Human factor VIII procoagulant activity and phospholipid interaction.

The interaction between purified human factor VIII and phospholipid vesicles was investigated. The binding of factor VIII to an equimolecular mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) was studied by sucrose gradient ultracentrifugation (10-40% w/v saccharose in 0.01 M Tris-HC1/0.15 M NaCl buffer (pH 7). In the absence of phospholipids all factor VIII activities (VIII R : WF and VIII R : AG) were found in the zone of highest sucrose density including the factor VIII related protein subunit (200 000 molecular weight). In the presence of an equimolecular mixture of PS/PC VIII R : WF activity, VIII R: AG and factor VIII related protein still migrated to the bottom of the tube, while VIII: C activity remained at the top where phospholipids were found. Thus a dissociation phenomenon between VIII : C and the other factor VIII related activities was apparent in the presence of phospholipids. These results also demonstrate the binding of factor VIII: C to certain active phospholipids.

Constitutive expression of GM-CSF mRNA by CML blast cells is correlated with endogenous megakaryocytic colony formation.

Purified blast cells from peripheral blood of 12 patients, with chronic myelogenous leukemia (CML) in chronic phase, were analyzed for the constitutive expression of the granulocyte-macrophage colony-stimulating factor (GM-CSF) transcript. Seven out 12 patients exhibited the specific 1.0 kb GM-CSF mRNA. Six from these patients presented an increased level of spontaneous megakaryocytic colony formation. Using an immunocytochemical procedure, the presence of GM-CSF was detected in a large proportion (52% to 72%) of the blast cells of the three patients studied, who were selected for the high expression of GM-CSF mRNA transcripts. Because the role of GM-CSF in the regulation of human megakaryocytopoiesis is well documented, we investigated the inhibiting effect of anti-GM-CSF antibodies on the spontaneous megakaryocytic colony growth of three of the patients expressing the GM-CSF transcript. Addition of anti-GM-CSF had a high neutralizing effect ranging from 60% to 70% inhibition of endogenous megakaryocytic colony growth. As the GM-CSF synthesis by leukemic cells is often induced by interleukin-1 (IL-1), we also investigated the effect of anti-IL-1 antibody on the spontaneous megakaryocytic colony growth of the same three patients. No significant inhibiting effect was observed, showing that the role of GM-CSF in the spontaneous colony formation is not mediated by IL-1. In addition, patients who constitutively expressed the GM-CSF transcript and showed endogenous megakaryocytic colony growth were those having a significantly higher platelet count than the group of patients without GM-CSF transcript and with no endogenous megakaryocytic colonies. In conclusion, these results suggest that GM-CSF, but not IL-1, participates in the production of spontaneous megakaryocytic colony formation observed in some CML patients. The true autocrine or paracrine nature of this stimulation remains to be established.

Resistance to CD95-mediated apoptosis through constitutive c-FLIP expression in a non-Hodgkin's lymphoma B cell line.

CD95 (Fas/Apo-1) is a transmembrane molecule that induces apoptosis and plays a central role in the regulation of the immune response. The present study describes two new B lymphoid cell lines, B593 and BR97, derived from non-Hodgkin's lymphoma, which differ in susceptibility to CD95-mediated apoptosis. While B593 cells are sensitive to CD95mediated apoptosis, BR97 cells are completely resistant. Activation of caspase-8 and caspase-3 proteases plays an important role in the CD95 signalling pathway. CD95 stimulation induced caspase-8 and caspase-3 activation in B593, but not in BR97 cells. However, activation of both caspase-8 and caspase-3 was achieved in BR97 cells treated with staurosporine. Furthermore, protein synthesis inhibition by cycloheximide restored sensitivity to CD95-mediated apoptosis and allowed activation of both caspase-8 and caspase-3 in BR97 cells. These results indicate that, in BR97 cells, both caspases are functional and suggest that CD95-apoptosis resistance may result from the presence of inhibitory factor(s). Constitutive high level expression of the apoptotic inhibitor c-FLIP was observed in the CD95-resistant BR97 cell line compared to B593. Moreover, downregulation of c-FLIP expression level by protein synthesis inhibition strictly correlated with restored sensitivity to CD95-mediated apoptosis in BR97 cells. Furthermore, we demonstrate that c-FLIP is recruited to the CD95 DISC in BR97 cells together with caspase-8 and FADD. The data presented in this study strongly suggests that, in a B-NHL-derived cell line, resistance to CD95-mediated apoptosis results from endogenous high level expression of apoptotic inhibitor c-FLIP.

Purification and cryopreservation of granulomonocytic colony forming cells (GM-CFC) from the blood of patients with chronic granulocytic leukemia (CGL) for autologous transplantation.

A simple density-cut separation technique is described for obtaining GM-CFC concentrates from the peripheral blood of patients with chronic granulocytic leukemia (CGL) for autografting at the acute blastic phase of the disease. Large numbers of granulomonocytic colony forming cells (GM-CFC) were recovered from a single procedure (68.4 X 10(6) GM-CFC). The cryopreservation of these concentrates in liquid nitrogen allowed the recovery of 46.6 +/- 33.8% viable GM-CFC. An improved yield of GM-CFC was obtained by avoiding washing the cells (65.2 +/- 41.1%). The separation technique resulted in a concentrated suspension of progenitors in a small volume of medium (mean = 15 ml) allowing a great reduction in the amount of the cryoprotective agent injected into the patient at autografting. Preliminary data obtained in 4 patients transfused in blastic crisis after chemotherapy, with or without TBI, indicated the capacity of stored concentrates to repopulate these patients.

Similarity of CSF of media conditioned by different human tissues.

Conditioned media prepared using human placenta, spleen, bone marrow and peripheral blood leucocytes revealed a common pattern of two distinct species of colony-stimulating factors (CSF) separable by gel filtration. The peak of greatest activity, active against both human and mouse marrow, had an apparent molecular weight (MW) of 24,000-28,000 Daltons. A peak of low activity detected only against mouse marrow had an apparent MW of approximately 150,000 Daltons. The type of progenitor cells stimulated by the crude medium, by the low MW CSF species, and by the high MW species were similar for the four conditioned media despite their different origins. No difference was found in either the MW of the human active CSF present or the type of progenitor cells stimulated by media conditioned with cells of leukemic origin.

Enrichment of high proliferation potential colony forming cells from mouse marrow by selecting low-density cells expressing receptors for wheat germ agglutinin.

Suspensions enriched for high proliferation potential colony forming cells (HPP-CFC's) were prepared from mouse marrow by selecting low-density cells stained with fluoresceinated wheat germ agglutinin on a fluorescence activated cell sorter. HPP-CFC's were 12-fold more concentrated in the final suspensions and co-enriched with a population of primitive CFUs detected in the spleens of irradiated mice reconstituted 13 days earlier with marrow. This is consistent with previous observations suggesting that these populations are closely related. The degree of enrichment for other haemopoietic progenitors was in the order HPP-CFC greater than day 8 CFUs greater than BFUe greater than GM-CFC greater than CFUe. "Extra" colonies developed when human placental conditioned medium (HPCM) was added to cultures of enriched suspensions already containing pregnant mouse uterus extract (PMUE). HPP-CFC's probably formed most of these and we discuss why counting these extra colonies may be more reliable than the conventional "size" assay for HPP-CFC's.

Biochemical fractionation of conditioned media containing factors which support in vitro proliferation of high proliferation potential colony forming cells.

Synergistic activity (SA), which is the capacity to support the in vitro proliferation of High Proliferation Potential Colony Forming Cells (HPP-CFC's), has been fractionated from conditioned media by standard biochemical techniques. On fractionation of both Wehi conditioned medium (Wehi CM) or human placental conditioned medium (HPCM) by several techniques, multiple peaks of SA were observed. Protein from HPCM expressing SA, ranged from 9,000-28,000 in molecular weight as determined by gel filtration. Chromatofocusing crude HPCM separated two activities possessing isoelectric points of 5.7 and 5.3. In crude Wehi CM, inhibitors or toxicity occasionally masked SA which partially adsorbed to Con-A sepharose and could be eluted with competing sugar. Further fractionation of this material on DEAE-sepharose separated three peaks of activity. Pooling the active fractions from the first two peaks gave a preparation that was up to 179 fold more active than the crude material.

Glanzmann thrombasthenia in children from Argentina.

PURPOSE: Glanzmann thrombasthenia is a well-defined inherited disorder of platelet function characterized by a decrease or absence of functional platelet glycoprotein (GP) GPIIbIIIa. The diagnosis must be considered in patients presenting with mucocutaneous bleeding, purpura, a normal platelet count, abnormal platelet aggregation, and a prolonged bleeding time. In most of the patients, the presence of small amounts of either GPIIb or GPIIIa was detected in their platelets. These observations could provide a basis for determining the clinical and laboratory heterogeneity of the disease. PATIENTS AND METHODS: We studied 10 patients of seven unrelated families with the usual methods and an immunoalkaline phosphatase technique (APAAP) to analyze the biosynthesis of GP in megakaryocytes. RESULTS: The results allowed us to classify six patients as GT type I, three as type II, and one as a variant. CONCLUSION: The nature and severity of the bleeding manifestations, in our patients, were not predictible by the laboratory findings. These confirm the clinical and laboratory heterogeneity of the disease.

cDNA clones for human platelet GPIIb corresponding to mRNA from megakaryocytes and HEL cells. Evidence for an extensive homology to other Arg-Gly-Asp adhesion receptors.

Platelet glycoprotein (GP) IIb is one of the two subunits of the common platelet adhesion receptor, GPIIb-IIIa. The isolation, characterization and sequencing of cDNA clones encoding for the two polypeptide chains of GPIIb are described. A number of clones were isolated from lambda gt11 libraries constructed with mRNA from an erythroleukemic cell line, HEL, and human megakaryocytes. Two of these clones, lambda IIb1, from HEL cells, and lambda IIb2, from megakaryocytes, cross-hybridized and were selected for detailed analysis. The identification of these as authentic GPIIb clones was based on immunological criteria and confirmed by the presence of nucleotide sequences in each insert encoding for known protein sequences of platelet GPIIb. These clones contained inserts of 1.54 kb and 1.39 kb, respectively, with an overlapping sequence of 801 bp. The nucleotide sequence of the overlapping region was identical indicating that HEL cells produce a protein closely related, if not identical, to platelet GPIIb. The determined nucleotide sequence of two inserts included a coding sequence for 648 amino acid residues, a TAG stop codon and 185 nucleotides of 3' non-coding sequence followed by a poly(A) tail. The coding sequence contained a portion of the heavy chain, the junction between the heavy and light chains and the entire light chain including a potential transmembrane-spanning domain and a short cytoplasmic tail. When these cDNA were used to probe for GPIIb mRNA, a single mRNA species of 3.9 kb was identified in both HEL cells and human megakaryocytes. A comparison of the deduced amino acid sequence for GPIIb with those of the alpha subunit of the vitronectin and the fibronectin receptors revealed extensive homologies. These homologies further establish that GPIIb-IIIa from platelets, together with the vitronectin and the fibronectin receptors, are members of a supergene family of adhesion receptors with a recognition specificity for Arg-Gly-Asp amino acid sequences.

Correlation between apoptosis microarray gene expression profiling and histopathological lymph node lesions.

AIMS: Microarray technology has recently led to the identification of molecular prognostic subgroups in non-Hodgkin's lymphomas. To determine the usefulness of ready made macroarrays as routine diagnostic tools in haematopathology, lymph node biopsies were analysed using a cDNA macroarray containing genes involved in apoptosis, including caspases. METHODS: Nine biopsy specimens were analysed using total frozen tissues: four samples of B cell follicular lymphoma, two of B cell diffuse large cell lymphoma, and three of non-neoplastic lymph nodes from benign lymphadenitis. Nine cell populations were sorted from fresh tissues: malignant B cells from two patients with follicular lymphoma and two with diffuse large cell lymphoma, reactive B cells from two benign lymph nodes, reactive T cells from one benign lymph node, and virgin (mantle zone) B cells and germinal centre B cells from benign tonsils. Immunohistochemistry (IHC) on paraffin wax sections was performed for the localisation of caspases 2, 3, 4, 7, 8, and 9. RESULTS: In the clustered array data, sorted cells from samples sharing common histological lesions were grouped together, whereas the array/histology correlation was less satisfactory for tissues. The expression profiles of both the array and IHC methods correlated for most caspases and samples. CONCLUSIONS: Variations in array profiles of sorted cell populations can be associated with specific histological features, suggesting a possible diagnostic application of ready made apoptosis macroarrays in haematopathology.

Role of autologous CD4+ T cell clones in human B non-Hodgkin's lymphoma: aborted activation and G1 blockade induced by cell-cell contact.

This article describes the study of the functional relationship between auto-tumor-reactive CD4(+) T cell clones (TCC) and autologous malignant B cells. Four auto-tumor-reactive CD4(+) TCC were derived from tumor-infiltrating T lymphocytes (TIL-T) from a freshly isolated human follicular lymphoma by the following technique: total CD4(+) TIL-T were negatively purified by an immunomagnetic procedure, then CD4(+) TCC were obtained by limiting dilution in the presence of IL-2 and autologous non-irradiated follicular lymphoma cells as feeders. After expansion, these CD4(+) TCC were co-cultured with non-irradiated autologous malignant B cells. All four TCC were activated by B lymphoma cells and proliferated, as assessed by CD25 expression and cell cycle analysis. Activation and proliferation of B lymphoma cells were studied in response to activated CD4(+) T cells. Although all four TCC were able to induce B lymphoma cell activation (Ki-67 antigen induction and CD40 up-regulation), cells were subsequently blocked in G1 phase. Activation of B-NHL cells was mediated by TCR-HLA class II interaction, as shown by a blocking experiment using an anti-CD4 monoclonal antibody (mAb). Since anti-CD40 mAb with or without IL-4 did not induce proliferation of B lymphoma cells in contrast to normal B cells, we suggest that the blockade in G1 phase is due to the presence of abnormalities in B lymphoma cells. This is the first evidence that autologous reactive CD4(+) TCC can engage follicular lymphoma B cells to enter the cell cycle and induce an aborted activation stage.