Heparin oligosaccharides bind L- and P-selectin and inhibit acute inflammation

Initial attachment of leukocytes to the vessel wall at sites of inflammation is supported by a family of carbohydrate-binding adhesion molecules called the selectins. Selectin ligands include sialyl-Lewis x (sLex, Neu5Ac alpha 2-3Gal beta 1-4[Fuc alpha 1-3]GlcNAc--) and related structures. We report here that defined heparin oligosaccharides interact with the selectins. Heparin chains containing four or more monosaccharide residues inhibited the function of L- and P-selectin, but not E-selectin, in vitro. In a competition enzyme-linked immunosorbent assay measuring inhibition of solution-phase selectin-Ig fusion proteins (selectin-Ig) binding to immobilized bovine serum albumin-sLex neoglycoprotein, a heparin-derived tetrasaccharide mixture inhibited 50% of L- and P-selectin-Ig binding (IC50) at 200 +/- 40 mumol/L and 850 +/- 110 mumol/L, respectively. A single hexasulfated tetrasaccharide (delta UA2S alpha 1-4GlcNS6S alpha 1-4IdoA2S alpha 1- 4GlcNS6S) was particularly active against L- and P-selectin-Ig (IC50 = 46 +/- 5 mumol/L and 341 +/- 24 mumol/L). By comparison, the tetrasaccharide sLex was not inhibitory at concentrations up to 1 mmol/L. In cell adhesion assays, heparin tetrasaccharides reduced binding of neutrophils to COS cells expressing P-selectin but not to COS cells expressing E-selectin. They also blocked colon cancer cell adhesion to L- and P-selectin but not E-selectin. In a model of acute inflammation, intravenously administered heparin tetrasaccharides diminished influx of neutrophils into the peritoneal cavities of thioglycollate-treated mice. We conclude that heparin oligosaccharides, including non-anticoagulant tetrasaccharides, are effective L- and P- selectin inhibitors in vitro and have anti-inflammatory activity in vivo.     

Retroviral-mediated transfer and amplification of a functional human factor VIII gene

Hemophilia A results from a deficiency in factor VII (FVIII), a cofactor in the intrinsic pathway of blood coagulation. As an approach toward genetic therapy of this disease, we constructed a retroviral vector encoding human FVIII and a selectable and amplifiable genetic marker, human adenosine deaminase (Ada). A retrovirus packaging line was transfected with this vector and stable transformants were selected for Ada expression. Isolated transformants produced both FVIII activity in the conditioned medium and retrovirus capable of transferring the Ada selectable marker and FVIII expression to the mouse 3T3 fibroblasts. Selection of virus-producer cell lines for increasing levels of Ada expression yielded a 20-fold increase in both FVIII expression and viral titer. Similarly, selection of infected 3T3 fibroblasts for Ada gene amplification yielded a 20-fold increase in FVIII expression. The results demonstrate the feasibility of retrovirus- mediated transfer of human FVIII, and also the utility of selection for gene amplification to increase retrovirus titers in producer cell lines as well as expression levels in infected cells.     

Role of the B domain for factor VIII and factor V expression and function

Factor V and factor VIII are homologous cofactors in the blood coagulation cascade that have the domain structure A1-A2-B-A3-C1-C2, of which the B domain has extensively diverged. In transfected COS-1 monkey cells, expression of factor VIII is approximately 10-fold less efficient than that of factor V, primarily because of inefficient protein secretion and, to a lesser extent, reduced mRNA expression. To study the functional significance and effect of the B domain on expression and activity, chimeric cDNAs were constructed in which the B domains of factor V and factor VIII were exchanged. Expression of a factor VIII chimera harboring the B-domain of factor V yielded a fully functional factor VIII molecule that was expressed twofold more efficiently than wild-type factor VIII because of increased mRNA expression. Thus, sequences within the factor VIII B domain were not responsible for the inefficient secretion of factor VIII compared with factor V. Expression of a factor V chimera harboring the B domain of factor VIII was slightly reduced compared with wild-type factor V, although the secreted molecule had significantly reduced procoagulant activity correlating with dissociated heavy and light chains and resistance to thrombin activation. Interestingly, the factor V chimera containing the factor VIII B domain was efficiently activated by Russell's viper venum (RVV). A factor V B domain deletion (residues 710- 1545) molecule also exhibited significantly reduced procoagulant activity caused by resistance to thrombin cleavage and activation, although this molecule was activatable by RVV. These results show that, in contrast to factor VIII, thrombin activation of factor V requires sequences within the B domain. In addition, thrombin activation of factor V occurs through a different mechanism than activation by RVV.     

Positively selected autologous blood CD34+ cells and unseparated peripheral blood progenitor cells mediate identical hematopoietic engraftment after high-dose VP16, ifosfamide, carboplatin, and epirubicin

To investigate the feasibility of peripheral blood CD34+ cell selection and to analyze CD34+ cell-mediated engraftment after high-dose chemotherapy, we performed a phase I/II trial in 21 patients with advanced malignancies. The rationale for the selection of CD34+ cells from peripheral blood progenitor cell (PBPC) collections is based on the observation that contaminating tumor cells can be depleted approximately 3 logs using this procedure. CD34+ cells from chemotherapy+granulocyte colony-stimulating factor-mobilized PBPCs were positively selected with an avidin-biotin immunoadsorption column (CEPRATE SC system). One leukapheresis product with a median number of 2.8 x 10(6) CD34+ cells/kg was labeled with a biotinylated anti-CD34 monoclonal antibody and subsequently processed over the column. The yield of selected CD34+ cells was 73% +/- 24.6%. The purity of the CD34+ cell fraction was 61.4% +/- 19.7%. CD34+ cells were shown to represent predominantly committed progenitors coexpressing CD33, CD38, and HLA-DR molecules (lin+). They gave rise to myeloid as well as erythroid and multilineage colonies in vitro. In addition, positively selected CD34+ cells also comprised early hematopoietic progenitor cells, as shown by the presence of CD34+/lin- cells. Transfusion of positively selected CD34+ cells (2.5 x 10(6) CD34+/kg; range, 0.45 to 5.1) after high-dose VP16 (1,500 mg/m2), ifosfamide (12 g/m2), carboplatin (750 mg/m2), and epirubicin (150 mg/m2) (VIC-E) in 15 patients resulted in a rapid and stable engraftment of hematopoiesis without any adverse events. As compared with 13 historical control patients reconstituted with a comparable number of unseparated PBPCs, time to neutrophil and platelet recovery was identical in both groups (absolute neutrophil count > 500/microL, day + 12; platelet count > 50,000/microL, day + 15). These data indicate that autologous peripheral blood CD34+ cells and unseparated PBPCs mediate identical reconstitution of hematopoiesis after high-dose VIC-E chemotherapy. Because positive selection of CD34+ cells from mobilized blood results in a median 403-fold depletion of T cells, allogeneic CD34+ cells from mobilized blood should be investigated as an alternative to bone marrow cells for allotransplantation.     

Immunohistochemical characterization of a 183 KD myeloid-specific-DNA- binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1

A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias. Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood. In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus. Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative. BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors. A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas. M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia. Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative. Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation. Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA. Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein. These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.     

Antisickling effects of 2,3-diphosphoglycerate depletion

Elevation of 2,3-bisphosphoglycerate (2,3-DPG) in sickle erthrocytes (SS RBCs) and concomitant acidification of the cell interior promote polymerization by decreasing the solubility (csat) of deoxyhemoglobin S. The antisickling effect of 2,3-DPG depletion was evaluated after activation of the 2,3-DPG phosphatase activity of bisphosphoglycerate mutase by glycolate-2-phosphate, leading to rapid loss of intracellular 2,3-DPG. To ensure its maximal reduction in a physiologic medium, isosmotic CO2/bicarbonate-buffered saline, pH 7.0, was used. Substitution of K+ for Na+ as the major extracellular cation suppressed K:Cl cotransport, prevented cell shrinkage, and allowed demonstration of the full antisickling effect of 2,3-DPG depletion. The modest effect on solubility per se of removing intraerythrocytic 2,3-DPG (delta Csat = 1.6 g/dL) was amplified into a much larger antisickling effect by interaction with three other cellular variables affecting solubility and polymer content (intracellular pH, O2 saturation, and mean cell hemoglobin concentration). Acting in concert, these four antisickling effects (three solubilizing, one osmotic) reduced polymer fraction of glycolate-treated SS RBCs by 32% to 63%, with a concomitant decrease in sickling of 46% to 95% at the nominal pO2 of the microcirculation (20 mm Hg). A decrement in sickling of this magnitude should significantly ameliorate the vasoocclusive severity of sickle cell disease.     

Role of upstream DNase I hypersensitive sites in the regulation of human alpha globin gene expression

Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells.     

A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells

BACKGROUND & AIMS: Constitutive expression of cyclooxygenase 2 (COX-2) has been found in 85% of colorectal cancers. Ras mutations are found in 50% of colorectal adenocarcinomas. The aim of this study was to determine the role of COX-2 in ras-induced transformation in rat intestinal epithelial (RIE) cells. METHODS: Cell growth was determined by cell counts. The expression of COX-2 was examined by Northern and Western analyses. For tumorigenicity assays, cells were inoculated into dorsal subcutaneous tissue of athymic nude mice. DNA-fragmentation assays were performed to detect apoptosis. RESULTS: The expression of COX-2 was increased in RIE-Ras cells at both messenger RNA (9-fold) and protein (12-fold) levels. Prostaglandin I2 levels were elevated 2.15-fold in RIE-Ras cells. Serum deprivation further increased COX-2 expression 3.8-fold in RIE-Ras cells. Treatment with a selective COX-2 antagonist (SC58125) inhibited the growth of RIE-Ras cells through inhibition of cell proliferation and by induction of apoptosis. SC-58125 treatment reduced the colony formation in Matrigel by 83.0%. Intraperitoneal administration of SC-58125 suppressed RIE-Ras tumor growth in nude mice by 60.3% in 4 weeks. SC-58125 treatment also induced apoptosis in RIE-Ras cells as indicated by increased DNA fragmentation. CONCLUSIONS: Overexpression of COX-2 may contribute to tumorigenicity of ras-transformed intestinal epithelial cells. Selective inhibition of COX-2 activity inhibits growth of ras-transformed intestinal epithelial cells and induces apoptosis. (Gastroenterology 1997 Dec;113(6):1883-91)     

Extracellular truncations of h beta c, the common signaling subunit for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and IL-5, lead to ligand-independent activation

The hypothesis that extracellular truncation of the common receptor subunit for interleukin-3 (IL-3), granulocyte-macrophage colony- stimulating factor, and IL-5 (h beta c) can lead to ligand-independent activation was tested by infecting factor-dependent hematopoietic cell lines with retroviruses encoding truncated forms of h beta c. A truncation, resembling that in v-Mpl, and retaining 45 h beta c-derived extracellular residues, led to constitutive activation in the murine myeloid cell line, FDC-P1. However, infection of cells with retrovirus encoding a more severely truncated receptor, retaining only 7 h beta c- derived extracellular residues, did not confer factor independence on these cells. These experiments show that truncation activates the receptor and define a 37-amino acid segment of h beta c (H395-A431) which contains two motifs conserved throughout the cytokine receptor superfamily (consensus Y/H XX R/Q VR and WSXWS), as essential for factor-independent signaling. The mechanism of activation was also investigated in less severe truncations. A receptor that retains the entire membrane-proximal domain (domain 4) also conferred factor independent growth on FDC-P1 cells; however, a retrovirus encoding a truncated form of h beta c having two intact membrane proximal domains did not have this ability, suggesting that domain 3 may have an inhibitory role in h beta c. The ability of these receptors to confer factor independence was cell specific as demonstrated by their inability to confer factor-independent growth when introduced into the murine IL-3-dependent pro-B cell line BaF-B03. These results are consistent with a model in which activation requires unmasking of an interactive receptor surface in domain 4 and association with a myeloid- specific receptor or accessory component. We suggest that in the absence of ligand intramolecular interactions prevent inappropriate signaling.     

High-dose therapy and autologous bone marrow transplantation in patients with follicular lymphoma during first remission

We report the results of a study in previously untreated advanced stage patients with follicular lymphoma (FL) who underwent uniform induction chemotherapy with cyclophosphamide, doxorubicin, vincristine, prednisone (CHOP) followed by myeloablative therapy and anti-B-cell monoclonal antibody purged autologous bone marrow transplantation (ABMT). Eighty-three patients with previously untreated, low-grade FL were enrolled. After CHOP induction, only 36% achieved complete remission (CR) and 77 patients underwent ABMT. Before BM harvest, 70 patients had a known t(14;18), as determined by polymerase chain reaction (PCR), and all remained PCR positive in the BM at harvest. After ABMT, the disease-free survival (DFS) and overall survival are estimated to be 63% and 89% at 3 years, respectively, with a median follow-up of 45 months. Patients whose BM was PCR negative after purging experienced significantly longer freedom from recurrence (FFR) than those whose BM remained PCR positive (P = .0006). Continued PCR negativity in follow-up BM samples was also strongly predictive of continued CR. This study suggests that a subset of patients with advanced FL may experience prolonged clinical and molecular remissions following high-dose ablative therapy, although longer follow-up will be necessary to determine potential impact on overall survival.