Artificial surface effect on red blood cells and platelets in laminar shear flow

Red blood cell (RBC) effects on platelet adhesion to a nonbiologic test surface (tetrafluoroethylene propylene copolymer) and platelet aggregation during laminar shear flow for shear rates to 5,680 s-1 (corresponding to shear stress to 200 dyne/cm2) were investigated. Results on hemoglobin (Hb) and adenosine diphosphate (ADP) release from RBCs, percent decrease of single platelets in the bulk, and percent of test surface covered with platelets were obtained in a cone-and-plate (CP) viscometer for samples of whole blood, suspensions of RBC ghosts in platelet-rich plasma (PRP), and suspensions of RBCs in either PRP or platelet-poor plasma. Results obtained over the shear rate range studied for samples of normal hematocrit indicated that low-stress shearing led to ADP and Hb release from intact RBCs; shear-induced release of ADP from RBCs was about twice that of platelets, and of the total ADP released, the ADP released from RBCs contributed about six times that of the platelets to single platelet reduction in the bulk and about twice that of the platelets to platelet adhesion, ie, coverage of the test surface with platelets. Results obtained for various hematocrits showed that above a threshold hematocrit of about 25% to 35% the RBCs (suspended in PRP) had a greater contribution to ADP release, platelet adhesion, and platelet aggregation than the platelets themselves. Single platelet reduction for samples of RBC ghosts suspended in PRP correlated with shear rate level and not with shear stress.     

A K-ras oncogene increases resistance to sulindac-induced apoptosis in rat enterocytes

BACKGROUND & AIMS: Mutations of c-K-ras occur commonly in colonic neoplasms. The aim of this study was to determine how c-K-ras mutations alter the responses to the chemopreventive agent sulindac. METHODS: The parental rat intestinal cell line IEC-18 and c-K-ras-transformed derivatives were treated with sulindac sulfide. Cell cycle distribution was determined by flow-cytometric analysis (fluorescence-activated cell sorter), apoptosis by DNA fragmentation (laddering), flow cytometry, and microscopy, and changes in gene expression by immunoblotting. RESULTS: Sulindac sulfide inhibited cell growth and induced apoptosis in a time- and dose-dependent manner more rapidly in and at lower concentrations in parental cells than ras-transformed cells. Expression of the sulindac sulfide arrested cells in G0/G1, but cells entered apoptosis throughout the cell cycle. Proapoptotic protein Bak was relatively high in untreated parental cells and increased markedly after sulindac sulfide but was low in untreated ras-transformed cells and did not increase after sulindac sulfide. Expression of other Bcl-2 family members was unchanged after sulindac sulfide. However, sulindac sulfide reduced levels of cyclin D1 protein and cyclin E- and cyclin D1- associated kinase activity. CONCLUSIONS: c-K-ras-transformed enterocytes are relatively resistant to sulindac sulfide-induced growth inhibition and apoptosis, which may result from specific reduction of bak expression. (Gastroenterology 1997 Dec;113(6):1892-900)     

Phorbol ester stimulation increases sickle erythrocyte adherence to endothelium: a novel pathway involving alpha 4 beta 1 integrin receptors on sickle reticulocytes and fibronectin

Sickle-cell adherence to endothelium has been hypothesized to initiate or contribute to microvascular occlusion and pain episodes. Adherence involves plasma proteins, endothelial-cell adhesion molecules, and receptors on sickle erythrocytes. It has previously been reported that sickle reticulocytes express the alpha 4 beta 1 integrin receptor and bind to cytokine-activated endothelium via an alpha 4 beta 1/vascular- cell adhesion molecule-1 (VCAM-1) interaction. To elucidate other roles for alpha 4 beta 1 in sickle-cell adherence, the ability of activated alpha 4 beta 1 to promote adhesion to endothelium via a ligand different than VCAM-1 was explored. Adherence assays were performed under dynamic conditions at a shear stress of 1 dyne/cm2. Preincubation of sickle erythrocytes with phorbol 12,13-dibutyrate (PDBu) increased adherence of sickle cells eightfold as compared with untreated sickle cells. Normal erythrocytes, whether treated with PDBu or not, did not adhere to the endothelium. Activating anti-beta 1 antibodies 4B4 and 8A2 also increased the adhesion of sickle, but not normal, red blood cell (RBC) adhesion to endothelium. Anti-alpha 4 antibodies HP1/2 and HP2/1, inhibitory antibody 4B5, or an RGD peptide inhibited sickle-cell adherence induced by PDBu. Additional studies were undertaken to examine if fibronectin, a ligand for activated alpha 4 beta 1, was involved in PDBu-induced sickle erythrocyte adherence. Adherence of PDBu-treated sickle cells was completely inhibited by the CS-1 peptide of fibronectin. Fibronectin was detected on the surface of washed endothelium using an antifibronectin antibody in enzyme-linked immunosorbent assays. Antifibronectin antibody pretreatment of endothelial cells inhibited PDBu-induced adherence by 79% +/- 17%. Incubation of sickle RBCs with exogenous fibronectin after PDBu treatment inhibited adherence 86% +/- 8%. Taken together, these data suggest that endothelial-bound fibronectin mediates adherence of PDBu- treated sickle cells. Interleukin-8 (IL-8), a chemokine released in response to bacterial infection, viral infection, or other injurious agents, and known to activate integrins, also increased adherence of sickle erythrocytes to endothelial cells via fibronectin. This novel adherence pathway involving sickle-cell alpha 4 beta 1 activated by PDBu or IL-8 may therefore be relevant in vivo at vascular sites that produce IL-8 or similar agonists in response to vascular injury or immune activation. These observations describe ways in which inflammation and immune responses cause vasoocclusive complications in sickle-cell disease.     

Overexpression of the major vault transporter protein lung-resistance protein predicts treatment outcome in acute myeloid leukemia

The monoclonal antibody LRP56 recognizes a 110-kD major vault protein (lung-resistance protein [LRP]) overexpressed in several P-glycoprotein- negative (Pgp-), multidrug resistant tumor cell lines. To determine the frequency of LRP overexpression, its prognostic significance, and its relation to Pgp, we analyzed bone marrow specimens from 87 consecutive patients with acute leukemia. Diagnoses included de novo acute myeloid leukemia (AML; 21 patients), leukemia arising from an antecedent hematologic disorder or prior cytotoxic therapy (secondary AML; 27 patients), AML in relapse (29 patients), and blast phase of chronic myeloid leukemia (CML-BP; 10 patients). A granular cytoplasmic staining pattern was detected by immunocytochemistry in 32 (37%) cases, including 7 (33%) de novo AML, 13 (48%) secondary AML, 11 (38%) relapsed AML, and 1 of 10 CML-BP. Among 66 evaluable patients with AML, LRP overexpression was associated with an inferior response to induction chemotherapy (P = .0017). Remissions were achieved in 35% of LRP+ patients as compared with 68% of LRP- patients. Although Pgp adversely affected response in univariate analysis (P = .0414), only LRP had independent prognostic significance when compared in a logistic regression model (P = .0046). Differences in remission duration (P = .075) and overall survival (P = .058) approached significance only for LRP. Sequential specimens from remitting patients receiving treatment with the Pgp modulator cyclosporin-A showed emergence of the LRP phenotype despite a decrease or loss of Pgp at the time of treatment failure (P =.0304). Significant associations were observed between LRP and age greater than 55 years (P = .017), Pgp (P = .040), and prior treatment with mitoxantrone (P = .020) but not with CD34. These findings indicate that overexpression of the novel transporter protein LRP is an important predictor of treatment outcome in AML.     

Trisomy 3 in low-grade B-cell lymphomas of mucosa-associated lymphoid tissue

Characteristic chromosomal aberrations have been associated with subtypes of non-Hodgkin's lymphoma with distinct clinicopathologic features. Low-grade B-cell lymphomas of mucosa-associated lymphoid tissue (MALT) form such a group and might be expected to be characterized by a specific cytogenetic abnormality. Metaphase analyses of MALT lymphoma are rare due to problems with fresh tissue collection and poor in vitro proliferation. However, the small number of published series suggests that chromosome trisomies, particularly trisomy 3, might be characteristic of these tumors. The application of interphase cytogenetic techniques to routinely processed material allows the examination of a large series of archival cases and is particularly useful for the demonstration of chromosome trisomies. We have used this technique to analyze 70 cases of low-grade MALT lymphoma from various sites and found trisomy 3 in 60%. This finding compares with 16% in low- grade nodal B-cell lymphoma and 27% in primary splenic lymphoma of marginal zone type (splenic lymphoma with villous lymphocytes). These results provide further evidence that low-grade MALT lymphomas from all sites form a single pathologic entity distinct from nodal B-cell lymphomas. Although MALT lymphoma and primary splenic lymphoma may arise from marginal zone B cells, they are genetically distinct.     

Column agglutination technology: the antiglobulin test

A new system for typing and screening blood, based on the sieving effect of glass bead microparticles, has been developed. The test is performed in a microcolumn in which the red cell agglutinates are trapped in the glass bead matrix during centrifugation, and unagglutinated cells form a pellet at the bottom of the column. Anti- human globulin reagents were incorporated in the diluent and the new test system, column agglutination technology, was compared to conventional tube tests and low-ionic-strength method. Sera and plasmas (228 samples) were screened for red cell antibodies with two anti-human globulin reagents: one containing only anti-IgG and the other containing both anti-IgG and anti-C3b, -C3d. After initial testing, there was 94-percent agreement between column agglutination technology and tube tests, and after repeat testing, there was 97-percent agreement. The column agglutination technology anti-human globulin test eliminates the need to wash red cells, which decreases the overall test time. The test is easy to perform, and the results are more objective than those with tube and microplate methods.