Expression of CD27 and its ligand, CD70, on chronic lymphocytic leukemia B cells

Crosslinking the CD27 antigen on T cells provides a costimulatory signal that, in concert with T-cell receptor crosslinking, can induce T- cell proliferation and cellular immune activation. We find that chronic lymphocytic leukemia (CLL) B cells from most patients coexpress both membrane-bound and soluble CD27, along with its newly identified ligand, CD70. The expression of soluble CD27 may preclude leukemic B cells from stimulating T cells via CD70, thereby potentially impairing their ability to function as effective antigen-presenting cells. We find that leukemic B-cell expression of soluble and membrane-bound CD27 can be downmodulated through a CD40-dependent signal. This signal also induces enhanced expression of CD70 on both normal and leukemic B cells. We find that tumor necrosis factor (TNF)-alpha, or the Th1 cytokine interferon (IFN)-gamma, also can induce downmodulation of CD27, whereas Th2-associated cytokines interleukin-4 (IL-4) or IL-10 can enhance leukemic B-cell expression of this accessory molecule. The modulation of CD27 induced by these conditions is accompanied by reciprocal changes in the expression levels of CD70, suggesting that these accessory molecules may be engaged in reciprocal receptor-ligand downmodulation. Consistent with this, we observe that co-culture of CLL B cells with transfected murine plasmacytoma cells that express human CD70 affects downmodulation of CD27 and enhanced expression of CD70 on leukemic B cells, but does not affect expression of CD27 mRNA. However, we find that CD40-crosslinking, in addition to reducing the level of CD27 protein, also reduces leukemic B-cell expression of CD27 mRNA. This argues that the changes in the expression levels of CD27 following CD40-signaling are not simply due to induced increases in the expression levels of CD70. Finally, we demonstrate that reciprocal changes in expression of CD27 and CD70 may contribute to the enhanced antigen-presenting capacity of CLL B cells after CD40-dependent leukemic B-cell activation. These findings expand the understanding of the regulation of costimulatory molecules important in antigen presentation and also have implications for the immunobiology of and therapy for CLL.     

Detection and viability of tumor cells in peripheral blood stem cell collections from breast cancer patients using immunocytochemical and clonogenic assay techniques [see comments]

Although peripheral blood stem cell collections (PBSC) are thought to have less tumor involvement than bone marrow (BM), the incidence of circulating tumor cells in patients with breast cancer has not been widely investigated. We prospectively investigated the incidence and viability of tumor cell involvement in PBSC and BM collections from breast cancer patients undergoing high-dose chemotherapy/hematopoietic stem cell transplantation. Paired samples of PBSC and BM from 48 patients were analyzed using an immunocytochemical technique that detects one epithelial-derived tumor cell per 5 x 10(5) mononuclear cells. Immunostained tumor cells were detected in 9.8% (13/133) PBSC specimens from 9/48 (18.7%) patients and in 62.3% (38/61) BM specimens from 32/48 (66.7%) patients, a significantly higher rate than in PBSC (P < .005). The geometric mean concentration of tumor cells in contaminated PBSC specimens was 0.8/10(5) mononuclear cells (range 0.33 to 2.0/10(5)) compared with 22.9/10(5) mononuclear cells in BM (range 1 to 3,000/10(5), P < .0001). In culture experiments, clonogenic tumor colonies grew in 21/26 immunocytochemically positive specimens. No tumor colony growth was detected in 30/32 immunocytochemically negative specimens. Immunocytochemical detection of tumor involvement in BM and PBSC correlated significantly with in vitro clonogenic growth (P < .0001). We conclude that PBSC contain fewer tumor cells than paired BM specimens from patients with advanced breast cancer and that these tumor cells appear to be capable of clonogenic growth in vitro.     

Visual identification of bacterially contaminated red cells

There have been increasing numbers of reports of transfusion-acquired Yersinia enterocolitica bacteremia (including several fatal cases). Fifteen units of whole blood were inoculated with various concentrations of Y. enterocolitica serotype 0:3 and processed into AS-3 preserved red cells (RBCs). Consistent growth of the organism was found at inoculum concentrations greater than or equal to 10 colony-forming units per mL. In all 13 units of RBCs that supported the growth of Y. enterocolitica, a darkening in color (due to hemolysis and a decrease in pO2) was observed in the bag. The attached sample segments, which were sealed from the main unit, remained sterile and did not darken. This color change was apparent in all the contaminated units by Day 35, which was 1.5 to 2 weeks after the bacteria were first detected in cultures of the blood. Hence, by comparison of the color of the segment tubing with that of the unit itself, units grossly contaminated with Y. enterocolitica can be identified prior to transfusion. Moreover, review of photographs on file at the Centers for Disease Control revealed this dramatic color change in 2 units of blood that caused transfusion-transmitted sepsis (Enterobacter agglomerans and an unidentified gram-negative bacillus, not Yersinia sp.), which demonstrated that the color change was not limited to Y. enterocolitica. This method of visual identification of contaminated units of blood could decrease the incidence of posttransfusion bacterial sepsis.     

Identification of Bakb, a new platelet-specific antigen associated with posttransfusion purpura

Baka is a platelet alloantigen whose putative allele, Bakb, has not been identified previously. By using a serum, "Har," obtained from a patient with posttransfusion purpura, we describe the platelet alloantigen Bakb. The Har serum reacted with an NP-40-extractable platelet membrane protein of 142 kd with mobility similar to platelet glycoprotein IIb alpha. We found that the antigen recognized by the Har serum is inherited in an autosomal dominant mode with an apparent gene frequency of .39. Chi-square analysis of observed and expected phenotype frequencies indicated that serum Har recognizes Bakb, the anticipated allele of Baka. Our findings provide new evidence for polymorphism of glycoprotein IIb and for the association of posttransfusion purpura with alloimmunization to determinants on this glycoprotein.     

Human platelet fibrinogen: purification and hemostatic properties

Conditions were developed in which 80% to 90% of platelet fibrinogen could be routinely purified in nondegraded form from the fluid phase of platelet suspensions stimulated with the calcium ionophore, A23187, in the presence of calcium, leupeptin, and prostaglandin E1. Fibrinogen was separated from other released proteins by chromatography on diethylaminoethanol (DEAE)-cellulose using a continuous pH and ionic strength gradient. Purified platelet fibrinogen, greater than 98% homogeneous by immunoelectrophoresis and sodium-dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), consisted of intact A alpha, B beta and gamma A chains, but not gamma' chains, and was 95% to 96% clottable. Platelet fibrinogen was shown to compete for the binding of radiolabeled plasma fibrinogen to ADP-activated platelets in a manner identical to that of unlabeled plasma fibrinogen itself. Also, at equivalent protein concentrations, platelet and plasma fibrinogens supported platelet aggregation to an equivalent extent. Based upon these results, we conclude that there is no significant difference between platelet and plasma fibrinogen with respect to their size, their clottability, their affinity for the activated platelet fibrinogen receptor, or their capacity to support subsequent platelet aggregation.     

Molecular cloning, chromosomal location, and tissue-specific expression of the murine cathepsin G gene

We previously have characterized a cluster of genes encoding cathepsin G (CG) and two other CG-like hematopoietic serine proteases, CGL-1 and CGL-2, on human chromosome 14. In this report, we clone and characterize a novel, related murine hematopoietic serine protease gene using human CG (hCG) cDNA as the probe. This murine gene spans approximately 2.5 kb of genomic DNA, is organized into five exons and four introns, and bears a high degree of homology to hCG at both nucleic acid (73%) and deduced amino acid (66%) levels. The predicted cDNA contains an open reading frame of 783 nucleotides that encodes a nascent protein of 261 amino acids. Processing of a putative signal (pre) peptide of 18 residues and an activation (pro) dipeptide would generate a mature enzyme of approximately 27 Kd that has an estimated pI of 12.0. Conserved residues at His44, Asp88, and Ser181 form the characteristic catalytic triad of the serine protease superfamily. The gene is tightly linked to the CTLA-1 locus on murine chromosome 14, where the serine protease genes mCCP1-4 are clustered. Expression of this gene is detected only in the bone marrow and is restricted to a small population of early myeloid cells. These findings are consistent with the identification of the gene encoding murine CG.     

Therapeutic efficacy of recombinant human leukemia inhibitory factor in a primate model of radiation-induced marrow aplasia

The therapeutic efficacy of recombinant human leukemia inhibitory factor (LIF) was examined in a nonhuman primate model of radiation- induced marrow aplasia. Rhesus monkeys received 450 cGy of total-body, 1:1 mixed neutron:gamma radiation. For 23 days thereafter, each monkey received a daily subcutaneous injection of LIF or human serum albumin (HSA) at a dose of 15 micrograms/kg body weight. Complete blood counts and white blood cell differentials were monitored for 60 days postirradiation. Administration of LIF significantly decreased (P < or = .05) the duration of thrombocytopenia (platelet count < 30,000 or 20,000/microL), ie, 9.3 days or 6.3 days, respectively, versus the HSA- treated control monkeys, 12.2 days or 10.2 days, respectively. Treatment with LIF did not alter the duration of neutropenia (absolute neutrophil count < 1,000/microL) as compared with the HSA-treated control monkeys. Cytokine administration did not exacerbate the radiation-induced anemia observed in the HSA-treated control monkeys.