TCR cases failed to indicate any consistent abnormalities, but there was some suggestion for the existence of a chronic reactive process in some of the patients with germline TCR. These findings suggest that expanded LGL/NKa+ components with phenotypic evidence of CD4/CD8 coexpression should be regarded as a distinct diagnostic category and that persistent CD4+CD8+ abnormalities with germline TCR should be monitored for possible clonal transition.
Mouse erythrocyte rosette formation with malignant human B‐lymphocytes re‐evaluated: still a useful marker for differentiating mature B‐cell malignancies
Authors: Hokland Peter , Geisler Chr. H. , Andersen Erik , Drivsnolm Aage , Hansen Mogens Mørk , Meyer Karin
Published: March 2008

Summary. We have re‐evaluated mouse rosette formation (MRF) as a marker for B‐CLL by estimating the fraction of mouse rosette forming B‐lymphocytes (identified by CD20 monoclonal antibodies) in normal donors and malignant CD20 + cell proliferations (ALL, AML, B‐NHL, B‐HCL and B‐CLL). Whereas this ratio was increased in B‐CLL, all other CD20 positive malignancies showed mean ratios of less than 0·1. As part of a Danish multi‐centre study, we furthermore prospectively analysed 86 patients and found that the mouse/CD20 ratio divided the 78 patients with monoclonal B‐cell populations suspected of B‐CLL in two distinct groups. In the low ratio group, three patients were categorized as leukaemized B‐NHL and one as PLL. The remaining three patients with low ratio were clinically and immunologically (by Smlg density and CD5 expression) B‐CLL patients suggesting a frequency of MR‐negative B‐CLLs of approximately 5%. In the high ratio group two of 70 patients were diagnosed as B‐NHLs. Thirdly, MRF was a valuable parameter in patients, where transformation of disease is suspected, since it preceded clinical changes by several months. Thus, MRF is still a useful marker in the age of monoclonal antibodies.
β2‐glycoprotein 1 (β2GP1) enhances cardiolipin binding activity but is not the antigen for antiphospholipid antibodies
Authors: Pierangeli S. S. , Harris E. N. , Davis S. A. , DeLorenzo G.
Published: March 2008

Summary. Some investigators have reported that a serum protein, β2‐glycoprotein 1 (β2GP1), either alone or in combination with negatively charged phospholipid, may be the antigen for anticardiolipin (aCL) antibodies. To examine these reports further, ELISA tests, inhibition experiments, Ouchterlony and Western blot techniques were used to examine anticardiolipin binding to β2GP1. Sera from patients with the antiphospholipid syndrome (APS) and syphilis were studied, as well as whole IgG immunoglobulin and affinity purified (a.p.) IgG aCL antibodies. Results showed no binding of aCL antibodies to β2GP1 in the absence of cardiolipin.

β2GP1 caused enhanced binding of aCL antibodies to cardiolipin, but this enhancement was not observed in inhibition experiments. Binding to cardiolipin occurred in the absence of β2GP1. Enhancement of cardiolipin binding activity by β2GP1 was observed for APS, but not for syphilis.

We conclude that β2GP1 is not the antigen for aCL antibodies, nor is it likely that the antibody recognizes shared β2GP1‐cardiolipin epitopes. Instead, this protein may make cardiolipin more available for aCL binding on solid surfaces by some yet undefined mechanism. This effect may not extend to aqueous suspensions.
Studies of oral neutrophil levels in patients receiving G‐CSF after autologous marrow transplantation
Authors: Lieschke Graham J. , Ramenghi Ugo , O'Connor Marian P. , Sheridan William , Szer Jeffrey , Morstyn George
Published: March 2008

Summary. Patients are at risk of mucositis and infections in the oral cavity during the neutropenic period after chemotherapy, which are significant causes of morbidity. In phase I/II studies with the haemopoietic growth factor granulocyte colony stimulating factor (G‐CSF), a reduction in post‐chemotherapy mucositis has been observed in addition to haematologic effects. To understand this phenomenon better in patients receiving G‐CSF following high‐dose chemotherapy with autologous bone marrow transplantation (ABMT), we studied the effects of G‐CSF on levels of neutrophils recoverable from the oral cavity using a quantitative mouthrinse assay. In normal subjects, mouthrinses contained 472 ± 329 ± 103 neutrophils/mouthrinse. After chemotherapy followed by ABMT, mouthrinse neutrophil levels decreased to undetectable levels during the neutropenic period, but recovered 1–2 and 3–9 d before circulating neutrophil levels reached 0 ± 1 and 1 ± 109/1 respectively, whether or not patients received G‐CSF. In patients who received G‐CSF, the mean cumulative mucositis score was reduced from 35 ± 9 to 21 ± 12 (P < 0 ± 05), and the maximum mean daily mucositis score was reduced from 2·8 ± 0·5 to l·7 ± 0·9 (P < 0·01), compared to patients who did not receive G‐CSF after ABMT. These studies provide in vivo evidence that neutrophils produced during G‐CSF therapy are available to leave the circulation and enter tissues where their function is required for host defence. Since the usual temporal relationship between oral and peripheral blood neutrophil recovery was preserved during G‐CSF administration after ABMT, these data support the hypothesis that the reduction in post‐ABMT mucositis observed with G‐CSF therapy may reflect a beneficial effect of G‐CSF on the kinetics of oral mucosal neutrophil recovery in addition to the effect of G‐CSF to accelerate peripheral blood neutrophil recovery.
Homozygosity for dominant form of hereditary spherocytosis
Authors: Duru Feride , Gürgey Aytemiz , ÖZtürk Gülyüz , Yörükan Selma , Altay Cišdem
Published: March 2008

Summary. A 6‐month‐old male infant with hereditary spherocytosis (HS) who was the first child of a cousin marriage is presented. The patient had splenomegaly and severe anaemia. Examination of the peripheral blood smear revealed spherocytes and the osmotic fragility of red blood cells was greatly increased.

Physical examination of the parents revealed that both parents had mild anaemia, jaundice and splenomegaly. Their peripheral blood smears showed spherocytes and a few acanthocytes. Osmotic fragility of red blood cells of both parents were increased. Red cell membrane electrophoresis indicated a deficiency of ankyrin in the propositus: mild deficiency was also detected in both parents. Electrophoretic patterns of red cell membrane proteins suggested that the child was homozygous for the dominant form of HS associated with ankyrin deficiency, while both parents had the simple dominant form of the disease.

Red blood cell transfusions were given to the patient starting at the age of 1 month until splenectomy was performed at the age of 1 year that resulted in complete haematological response. This observation indicates that homozygosity for dominant type of HS associated with ankyrin deficiency is life compatible and splenectomy may cure the anaemia.
The production of Steel factor mRNA in Diamond‐Blackfan anaemia long‐term cultures and interactions of Steel factor with erythropoietin and interleukin‐3
Authors: Sieff C. A. , Yokoyama C. T. , Zsebo K. M. , Trammell Joel , Andersen J. W. , Nathan D. G. , Williams D. A.
Published: March 2008

. Diamond‐Blackfan anaemia (DBA) is a congenital macrocytic anaemia. To investigate whether DBA is due to hyporesponsiveness to or hypoproduction of Steel factor (SF). we compared the in vitro responsiveness of the BFU‐E contained in the Ficoll‐Hypaque non‐adherent cell fraction of six DBA marrows with that of four normal marrows and one transient erythroblastopenia of childhood (TEC) marrow. In addition, we studied the effect of soluble SF on long‐term marrow cultures (LTMC) and analysed the stromal cells from these cultures for SF mRNA transcripts. All the patients showed an erythropoietin dose‐related increase of small BFU‐E. The number and size of BFU‐E was increased with the addition to the epo of IL‐ 3 or SF: IL‐ 3 + SF was not synergistic. The addition of soluble SF to LTMC of DBA patients was associated with a small but consistent increase in non‐adherent cell production and an increase in the number of progenitors. Messenger RNA from immortalized stromal cell lines of three patients and from primary bone marrow stromal cells of one patient showed the presence of expected SF transcripts by PCR analysis. These results demonstrate that this group of DBA patients responds to SF and produces SF mRNA normally, indicating that SF itself is not involved in DBA pathophysiology. The effects observed suggest that, despite the lack of evidence for a causative role. SF may prove to be effective treatment for such patients.
Erythropoiesis in multiple myeloma: defective red cell production due to inappropriate erythropoietin production
Authors: Beguin Yves , Yerna Michèle , Loo Martine , Weber Mireille , Fillet Georges
Published: March 2008

We investigated the pathophysiology of erythropoiesis in 62 patients with multiple myeloma and examined whether it would establish a rational basis for the treatment of their anaemia with recombinant human erythropoietin. Erythropoietin (Epo) production was evaluated by serum levels and erythropoiesis was quantitated by serum transferrin receptor (TfR) levels. both assessed relative to the degree of anaemia. Instead of the expected stimulation of erythropoiesis in response to anaemia. haematocrit correlated positively with marrow erythropoietic activity. indicating that the mechanism of anaemia was primarily defective red cell production. Erythropoiesis decreased and anaemia worsened significantly with advancing clinical stage. 2 5% of the patients had inadequate Epo production and this proportion increased to 50% in stage 3. Inappropriate Epo production was seen in 60% of patients with renal impairment but was also observed in a number of patients with normal renal function. Erythropoiesis correlated strongly with the adequacy of Epo production particularly in advanced disease. Ve conclude that most myeloma patients have defective red cell production even in the absence of massive marrow infiltration and that inappropriate Epo production contributes to their anaemia.
Quantitative determination of the hybrid Bcr‐Abl RNA in patients with chronic myelogenous leukaemia under interferon therapy
Authors: Malinge Marie‐Claire , Mahon Francois Xavier , Delfau Marie Hélène , Daheron Laurence , Kitzis Alain , Guilhot François , Tanzer Joseph , Grandchamp Bernard
Published: March 2008

In vitro amplification of the Bcr‐Abl cDNA has been widely used to assess for the presence of minimal residual disease in patients with chronic myelogenous leukaemia (CML) presenting with complete clinical and cytogenetic remission. However, the level of sensitivity achieved in different laboratories remains largely unknown. Moreover, the results are usually expressed as positive or negative, making a precise follow‐up of the patients difficult. In an attempt to overcome these limitations, we devised a quantitative method to measure the amount of Bcr‐Abl mRNA in clinical samples. This methodology involves a single reverse transcription step, followed by separate amplification of Bcr‐Abl and total Abl mRNA. These two amplifications are performed in the presence of different dilutions of a same internal standard. This standard consists of Bcr‐Abl sequences with an eight bases deletion in exon 2 of Abl. One of the primers used in each separate reaction is labelled with fluorescein. Following amplification, PCR products derived from cellular RNA and those from the internal standard are separated and their relative fluorescence is determined using a laser fluorescent DNA sequencer (ALF. Pharmacia). The number of Bcr‐Abl and total Abl mRNA molecules initially present in each sample is then calculated. The accuracy and reproducibility of this method was assessed by studying serial dilutions of K562 RNA into normal RNA. Blood samples from 10 patients in cytogenetic remission under interferon therapy were studied. Only one sample was found negative while the others contained between 0.05 and 17 hybrid Bcr‐Abl mRNA molecules per 1000 molecules of Abl mRNA. These results suggest that a variable number of malignant cells are present in the majority of CML patients in cytogenetic remission following interferon therapy.
Constitutive expression of IL‐1β, M‐CSF and c‐fms during the myeloid blastic phase of chronic myelogenous leukaemia
Authors: Specchia Giorgina , Liso Vincenzo , Capalbo Silvana , Fazioli Francesca , Bettoni Stefania , Bassan Renato , Viero Piera , Barbui Tiziano , Rambaldi Alessandro
Published: March 2008

During the myeloid blast crisis (BC) of chronic myelogenous leukaemia (CML) non‐random additional chromosome abnormalities occur in over 80% of patients. However, these cytogenetic changes have been reported to precede the clinical signs of CML‐BC by several months to years suggesting that other biological events may participate in the multistep process of acute transformation of CML. The autocrine production of growth factors has been recently shown to occur in several haematological malignancies and particularly in acute myeloblastic leukaemia (AML). In the present report we demonstrate that IL‐1β gene is expressed in almost all cases of CML in myeloid blast crisis. The secretion of IL‐1 from CML blasts in culture supernatants was confirmed in all five of the patients we studied. A high proportion of cases showed constitutive expression of the M‐CSF gene and many of the same patients often had a simultaneous co‐expression of the proto‐oncogene c‐fms which encodes for the M‐CSF receptor. After exposure of leukaemic cells to phorbol myristate acetate (PMA), release of M‐CSF protein was documented in three of five patients studied. No significant interleukin‐3 (IL‐3), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) or granulocyte colony‐stimulating factor (G‐CSF), was detected in these patients demonstrating that a different pattern of growth factors secretion exist in AML and CML, where distinct molecular events are likely involved in the control of leukaemic proliferation.
Autotransplantation of peripheral blood stem cells mobilized by chemotherapy and recombinant human granulocyte colony‐stimulating factor in childhood neuroblastoma and non‐Hodgkin's lymphoma
Authors: Fukuda Minoru , Kojima Seiji , Matsumoto Kimikazu , Matsuyama Takaharu
Published: March 2008

Seven children with advanced neuroblastoma and non‐Hodgkin's lymphoma were treated with myeloablative chemoradiotherapy (180 mg/m2 melphalan plus 12 Gy fractionated total body irradiation), followed by autotrans‐plantation of peripheral blood stem cells (PBSC). Sufficient PBSC to restore bone marrow function were collected by a small number of leukaphereses during haematopoietic recovery after chemotherapy and recombinant human granulocyte colony‐stimulating factor (rhG‐CSF). Furthermore, rapid recovery of neutrophils was found in all patients by the administration of rhG‐CSF following transplantation: median 10 d (range 8–12) to attain more than 0.5 × 109/1 neutrophils, and 27 d (range 14–73) to attain more than 50 × 109/1 platelets, respectively. Haematopoietic reconstitution has been maintained throughout the follow‐up period (median 15 months; range, 6–22). Peripheral blood stem cells mobilized by chemotherapy and rhG‐CSF can induce complete haematopoietic reconstitution after myeloablative chemoradiotherapy.
Intramuscular anti‐D immunoglobulins for home treatment of chronic immune thrombocytopenic purpura
Authors: Gringeri Alessandro , Cattaneo Marco , Santagostino Elena , Mannucci Pier Mannuccio
Published: March 2008

To assess the efficacy of home treatment with intramuscular anti‐D immunoglobulins (anti‐D IgG) in patients with idiopathic or HIV‐related chronic immune thrombocytopenic purpura (ITP), we conducted an open label, non‐controlled, prospective study with a follow‐up of 6 months in 51 consecutive patients with HTV‐related (n= 24) or idiopathic ITP (n= 27). Anti‐D IgG (13 μg/kg) were infused intravenously for three consecutive days (induction therapy). Patients whose platelet counts tailed to increase above 50 × 109/l were given a second course of the same therapy. If their platelet counts remained lower than 50 × 109/l, they were considered non‐responsive. Patients who responded to induction therapy were injected intramuscularly with 6 μg/kg/week anti‐D IgG if platelets were between 50 and 100 × 109/l or 13 μg/kg/week if they dropped to less than 50 × 109/l. After induction therapy, responses occurred in 20/24 HIV‐related chronic ITP patients (83%) and in 14/27 idiopathic chronic ITP patients (52%). During follow‐up, platelets remained above 50 × 109/l in 17/20 HIV‐related chronic ITP patients (85%) and in 8/14 (57%) idiopathic chronic ITP patients. We conclude that anti‐D IgG are a valid therapeutic option for treatment of idiopathic and HIV‐related chronic ITP, since they are safe, cheap and can be given intramuscularly at home.
Two distinct patterns of glycosylphosphatidylinositol (GPI) linked protein deficiency in the red cells of patients with paroxysmal nocturnal haemoglobinuria
Authors: Hillmen Peter , Hows Jill M. , Luzzatto Lucio
Published: March 2008

We have studied three glycosylphosphatidylinositol (GPI) linked proteins on the erythrocytes of 14 patients with paroxysmal nocturnal haemoglobinuria (PNH). The pattern observed was bimodal in 12 of the patients and trimodal in two. Ten patients had a red cell population with normal CD59 antigen (membrane inhibitor of reactive lysis, MIRL), decay accelerating factor (DAF or CD55) and lymphocyte function‐associated antigen (LFA‐3 or CD58) and a second abnormal PNH population with absent CD59 antigen, DAF and LFA‐3. The other two patients with a bimodal pattern had a red cell population with normal CD59 antigen, DAF and LFA‐3 and an abnormal population with reduced, but not absent, CD59 antigen and DAF. The LFA‐3 on the abnormal red cells in these two patients appeared to be only slightly reduced. The two patients with a trimodal pattern had a normal population, a population with reduced, not absent, CD59 antigen and DAF, and a population with complete absence of CD59 antigen, DAF and LFA‐3. The accuracy of the Ham test in estimating the proportion of red cells with the PNH defect in the two types of PNH was assessed. The case of one patient who appeared to be ‘rescued’ from severe aplastic anaemia by the development of PNH is described.