bluuebunny/crossref_metadata_2025 huggingface dataset:
The placental transport of [3H] vitamin K1 in rats
Authors: Hamulyák Karly , De Boervanden Berg Marian A. G , Thijssen Henk H. W. , Hemker H. Coenraad , Vermeer Cees
Published: March 2008

In this paper we describe the placental transport of [3H]vitamin K1 in pregnant rats during the first 24 h after the oral administration of the vitamin. Vitamin K1 in the fetal livers ranged from 0.13% (3 h) to 2% (24 h) of the values found in the corresponding maternal livers. In spite of the low placental transfer of vitamin K, we found no accumulation of coagulation factor precursors in the fetal rat liver microsomes as could be expected in vitamin K deficiency. Moreover, we could not demonstrate any difference between adult and fetal rat liver microsomes with regard to the sensitivity for warfarin. From these results we conclude that a substantial placental barrier exists for the transport of pharmacological amounts of vitamin K1 but that under physiological conditions sufficient vitamin K1 appears to be present in the fetal liver to ensure a full carboxylation reaction. The vitamin K‐dependent carboxylase activity rate of adult and fetal rat liver microsomes was comparable, indicating that the newborn rat has an adequate carboxylating system.
Decreased sialic acid content of erythrocytes in patients with aplastic anaemia
Authors: Yoshida Minore , Takaku Fumimaro , Nojiri Hlsao , Miura Yasusada , Saito Masaki
Published: March 2008

The sialic acid content of erythrocytes from patients with aplastic anaemia was determined and compared with those in patients with several haematological disorders and healthy individuals. The sialic acid was released enzymatically with Vibrio cholerae sialidase and quantitated by the thiobarbituric acid method (Aminoff, 1961). The sialic acid content of normal erythrocytes was 538 ±31 nmol/ml of packed erythrocytes. That of erythrocytes from patients with aplastic anaemia was 480±35 nmol/ml of packed erythrocytes, which was significantly lower than normal (P<0.01).

In contrast, erythrocytes from patients with myeloproliferative disorders showed significantly (P<0.05) higher sialic acid contents (564±45 nmol/ml of packed erythrocytes). These results suggest that some membrane changes occur in erythrocytes in patients with these disorders.
Fluorouracil (5‐FU) induced acute immune haemolytic anaemia
Authors: Sandvei P. , Nordhagen R. , Michaelsen T. E. , Wolthuis K.
Published: March 2008

A female patient on Fluorouracil (5‐FU) therapy for rectal carcinoma developed acute intravascular haemolysis with her fifteenth drug injection; a similar episode occurred later after a controlled challenge with the drug. A 5‐FU‐dependent complement‐activating IgM antibody, which reacted with RBC in an indirect antiglobulin test, was detected in her serum. The antibody did not react with cord RBC, but a blood group specificity could not be determined. An excess of 5‐FU in the indirect antiglobulin tests resulted in inhibition of these reactions.
Long survival in B‐CLL correlates with surface IgMK phenotype
Authors: Hamblin T. J. , Oscier D. G. , Stevens J. R. , Smith J. L.
Published: March 2008

200 patients with B‐cell chronic lymphocytic leukaemia (B‐CLL) presenting to a single centre over 13 years have been studied. In 72.2% the diagnosis was made on an incidental blood count, and 70.1% were stage A at presentation. Those patients whose cells expressed surface IgMK were significantly more likely to be stage A at presentation and significantly less likely to have a lymphocyte count >50x 109/1, to have progressive disease or to require treatment than those with other classes of surface Ig. Patients whose cells express surface IgMK have a significantly longer actuarial survival than others and this is also so when only patients in Binet stage A or when only patients presenting below the age of 75 are studied. By studying all of the cases presenting in a single catchment area we have attempted to avoid the bias against trivial disease likely to be seen in specialist referral centres.
Relationship of serum lactate dehydrogenase level with first remission length in adult acute lymphocytic leukaemia
Authors: Fasola Gianpiero , Fanin Renato , Baccarani Michele , Zuffa Eliana , Haanen Clemens , Comotti Benedetto , Scapoli Gianluigi , Alghisi Alberta , Battista Raffaele , Aieta Michele , Fiacchini Mauro , Tura Sante
Published: March 2008

Several features have a prognostic value in adults with acute lymphocytic leukaemia (ALL). However, in about two‐thirds of all cases prognosis remains quite variable, with a substantial number of early relapses. This study shows in 118 adult patients who attained complete remission (CR) between 1978 and 1984 that pretreatment serum total lactate dehydrogenase (LDH) activity was inversely correlated with first CR length. The prognostic value of LDH was higher than that of any other features both in univariable and in multivariable analysis. The value was significant in the whole series as well as in patients who lacked other high risk features. Among non‐high risk and low‐WBC count cases, patients with LDH > 500 U/l had a median first CR duration of 27 months, and a projected 5‐year relapse free survival of 36%, versus 9 months and 12% of patients with LDH > 500 U/l. These results fit well with the results of a study of ALL in children, and suggest that pretreatment serum total LDH activity is an important risk determinant in adult ALL.
Functional studies of cell‐mediated immunity in haemophilia and other bleeding disorders
Authors: Mahir Walla S. , Millard Rosemary E. , Booth J. C. , Flute P. T.
Published: March 2008

Defects of immunoregulation which occur in haemophilia, reflected by numerical changes in T lymphocyte subsets, have been further investigated in functional studies. Polyclonal T‐cell activation by the mitogen phytohaemagglutinin (PHA) and specific stimulation by cytomegalovirus (CMV) or herpes simplex type 2 (HSV‐2) in previously sensitized subjects were studied in peripheral blood lymphocyte and in T4‐cell‐enriched cultures. Compared with 12 controls, the responses of 11 patients (nine with haemophilia A and two with von Willebrand's disease) to PHA were significantly reduced both in unfractionated and in T4‐cell‐enriched peripheral blood lymphocyte cultures. Reduced responses to PHA were found in HIV (HTLV III)‐seronegative as well as ‐seropositive patients. There were no significant differences between the response of patients’unfractionated and T4‐enriched peripheral blood lymphocytes to CMV/HSV‐2 recall antigen and the control subjects, although there was evident variation in the magnitude of patients’unfractionated and T4‐enriched lymphocyte responses.
Cocoa: a new mouse model for platelet storage pool deficiency
Authors: Novak Edward K. , Sweet Hope O. , Prochazka Michal , Parentis Michael , Soble Rebecca , Reddington Madonna , Cairo Alfred , Swank Richard T.
Published: March 2008

We describe genetic, haematological and biochemical properties of a new mouse pigment mutant, cocoa (coa). Cocoa is a recessive mutation located on the centromeric end of chromosome 3 near the Car‐2 locus. The mutation causes increased bleeding time accompanied by symptoms of platelet storage pool deficiency (SPD), including decreased platelet serotonin and decreased visibility of dense granules as analysed by electron microscopy of unfixed platelets. Dense granules were visible in normal numbers when platelets were incubated with the fluorescent dye, mepacrine. The intragranular environment, however, was abnormal as indicated by decreased flashing of mepacrine‐loaded dense granules after exposure to ultraviolet light. Unlike the previously described seven mouse pigment mutations with SPD in which pigment granules, platelet dense granules and lysosomes are affected, the cocoa mutant had normal secretion of lysosomal enzymes from kidney proximal tubule cells and platelets. The cocoa mutation thus represents an example of a single gene which simultaneously affects melanosomes and platelet dense granules but probably does not affect lysosomes. The results indicate that melanosomes and platelet dense granules share steps in synthesis and/or processing. Cocoa may be a model for cases of human Hermansky‐Pudlak syndrome in which functions of melanosomes and platelet dense granules, but not lysosomes, are involved.
Ristocetin induces platelet aggregation: a morphological demonstration
Authors: Escolar G. , Monteagudo J. , Villamor N. , Garrido M. , Castillo R. , Ordinas A.
Published: March 2008

Changes in the morphology of human platelets induced by ristocetin in platelet‐rich plasma (PRP) have been analysed at the ultrastructural level by means of a tannic acid procedure. Studies were also undertaken to measure the release of serotonin. Modifications of the aggregation tests induced by apyrase, a monoclonal antibody (Mab) to GPIIb/ IIIa and by EDTA were also investigated. Transmission electron microscopy revealed that ristocetin precipitated adhesive proteins on the platelet membrane. An electron‐dense deposit was seen within 20s after ristocetin was added. When experiments were carried out in the aggregometer cuvette during stirring, groups of platelets became activated, changed their shape, and finally aggregated releasing part of their contents. The morphology of aggregates did not differ from those formed in the presence of ADP. Aggregation studies demonstrated that a Mab to GPIIb/IIIa modified the extent and the rate of the aggregation curve when RIPA was performed in citrated platelet‐rich plasma (c‐PRP), while apyrase modifies the extent, but not the slope, of the curve. Neither the antibody nor apyrase modified RIPA when it was performed in PRP obtained in the presence of EDTA. All this evidence suggests that RIPA in c‐PRP, besides reflecting the interaction of GPIb with vWF, may also test other mechanisms of the platelet function including: assembly of GPIIb/IIIa complex, interaction of fibrinogen with this glycoprotein complex, and possibly the release reaction.
Erythrocyte pyruvate kinase deficiency: 11 new cases
Authors: Zanella A. , Colombo M. B. , Miniero R. , Perroni L. , Meloni T. , Sirchia G.
Published: March 2008

Eleven new cases of red cell pyruvate kinase (PK) deficiency with congenital haemolytic disease from 10 unrelated Italian families were characterized using the methods recommended by the International Committee for Standardization in Haematology (ICSH). All patients were double heterozygotes for the PK gene. The 10 variants were designated PK ‘Lecce,’‘Parma,’‘Verona,’‘Milano,’‘Soresina,’‘Macerata,’‘Sassari,’‘Genova,’‘Mantova’and ‘Brescia.’PK ‘Sassari’was associated with glucose‐6‐phosphate dehydrogenase deficiency in two siblings. All mutants displayed multiple biochemical abnormalities except for PK ‘Lecce’that only showed decreased red cell PK activity. No relation was found between the severity of anaemia and either the residual PK activity or specific biochemical enzyme abnormalities.

Increased serum ferritin levels were detected in most of the patients, suggesting the need for systematically monitoring iron status in this disease.
Transplantation of embryonic haemopoietic stem cells without prior recipient X‐irradiation
Authors: HOLLANDS P.
Published: March 2008

Allogeneic day 7 mouse embryonic cells can colonize the haemopoietic system of normal, non‐irradiated recipient mice. Donor embryonic cells are disaggregated and injected intravenously resulting in colonization in 40% of recipients, as shown by the presence of electrophoretic markers, characteristic of the donor cells. Donor type haemoglobin (Hb) and donor type glucose phosphate isomerase (GPI) demonstrates the presence of donor type erythrocytes and lymphocytes respectively. Repeat grafts in recipients not showing donor markers did not result in colonization. Recipient type haemopoiesis was dominant in all types of recipient. Skin grafts of allogeneic donor type skin onto successfully grafted embryonic cell recipients did not survive. Allogeneic donor embryonic cells therefore survive in recipients where adult skin allografts do not.

Donor embryonic cells, homozygous for T6 marker chromosomes, were used to assess the site of colonization of intravenously grafted cells. Donor chromosomes were seen in recipient liver and bone marrow at low levels. This distribution of donor cells persists for up to 64 d post‐graft.
Investigation of a large kindred with type IIB von Willebrand's disease, dominant inheritance and age‐dependent thrombocytopenia
Authors: Mazurier C. , Parquet‐Gernez A. , Goudemand J. , Taillefer M. F. , Goudemand M.
Published: March 2008

Twenty‐six members of four generations of one family in which a man was diagnosed in 1961 as having von Willebrand's disease (vWD) have been studied. Subtype IIB vWD and autosomal dominant inheritance was identified in 19 individuals with bleeding signs varying in severity and frequency. The absence of high molecular weight multimers of plasma von Willebrand factor (vWf) and the heightened interaction of plasma vWf with platelets in the presence of low ristocetin concentrations were consistent in all affected subjects. Nevertheless the degree of these abnormalities was variable without clearcut linkage to the severity of clinical symptoms. Thrombocytopenia was present only in the adult affected family members and the platelet count appeared to be age‐dependent. The investigation of this family provides further evidence of the phenotypic variability in the vWf‐platelet interactions within this vWD subtype.
Haemostatic disturbances in patients bitten by Russell's viper (Vipera russelli siamensis) in Burma
Authors: Than Than , Hutton R. A. , Lwin Myint , Han Khin‐Ei , Soe Soe , Swe Tin‐Nu , Phillips R. E. , Warrell D. A.
Published: March 2008

Patients who are severely envenomed by Russell's viper develop DIC which is frequently associated with spontaneous bleeding and incoagulable blood. These haemostatic disturbances may be responsible for death or organ/tissue damage both through haemorrhage and microvascular occlusion by fibrin thrombi. The most striking laboratory features of the coagulopathy developing after Russell's viper bite in the 42 patients studied were depletion of fibrinogen (mean 0.09 g/l, range 0–0.6), factor V (6.5 u/dl, range 0–17), factor × (35 u/dl, range 1–85), factor XIIIa (57 u/dl, range 15–82), plasminogen (61 u/dl, range 10–92), antiplasmin (36 u/dl, range 14–62), Protein C (49 u/dl, range 15–100) and platelets (104 × 1099/l, range 25–197). Intense fibrinolytic activity was detected in all cases with marked elevation of FDPs (1614 μg/ml, range 350–3000), a large proportion of which were cross‐linked (1058 μg/ml, range 38–3000). The monospecific Burmese antivenom appeared to be very effective in neutralizing the venom procoagulants and in restoring blood coagulability. Moreover, the unexpectedly normal level of AT III provides a theoretical basis for the use of heparin to enhance the inactivation of those serine proteases present before antivenom administration.
Cytogenetic studies of haemopoietic colonies from patients with an initial diagnosis of acute lymphoblastic leukaemia
Authors: Kalousek Dagmar K. , Dubé Ian D. , Eaves Connie J. , Eaves Allen C.
Published: March 2008

For patients with an initial diagnosis of Philadelphia chromosome (Ph1)‐positive acute lymphoblastic leukaemia (ALL) and no documented history of Ph1‐positive chronic myeloid leukaemia (CML), the cell of origin and extent of lineage involvement of the disease is often unclear. This is largely due to the fact that cytogenetic analysis of direct marrow preparations cannot distinguish between the presence of Ph1‐positive myeloid metaphases and infiltration of the marrow with dividing Ph1‐positive blasts. Cytogenetic analysis of cultured haemopoietic colonies allows more precise lineage assignment. We have used this approach to study five adult patients who presented with Ph1‐positive ALL and subsequently showed a significant increase in normal metaphases (to 18‐100% of all metaphases examined) following remission induction. In four of these five patients, some Ph1‐positive erythroid and granulopoietic cells could be demonstrated. In the other, as in three patients with childhood ALL who presented with a Ph1‐negative but cytogenetically abnormal clone, only chromosomally normal erythroid and granulopoietic progenitors were detected. Two Ph1‐positive CML patients who entered a lymphoid blast crisis following a recognized chronic phase were also studied. In both of these latter two patients all erythroid and granulocyte‐macrophage colonies analysed were Ph1‐positive. These findings support the concept that patients presenting with Ph1‐positive ALL comprise a heterogeneous group. In some cases the disease may arise in a restricted lymphopoietic cell not capable of myelopoietic differentiation. However, in others involvement of myeloid cells suggests these may be variant forms of CML in spite of an unusual initial response of the Ph1‐positive clone to therapy.
Difference of cell lineage expression of haematopoietic progenitor cells in Philadelphia‐positive acute lymphoblastic leukaemia and chronic myelogenous leukaemia
Authors: Kitano Kiyoshi , Sato Yuko , Suda Toshio , Miura Yasusada
Published: March 2008

It is still difficult to clinically distinguish Philadelphia (Ph1)‐positive acute lymphoblastic leukaemia (ALL) from Ph1‐positive chronic myelogenous leukaemia (CML) in lymphoid crisis. In this study we tried to discriminate between these two disorders by simultaneous analyses of cell morphology and karyotype in single in vitro colonies. We studied three patients with Ph1‐positive ALL and four with Ph1‐positive CML in various phases of the disease. Bone marrow and peripheral blood cells obtained directly from all seven patients showed abnormal karyotypes including Ph1‐chromosomes. Normal karyotypes were found in a small proportion of cells from two ALL patients, but none were found in any from the CML patients. The patients’mononuclear cells (MNCs) were plated at 1‐5 × 104/ml in semi‐solid medium containing methylcellulose plus phytohaemagglutinin‐stimulated leucocyte conditioned medium and erythropoietin. After 9‐14 d cultivation, granulocyte‐macrophage, erythroid and mixed colonies obtained were used for simultaneous analysis of cell morphology and karyotype. Morphological examination showed that these colonies contained neutrophils, eosinophils, basophils, macrophages and/or erythroblasts in various combinations. No lymphoblast colonies were obtained under the culture conditions used. Cytogenetic examination revealed that all metaphase cells observed in colonies obtained from Ph1‐positive ALL patient MNCs had a normal karyotype, whereas those in colonies from Ph1‐positive CML patient MNCs had abnormal karyotypes, including Ph1 chromosomes, suggesting that the difference between the two disorders involved a difference in cell lineage. Our results showed that this method was a practicable method for distinguishing Ph1‐positive ALL from Ph1‐positive CML in lymphoid crisis.
Plasma levels of highly sulphated glycosaminoglycans are raised in patients with chronic myeloid leukaemia
Authors: Craig J. I. O. , Reilly K. M. , Yap P. L. , Dawes J. , Allen L. , Parker A. C.
Published: March 2008

A study of absolute basophil counts and plasma levels of highly sulphated glycosaminoglycans in 30 peripheral blood samples from 21 patients with different leukaemias was performed. This revealed significantly raised levels of both plasma highly sulphated glycosaminoglycans and basophils in those patients with chronic myeloid leukaemia, as compared to those with other types of leukaemia and 35 normal controls. A strong correlation (r, = 0.83) was observed between the levels of highly sulphated glycosaminoglycans and basophil counts in the group as a whole, supporting a direct relationship between the two. The elevated plasma levels of highly sulphated glycosaminoglycans may contribute to the bleeding tendency reported in some patients with chronic myeloid leukaemia.
Acquired alpha‐2‐antiplasmin deficiency in acute promyelocytic leukaemia
Authors: Avvisati Giuseppe , Ten Cate Jan W. , Sturk Augueste , Lamping Roy , Petti Maria C. , Mandelli Franco
Published: March 2008

We report a prospective study in nine consecutive adult patients with acute promyelocytic leukaemia (APL). The study objective was to assess the prevalance of activation of blood coagulation and/or activation of fibrinolysis in APL. Coagulation and fibrinolytic parameters relevant to the objective included antithrombin III, plasminogen, fibrin/fibrinogen degradation products and alpha‐2 antiplasmin activity and antigen levels. The results of this study revealed consistently normal antithrombin III levels, both before and in the course of antileukaemic treatment. Plasminogen levels were slightly decreased or normal. However, a distinct alpha‐2 antiplasmin activity deficiency in all patients was observed with levels even reaching zero in three patients, during chemotherapy. Alpha‐2 antiplasmin activity levels were consistently lower than the alpha‐2 antiplasmin antigen levels. The in vitro binding of alpha‐2 antiplasmin activity to fibrin clots was severely reduced which appeared to be due to the reduced alpha‐2 antiplasmin plasma levels. Upon crossed‐immunoelectrophoresis against alpha‐2 antiplasmin antiserum two alpha‐2 antiplasmin antigen peaks were observed in the plasma of all nine patients. All abnormalities were reversible 4 d after completion of chemotherapy.

In a second series of 12 consecutive APL patients we confirmed the consistency of the alpha‐2 antiplasmin activity deficiency and normal antithrombin III plasma levels. In addition Protein C activity and antigen levels were normal or near normal in 10 and reduced in two patients. Thrombinantithrombin III complexes were increased in 10 and normal in two patients.

We conclude that some activation of blood coagulation is present in APL (increased thrombin‐antithrombin III complex levels) but its contribution to the coagulopathy seems to be minor (normal antithrombin III and only slightly reduced protein C levels). The observed reduced alpha‐2 antiplasmin content of the fibrin clot in vitro may result in vivo in a fibrin clot that is highly susceptible to fibrin degradation, thus aggravating the coagulopathy in APL.
Hb S(C)‐β+‐thalassaemia: different mutations are associated with different levels of normal Hb A
Authors: Gonzalez‐Redondo J. M. , Kutlar F. , Kutlar A. , Stoming T. A. , De Pablos J. MA. , Kilinç Y. , Huisman T. H. J.
Published: March 2008

Analysis of amplified DNA through hybridization with 32P‐labelled synthetic oligonucleotide probes has provided data about the molecular abnormality for β‐thalassaemic globin genes present in 32 Black and eight Mediterranean patients with Hb S(C)‐β+‐thalassaemia. The patients were categorized according to these β‐thalassaemia mutations, and average haematological and haemoglobin composition data were compared for each of four different groups. Twenty‐eight Black patients had the ‐29 AG substitution and four had the ‐88 CT substitution; all had mild disease with comparable haematology and an average Hb A level of 20%. Six Mediterranean patients had the IVS‐1, 110 GA mutation; their haematological data were nearly the same as that for the Black patients except for a lower Hb A value of 11%. Two Turkish patients with the IVS‐2, 745 CG mutation were more severely affected with mild sickling disease and low Hb A levels of 5%. Hb F levels varied greatly because of age differences; high Gγ values were observed only in patients with a β‐thalassaemia chromosome having an Xmn I site 5’to Gγ.

The data readily explain the variability in Hb A level that has been repeatedly noted in patients with Hb S(C)‐β+‐thalassaemia.
Tumour necrosis factor may contribute to the anaemia of malaria by causing dyserythropoiesis and erythrophagocytosis
Authors: Clark I. A. , Chaudhri G.
Published: March 2008

Among the unexplained changes caused by malaria in several host species, including man and mouse, are erythrophagocytosis and dyserythropoiesis. In order to see whether tumour necrosis factor (TNF) could contribute to these changes we injected recombinant human TNF intravenously into mice made very susceptible to this monokine by low density infection with a mouse malaria (Plasmodium vinckei) or prior injection of an extract of Coxiella burneti. Appreciable erythrophagocytosis, involving nucleated erythroblasts as well as mature red cells, was observed in bone marrow preparations from TNF‐treated mice and those with severe illness due to P. vinckei, but in no other group. Dyserythropoiesis, involving irregularly‐shaped nuclei and karyorrhexis, had the same distribution. TNF at concentrations up to 8 ng/ml was detected in the serum of all mice with severe malaria, but not uninfected animals or those with light infections. These findings are consistent with TNF making an important contribution to erythrophagocytosis and dyserythropoiesis, and thus to anaemia, in malaria and other conditions.
Membrane studies on rod‐shaped red cells in hereditary elliptocytosis: least haemolysis and normal sodium influx with decreased membrane lipids
Authors: Kanzaki Akio , Ikeda Akiyo , Yawata Yoshihito
Published: March 2008

The clinical features of hereditary elliptocytosis were studied in 25 cases and compared with 20 normal individuals. Based on morphological features, these patients were classified into two groups; those with a rod‐shaped type of elliptocytosis (nine cases), and those with a non‐rod type (16 cases). Most of the cases with overt haemolysis were detected among cases of the non‐rod type. Overt haemolysis typically tended to be accompanied by stomatocytic changes, which appear to be superimposed on elliptic transformation of the red cells. Sodium influx increased in eight of nine HE patients with overt haemolysis, but in none of the HE with rod‐shaped red cells. Significant quantitative and qualitative differences in red cell membrane lipids were observed between the two types of HE. No spectrin abnormalities in domain composition, dimer‐dimer association, and thermal stability were observed in 11 of the HE patients studied, including four cases with overt haemolysis.
Embryonic haemopoietic stem cell grafts in the treatment of murine genetic anaemia
Authors: Hollands P.
Published: March 2008

Embryonic haemopoietic stem cells obtained from early post‐implantation mouse embryos can be successfully used in the treatment of murine genetic anaemia. Anaemic recipients had either chronic macrocytic anaemia, which was lethal without treatment, or mild anaemia without increased mortality. All successfully grafted recipients developed donor haemoglobin and glucose phosphate isomerase electrophoretic markers, indicating the presence of donor erythrocytes and lymphocytes. The minimum number of embryonic cells resulting in a successful graft in chronic macrocytic anaemia recipients was 0.8 × 106 nucleated cells. Athymic (nude) mice were also colonized by embryonic haemopoietic stem cells. Donor electrophoretic markers were seen but all recipients soon died, possibly of pneumonia.

Bone marrow grafts into recipients with chronic macrocytic anaemia were not successful. Bone marrow grafts into recipients with mild anaemia resulted in some recipients showing donor markers. These recipients later died, showing symptoms of graft‐versus‐host disease.
Methods and theory for analysis of flow of white cell subpopulations through micropore filters
Authors: Nash G. B. , Jones J. G. , Mikita J. , Dormandy J. A.
Published: March 2008

Methods have been developed for analysing the resistance of WBC to flow, by measuring their transit rates through 5 and 8 μm pore filters at constant pressure. Unfractionated WBC and separated subpopulations have been compared. For either pore size, lymphocytes exhibited least resistance to flow, followed by granulocytes, with monocytes being most resistant. A theoretical model, which represents WBC suspensions as made up of three particle types (a relatively fast and a relatively slow population, plus a pore blocking population) adequately describes the data for flow rate versus volume filtered. For 5 μm pores, this theory indicates that a majority of WBC have transit times ±0.5 s. Unfractionated and mixed mononuclear samples contained a proportion of particles with transit times an order of magnitude longer, whereas, for granulocytes, no slow flowing population was evident. Removal of monocytes by plating out, reduced the proportion of the slow particles in the mononuclear preparation. Unique values for transit times could not be determined for 8 μm pores, but it could be concluded that the great majority of WBC made a very quick transit, with the flow becoming dominated by a small number of much more resistant cells. Simple flow parameters (initial relative flow rate and slow particle resistance) are described which characterize these two populations. Both 5 and 8 μm pore data indicated that few cells became permanently trapped within pores.
Procoagulant induction by human lymphokine and interferon γ/PMA on a myelomonocytic cell line, RC2a
Authors: Geczy Carolyn L. , Jones Annette
Published: March 2008

The human myelomonocytic cell line RC2a expressed procoagulant activity following stimulation with human lymphokine (LK) prepared by stimulating peripheral blood mononuclear cells with either mitogens (Concanavalin A, phytohaemagglutinin or antigen (tuberculin). Induction was rapid, with optimal activity observed between 6 and 8 h, was decreased in cultures containing serum and was not inhibited by actinomycin D or cycloheximide. The LK activity was not inhibited by an anti‐interferon γ (IFNγ) antibody. IFNγ and phorbol myristate acetate (PMA) had no activity but acted in synergy to induce procoagulant expression; interferon α plus PMA were without effect. In contrast to the LK‐induced response, procoagulant induced by IFNγ/PMA was not detected for up to 8 h and steadily rose over 24–48 h culture and was dependent on new protein and RNA synthesis. Bacterial lipopolysaccharide, a potent inducer of thromboplastin on normal human monocytes, failed, either alone or in combination with LK, IFNγ. PMA or IFNγ plus PMA, to activate procoagulant expression on RC2a cells. Both LK and IFNγ/PMA‐induced procoagulant had properties of thromboplastin expressed both intracellularly and on intact, viable cells. This study shows that RC2a cells are responsive to factors produced as the result of an activated cell‐mediated immune response which may therefore contribute to the coagulopathies common to this form of malignancy.
The molecular basis of thalassaemia major and thalassaemia intermedia in Asian Indians: application to prenatal diagnosis
Authors: Thein S. L. , Hesketh C. , Wallace R. B. , Weatherall D. J.
Published: March 2008

A study of the molecular pathology of β thalassaemia in the Asian Indian immigrant population in the U.K. included 37 patients with thalassaemia major and 14 with thalassaemia intermedia. Using a combination of oligonucleotide probe hybridization and restriction endonuclease analysis the mutations in 100/102 (98%) of the β thalassaemia genes were characterized. Nine different types were found, of which six are associated with β, one with severe β+ and two with mild β+ thalassaemia. Comparison of the β‐globin gene cluster haplotypes, α globin genotypes and β gene mutations of the thalassaemia major group with the thalassaemia intermedia group suggests that the co‐inheritance of a high Hb F determinant associated with the ‐ + ‐ ++ 5’β haplotype and the inheritance of a mild β‐thalassaemia mutation are the major ameliorating factors of disease severity in Asian Indians. In comparison with other population groups, β thalassaemia in Asian Indians is not associated with one or two predominant mutations. Despite this, prenatal diagnosis by direct detection is possible in the majority of families by restriction analysis and a limited number of oligonucleotide probes since the majority of severely affected individuals are homozygous for a single mutation. The characterization of these mutations should be useful for the planning of prenatal diagnosis programmes for β thalassaemia in other Asian Indian communities.
Enhancement of erythropoiesis in vitro by human growth hormone is mediated by insulin‐like growth factor I
Authors: Merchav Shoshana , Tatarsky Ilana , Hochberg Zeev
Published: March 2008

Insulin‐like growth factor I (IGF‐I) is the presumed paracrine or autocrine growth‐promoting mediator of growth hormone in peripheral tissues. In order to evaluate the role of IGF‐I as mediator of human growth hormone (hGH) in erythropoiesis, we compared the effects of both peptides upon in vitro colony formation by primitive (BFU‐E) and relatively mature (CFU‐E) human erythroid precursors. Biosynthetic IGF‐I (2 ng/ml) and hGH (25 ng/ml) induced a significant increase in the growth of both BFU‐E and CFU‐E. BFU‐E growth was maximally enhanced by 6 ng/ml IGF‐I and by 50 ng/ml hGH, resulting in an increase in burst numbers of 62 ± 12% and 52 ± 12%, respectively. Maximal enhancement of CFU‐E growth was detected at higher concentrations of IGF‐I (20 ng/ml) and hGH (150 ng/ml), with respective increases of 121 ± 35% and 137 ± 18% in colony numbers. Enhancement of bone marrow and peripheral blood erythroid progenitor cell growth by hGH required the presence of monocytes and was abrogated by specific monoclonal antibodies directed against IGF‐I membrane receptors. The in vitro growth‐promoting effect of hGH upon human erythroid precursors thus appears to be mediated by paracrine IGF‐I.
Relationship between internalization and cytotoxicity of ricin A‐chain immunotoxins
Authors: Preijers F. W. M. B. , Tax W. J. M. , Witte T. De , Janssen Anke , Heijden Hélènev.d. , Vidal H. , Wessels J. M. C. , Capel P. J. A.
Published: March 2008

Immunotoxins (ITs) appear to vary considerably in their killing efficiency towards antigen positive cells. In order to unravel the mechanisms underlying these differences, the parameters responsible for these variations were studied. The efficacy of the monoclonal antibodies (MoAbs) WT32 (CD3), OKT4 (CD4), T101 (CD5), WT1 (CD7) and WT82 (CD8) conjugated to ricin A‐chain was expressed by the extent of protein synthesis inhibition of four leukaemic T cell lines (CEM, GH1, Jurkat and HPB‐ALL). Large differences in cytotoxicity were observed. Efficient inhibition of protein synthesis was seen with anti‐CD3 IT, anti‐CD5 IT and anti‐CD7 IT. In these cases the cytotoxicity was related to the antigen density on the target cell membrane. Anti‐CD4 IT inhibited poorly and anti‐CD8 IT was ineffective, even on cell lines with a high expression of the corresponding antigen. When antigen density and cytotoxicity were plotted for all CD antigens, no correlation could be found. Subsequently, internalization was studied with 125Iodine‐labelled antibodies. Anti‐CD7 showed the fastest internalization rate, followed by anti‐CD3 and anti‐CD5. Anti‐CD4 and anti‐CD8 antibodies were respectively moderately and hardly internalized. When the absolute amount of internalized MoAb was calculated, a highly significant correlation with cytotoxicity was found. We conclude that the degree of antigen expression is not so important as the absolute amount of antibody internalized in predicting the efficacy of ITs.
The mixed epidermal cell lymphocyte‐reaction is the most predictive factor of acute graft‐versus‐host disease in bone marrow graft recipients
Authors: Bagot Martine , Mary Jean‐Yves , Heslan Michèle , Kuentz Mathieu , Cordonnier Catherine , Vernant Jean‐Paul , Dubertret Louis , Levy Jean‐Paul
Published: March 2008

Risk factors for acute graft‐versus‐host disease (GvHD) remain controversial. We performed uni‐ and multivariate statistical analyses on a series of 37 patients receiving a non‐depleted allogeneic bone marrow transplant from an HLA‐identical sibling donor for a haematological malignancy, in order to identify risk factors for GvHD. Three factors were associated with development of moderate to severe GvHD: a positive mixed epidermal cell‐lymphocyte reaction (MECLR) between donor and recipient, previous pregnancies in female donors and chronic myeloid leukaemia diagnosis. The MECLR was the most important predictive factor, selected in first rank by the stepwise linear discriminant analysis. Combining these three prognostic factors in the jackknifed procedure, we could correctly classify 33/37 patients in two groups: grade 0–I versus grade II–IV acute GvHD. These results should apply to donor selection and to predict donor/recipient pairs at high risk of GvHD who might benefit of bone marrow T‐cell depletion and those at low risk for whom depletion could be avoided.
An acquired Bernard‐Soulier‐like platelet defect associated with juvenile myelodysplastic syndrome
Authors: Berndt Michael C. , Kabral Arnold , Grimsley Phillip , Watson Neville , Robertson Thomas I. , Bradstock Kenneth F.
Published: March 2008

Summary Bernard‐Soulier syndrome is an inherited bleeding abnormality characterized by thrombocytopenia with large platelets and deficiency of the platelet membrane glycoprotein (GP) Ib‐IX complex. We have identified a young female with an acquired Bernard‐Soulier‐like platelet defect and a coexisting primary myelodysplastic disorder. Abnormal bruising had developed at age 5. A normal platelet count with some giant platelets was noted at age 7. At age 9 she developed a large haematoma following surgery. Laboratory investigation revealed thrombocytopenia and large platelets. Platelet membrane glycoprotein analysis showed a marked deficiency of the components of the GP Ib‐IX complex (25% of normal). Flow cytometry revealed two populations of platelets: a predominant population of large platelets lacking the GP Ib‐IX complex and a minor population of normal‐sized platelets with normal GP Ib‐IX expression. The patient developed progressive anaemia, more severe thrombocytopenia and neutropenia, and circulating blast cells were seen. A bone marrow showed gross hypercellularity with marked dysplasia of all three lineages and increased blasts. Marrow cytogenetic studies showed the presence of monosomy 7 in all metaphases, with an additional trisomy 21 in 10%. Peripheral blood cells were normal 46XX. The above data are consistent with an acquired myelodysplastic syndrome associated with a Bernard‐Soulier‐like platelet defect.
Human anti‐PlEl antibody recognizes epitopes associated with the alpha subunit of platelet glycoprotein Ib
Authors: Furihata Kenichi , Hunter Jay , Aster Richard H. , Koewing Gale R. , Shulman N. Raphael , Kunicki Thomas J.
Published: March 2008

Summary Together, a platelet‐reactive antibody in the serum of a polytransfused patient (proband) and a plateletreactive antibody in the serum of a mother of an infant with neonatal thrombocytopenia have served to establish the diallelic, platelet‐specific alloantigen system, PlE. We now provide evidence that the platelet‐specific antibody in the serum of the proband, anti‐PlEl, recognizes epitopes associated with the alpha subunit of glycoprotein (GP) Ib. By 51Cr release, platelets from two of three patients with the Bernard‐Soulier syndrome (BSS) responded sub‐normally to anti‐PlEl, and the apparently normal response of platelets from the last BSS patient was attributable to anti‐HLA‐A2 antibodies in the proband serum. These results suggested that the PlEl antigen is associated with the GPIb complex (glycoproteins Ib+IX) known to be absent from BSS platelets. This possibility was confirmed by ELISA using the purified GPIb complex or glycocalicin, the N‐terminal fragment of GPIb alpha produced by proteolysis with endogenous platelet calpain. as solid‐phase antigen. Anti‐PlEl antibody bound specifically to both the GPIb complex and glycocalicin. 3H‐labelled platelet membrane glycoproteins with apparent molecular weights of 130k, 25k, and 21k (under reduced conditions) corresponding to GPIb alpha, GPIb beta, and GPIX were immunoprecipitated by anti‐PlEl plasma. Finally, at a titre of 1:16, anti‐PlEl completely inhibited ristocetin‐induced platelet agglutination, a property of platelets mediated by GPIb.
Red cell aggregation during normal pregnancy
Authors: Huisman A. , Aarnoudse J. G. , Krans M. , Huisjes H. J. , Fidler V. , Zijlstra W. G.
Published: March 2008

Summary Red cell aggregation (RCA) is responsible for the increase in whole blood viscosity at lower shear rates. RCA depends on the concentrations of red cells and plasma proteins with a high molecular weight and a large and asymmetrical spatial structure such as fibrinogen, immunoglobulin M and α2‐macroglobulin. During normal pregnancy, changes occur in all these concentrations. In a prospective study these changes and their influence on the resulting RCA were investigated in 24 healthy women with normal pregnancies. RCA was determined by light reflection measurement (syllectometry).

RCA considerably increased during normal pregnancy in spite of the physiological haemodilution. The aggregation half time, used as a measure for RCA, decreased from an average non‐pregnant value of 5.6 s to 3.3 s at 37 weeks. Multiple regression analysis showed that the increase in RCA could be mainly attributed to the raised fibrinogen concentration. However, at 37 weeks other factors, in addition to fibrinogen, contribute significantly to the increase in RCA.
Biochemical studies on McLeod phenotype red cells and isolation of Kx antigen
Authors: Redman C. M. , Marsh W. L. , Scarborough A. , Johnson C. L. , Rabin B. I. , Overbeeke M.
Published: March 2008

Summary Red cells of the McLeod blood group phenotype have weak Kell antigens, lack Kx antigen and have acanthocytic morphology. We have immunoprecipitated Kell antigens from McLeod red cells and show that they are markers on the same 93 kDa membrane protein that carries Kell antigens on normal red cells. However, as determined by Western immunoblotting, McLeod red cells have a marked deficiency of this protein. We have also studied the near‐neighbour relationship of McLeod and common Kell red‐cell membrane proteins by cross‐linking intrinsic sulphydryl groups by oxidation, catalysed with orthophenanthroline and copper, or by cross‐linking amino groups with dimethyl 3,3‐dithiobispropionimidate. Results were analysed by diagonal mapping in two‐dimensional gels. No abnormalities of membrane protein inter‐relationship were detected in McLeod red cells.

We have isolated Kx antigen from K0 red cells by immuno‐precipitation with human alloimmune anti‐Kx serum, isolation of immune complexes from detergent‐solubilized cell membranes with protein A‐Sepharose and analysis of the eluted immune complex by SDS‐PAGE under reducing conditions. Kx antigen is a marker on a red‐cell membrane protein of approximately 37 kDa. K0 (Knull) red cells have about twice the amount of Kx antigen as do red cells of common Kell type. McLeod red cells have no detectable Kx antigen by serological tests or by immunoprecipitation.
Beneficial effect of heparin in the management of patients with APL
Authors: Hoyle C. F. , Swirsky D. M. , Freedman L. , Hayhoe F. G. J.
Published: March 2008

Summary 115 patients with acute promyelocytic leukaemia (APL) were studied retrospectively to evaluate prognostic factors and assess therapeutic approaches, particularly the use of heparin in the management of disseminated intravascular coagulation (DIC). The remission rate was 86% (30/35 patients) in those who received heparin and 49% (39/80 patients) in those who received no heparin (P= 0.0002). This difference in remission rates was accounted for by a marked decrease in the number of haemorrhagic deaths, especially those due to intracranial haemorrhage (ICH), in the heparin treated group. Other factors associated with a poor remission rate were prothrombin ratio (PTR) > 1.3 (P=0.008), fibri‐nogen < 1.5 g/l (P=0.02) and WCC >2.0x109/1 (P=0.03).
A phorbol ester tolerant (PET) variant of HL‐60 promyelocytes
Authors: Macfarlane Donald E. , Gailani David , Vann Kenneth
Published: March 2008

Summary The promyelocytic leukaemia cell line HL‐60 differentiates to a macrophage‐like cell when exposed to the phorbol ester 12–0‐tetradecanoylphorbol 13‐acetate (TPA) and other agents which activate protein kinase C. To investigate this phenomenon we developed an HL‐60 variant which does not differentiate when exposed to TPA. HL‐60 cells were exposed to the mutagen ethyl methanesulphonate and were cloned in soft agar in the presence of a normally lethal concentration of TPA. One colony of cells that proliferated in TPA was obtained. The cells of this phorbol ester tolerant (PET) line have retained their resistance to TPA for several years without selective pressure. They are some‐what larger than their phorbol ester sensitive (S) parent, but they are otherwise morphologically similar.

When PET‐cells are exposed to TPA their growth is arrested for approximately 48 h. Thereafter, they resume their original rate of replication at all concentrations of TPA tested. S‐cells undergo changes typical of HL‐60 when exposed to TPA; they aggregate, stop growing, adhere to the flask and die. The PET‐cells appeared to be as sensitive as S‐cells to other agents which differentiate HL‐60 such as retinoic acid, dimethysulphoxide, and 1,25‐dihydroxyvitamin D3, as determined by rate of proliferation in culture, Wright's stain, nitroblue tetrazolium reduction, and induction of the ectoenzyme NAD‐glycohydrolase.

TPA‐induced protein phosphorylation was studied using one‐and two‐dimensional polyacrylamide gel electrophoresis. Several proteins increased their incorporation of 32P when S‐ and PET‐cells were exposed to TPA, the most prominent of which were the two previously described nuclear matrix proteins of 80 kd and 33 kd. There was no difference in the protein phosphorylation pattern in S‐ and PET‐cells,3 nor in how this pattern changed on TPA exposure.

Fluorescent activated cell sorting and karyotypic analysis revealed PET‐cells to be a hypotetraploid variant of S‐cells, with approximately 80 chromosomes, including a marker chromosome iso(1p) not found in the S‐cells.

Identification of the biochemical lesion responsible for this TPA resistance in PET cells will provide clues concerning the mechanism of this important pathway for the induction of cell differentiation.
Bleeding tendency caused by IgG inhibitor to factor XIII, treated successfully by cyclophosphamide
Authors: Nakamura Satoko , Kato Atsushi , Sakata Yoichi , Aoki Nobuo
Published: March 2008

Summary A case exhibiting bleeding tendency caused by an acquired inhibitor to factor XIII is reported. The patient, a hitherto healthy 87‐year‐old Japanese man, presented with a massive subcutaneous bleeding, leading to severe anaemia. The routine coagulation study was normal except for a decreased plasma factor XIII level, which was 3% of the control level by the dansylcadaverine incorporation assay. An inhibitor of factor XIII was demonstrated to be present in the IgG fraction of the patient's plasma; by immunoblotting this inhibitor was shown to bind specifically the a and a ‘subunits of factor XIII. The IgG fraction suppressed the transglutaminase activity of activated factor XIII, but did not inhibit the molecular transformation of subunit a to a’ in the activation process. Massive infusion of plasma and factor XIII concentrate was effective for controlling the bleeding temporarily. In the long‐term prednisolone was ineffective for suppressing the plasma inhibitor level and bleeding episodes recurred. A small daily dose (50 mg) of cyclophosphamide, however, effectively decreased the inhibitor level and controlled bleeding.