ALBERT

Description: A 42-yr-old woman with systemic lupus erythematosus without bleeding diathesis developed a prolonged activated partial thromboplastin time that was not corrected by normal plasma. An inhibitor that acted rapidly and inactivated 0.5 U/ml plasma thromboplastin antecedent (PTA, factor XI) at a 1:200 plasma dilution was demonstrated. In addition to a low titer of PTA (less than 0.01 U/ml), plasma assayed at 20-fold dilution also showed low titers of Hageman (factor XII, 0.02 U/ml), Fletcher (plasma prekallikrein, 0.02 U/ml), and Fitzgerald (high molecular weight kininogen, less than 0.01 U/ml) factors. The titer of these factors, except PTA, returned to normal upon further plasma dilution or upon removal of the inhibitor by protein A adsorption. Thus, the inhibitor appeared to interfere with these clotting factor assays, possibly by inactivating PTA in the substrate plasmas in the test system. Its specificity was further confirmed. The inhibitor did not interfere with surface-induced proteolytic cleavage of Hageman factor. Surface-induced generation of plasma kallikrein activity (amidolysis of H-D-pro-phe-arg-pNa and cold-promoted factor VII activity enhancement) requires only Hageman, Fletcher, and Fitzgerald factors and was normal. Reactions requiring all 4 contact phase factors, including PTA, such as surface-induced generation of plasmin activity (amidolysis of H-D-val-leu-lys-pNa) and activated Christmas factor (factor IXa) activity, were defective. Furthermore, the inhibitor bound to agarose-protein A inactivated and removed PTA selectively from normal plasma. The inhibitor was an IgG-lambda autoantibody that precipitated PTA. The inactivated activated PTA (factor XIa) without the requirement for an additional cofactor. Furthermore, it inhibited surface-induced activation of PTA by interfering with its proteolytic cleavage upon glass surface exposure and with its binding onto the reactive surfaces.

Description: A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

Description: The effect of differentiation induction by a tumor-promoting phorbol diester, 12-O-tetradecanoylphorbol-13-acetate (TPA) on a clonal human megakaryoblastic cell line, MEG-O1s, was investigated, and a prominent response was demonstrated. The cells became weakly adherent, developed conspicuous cytoplasmic blebs, and displayed mature megakaryocytic characteristics by light microscopy such as the development of azurophilic cytoplasmic granules and a mosaic pattern of oxyphilic patches, multiplication of nuclei, and enhancement of the PAS reaction and alpha-naphthyl acetate esterase staining. Ultrastructural studies demonstrated the development of prominent cytoplasmic blebs, budding of blebs, and multiplication of nuclei. Numerous granules with central nucleoids that are similar to alpha granules had developed as well as granules with high electron density and clearly demarcated zones. Surface marker analysis revealed a moderate increase in IgG Fc receptor levels and a profound decrease in C3 receptor sites. By an immunofluorescent technique using monoclonal and polyclonal antibodies, a dramatic change in the expression of several megakaryocyte-platelet- specific proteins was demonstrated. All the proteins that had been expressed before induction such as platelet glycoprotein (GP) IIb/IIIa, fibrinogen, von Willebrand factor, factor XIIIA, beta-thromboglobulin (beta-TG), and HLA class 1 antigen were profoundly enhanced after induction by TPA. Induction by TPA led to the expression of fibronectin and factor V, which were not detected on nontreated cells. An ultrastructural immunoperoxidase study demonstrated platelet GPIb and GPIIb/IIIa in both plasma membranes and protein synthesis areas such as perinuclear cisternae and endoplasmic reticulum after TPA induction. beta-TG was also observed in some cytoplasmic granules of TPA-treated cells. TPA remarkably increased the secretion of beta-TG into the culture medium of MEG-01s. Ploidy was also increased from 2C to 4C to 4C to 8C. Similar maturation of MEG-01s was induced by other phorbol diester analogues such as phorbol-12,13-dibutyrate, but not by phorbol itself. These results indicate that phorbol diester, TPA, can bring about differentiation and maturation of a human megakaryoblastic cell line (MEG-01s) and that MEG-01s cells will provide a useful model for studying megakaryocytic differentiation and numerous megakaryocyte- platelet-specific proteins.

Description: Four distinct intragenic polymorphisms in the coagulation factor IX gene which have been reported to be important for family diagnosis of Caucasian hemophilia B were studied in 51 normal Japanese subjects (21 males and 30 females). High-molecular-weight DNA prepared from peripheral blood lymphocytes were digested with endonuclease, Ddel, Mspl, Taql or Xmnl, and were studied by Southern blot analysis with factor IX complementary DNA as a probe. None of the minor fragments produced by these enzymes was found in the normal Japanese DNA samples tested, although the probe detects minor allelic forms in control Caucasian DNA samples. Our data suggest that the frequent polymorphic sites found in Caucasians are possibly absent in the Japanese population.

Description: A murine hybridoma cell line that produces a monoclonal antibody to human Hageman factor (HF, factor XII) is described. The antibody (P 5–2– 1) consists of mouse IgG2b heavy chains and lambda light chains, selectively neutralizes HF procoagulant activity, and prevents the proteolytic cleavage of HF during contact activation in plasma. When HF is exposed to P 5–2–1 before the absorption of HF to kaolin, HF procoagulant activity is markedly inhibited. In contrast, P 5–2–1 does not interfere with HF activity after the adsorption of HF to kaolin. P 5–2–1 does not inactivate the prekallikrein–activating activity of 28,000–mol wt HF fragments (HFf). P 5–2–1 binds exclusively to the 40,000mol wt portion of a heavy chain of HF and inhibits the adsorption of HF to negatively charged surfaces. P 5–2–1 immobilized on Sepharose can be used to deplete HF from normal human plasma. This immunoaffinity-depleted plasma is indistinguishable from congenital HF- deficient plasma and can be used as the substrate for HF procoagulant activity assay.

Description: A specific, sensitive, and reproducible radioimmunoassay for human plasma thromboplastin antecedent (PTA, factor XI) has been developed with purified PTA and monospecific rabbit antiserum. Precise measurements of PTA antigen were possible for concentrations as low as 0.3% of that in normal pooled plasma. Normal plasma contained approximately 6 microgram PTA/ml. A good correlation (correlation coefficient 0.68) existed between the PTA procoagulant assays and radioimmunoassays among 50 normal adults (25 males and 25 females). PTA antigen was markedly reduced in plasma of 13 patients with congenital homozygous PTA deficiency (range less than 0.003–0.128 U/ml) and 9 patients with hepatic cirrhosis (0.35+/-0.17 U/ml), but was normal in those of 9 patients under treatment with warfarin, 8 patients with disseminated intravascular coagulation and 16 patients with other congenital clotting factor abnormalities, including prekallikrein deficiency (Fletcher trait) and high molecular weight kininogen deficiency (Fitzgerald trait).

Description: A case of WT31-, CD3+ large granular lymphocyte leukemia is reported. On surface marker analysis, the proliferating cells were found to be CD3+4–8–16+ and WT31-. By two-color immunofluorescence staining, CD3+4- 8- cells were found to be WT31-, and a small population of WT31+ cells expressed either CD4 or CD8. WT31-, CD3+ cells were also identified in a bulk culture of lymphocytes expanded in vitro. Because WT31 monoclonal antibody (MoAb) reacts with the nonpolymorphic epitope of the disulfide-linked heterodimer of the T cell antigen receptor (Ti), the absence of the WT31-reactive Ti determinant may represent an expression of different CD3-associated polypeptides. The rearrangement of the Ti-beta and Ti-gamma genes but not the immunoglobulin gene was demonstrated, and the single pattern of rearrangement indicated the monoclonal origin of the lymphocytes. When the lymphocytes were assayed for their cytotoxicity against K562, MOLT-4, Daudi, and Raji tumor cell lines, a broad spectrum of cytotoxicity for these tumor cells was observed, and the lymphocytes also exhibited antibody- and lectin- dependent cellular cytotoxicity and lymphokine-activated killer activity. Treatment with anti-CD2 and anti-CD3 MoAbs inhibited their nonspecific cytotoxicity. The anti-CD3-mediated inhibition of nonspecific cytotoxicity suggested that an as yet unidentified Ti, present in association with the CD3 molecule on these lymphocytes, serves as a specific receptor for target tumor cell recognition.

Description: The alteration of p53 tumor suppressor gene was studied in 48 patients with B-cell lymphoma. A sequential combined technique of polymerase chain reaction-mediated single-strand conformational polymorphism (PCR- SSCP) or reverse transcription (RT)-PCR-SSCP and direct sequencing were used as a simple and sensitive approach to analyze nucleotide changes. By these methods, we identified 8 missense point mutations and 2 codon deletions in 9 of the 48 patients. These mutations were located in or close to the evolutionally highly conserved regions of the p53 gene. Eight of nine patients having p53 gene alterations were in advanced clinical stage (IV). It is the first report of p53 gene mutations in follicular and diffuse lymphoma. These observations suggest that the p53 gene alteration may play an important role in lymphomagenesis and/or disease progression in some types of B-cell lymphoma.

Description: The early event of thymocyte maturation has been analyzed using acute lymphoblastic leukemia (ALL) cells. A group of ALL cells whose cell surface phenotype was CD2 (SRBC receptor) negative and CD7 (T cell antigen) positive has been considered as precursor thymocyte ALL (pre-T- ALL). No rearrangements of the T cell receptor beta-gene (TCR beta) and gamma-gene (TCR gamma) were found in three of four pre-T-ALL patients. Stimulation of these pre-T-ALL cells with 12–0-tetradecanoylphorbol-13- acetate (TPA) induced only CD25 (Tac) antigen but no other T cell antigens. These findings suggest that the activation pathway of interleukin 2 (IL-2) receptor already exists in the most immature precursor thymocytes. Pre-T-ALL cells from the fourth patients showed the expression of CD3 antigen, and both TCR beta and TCR gamma rearrangement. TPA induced the differentiation of the more mature pre-T- ALL cells of this case in vitro, and not only CD25 (Tac) antigen but also CD4 and CD8 antigens appeared on the cell surface. The low affinity binding of 125I-IL-2 to TPA-stimulated leukemia cells was observed in the three cases of pre-T-ALL tested, and the addition of recombinant IL-2 to TPA-stimulated cells showed no effect on cell proliferation.

Description: Clonality of marrow hematopoietic progenitor cells in myelodysplastic syndromes (MDS) was analyzed by X-chromosome inactivation pattern using polymerase chain reaction (PCR). Five female patients were included in this study; two with refractory anemia (RA) and three with RA with excess blasts (RAEB). They were heterozygous for BstXI restriction fragment length polymorphisms (RFLP) of the X-chromosome-linked phosphoglycerate kinase (PGK) gene. In each patient, erythroid and nonerythroid colonies, grown in the presence of erythropoietin and granulocyte-macrophage colony-stimulating factor (GM-CSF), exhibited no remarkable difference in clonal constitution. Two patients showed only one methylation pattern, suggesting the monoclonal origin of hematopoietic progenitor cells. Colonies of two other patients exhibited predominant and minor methylation patterns in PGK gene, indicating that nonclonal progenitor cells remain a minor population. The bone marrow of one patient appeared to contain a greater proportion of nonclonal progenitors. Stem cell factor (SCF), a potent colony- stimulating factor, enhanced both erythroid and nonerythroid colony formation. However, it did not notably alter the clonal constitutions. We conclude that nonclonal hematopoietic progenitor cells can persist in a substantial number of MDS patients.