Point mutations in the conserved box 1 region inactivate the human granulocyte colony-stimulating factor receptor for growth signal transduction and tyrosine phosphorylation of p75c-rel

The human granulocyte colony-stimulating factor receptor (hG-CSFR) belongs to the cytokine receptor superfamily. As with other members of this family, the cytoplasmic domain of hG-CSFR lacks intrinsic tyrosine kinase activity. To identify critical regions mediating growth signal transduction by hG-CSFR, deletions or site-directed amino acid substitutions were introduced into the cytoplasmic domain of hG-CSFR, and the mutant cDNAs were transfected into the murine interleukin-3 (IL- 3)-dependent Ba/F3 and FDCP cell lines. Truncation of the carboxy- terminal end of the receptor to the membrane-proximal 53 amino acids of the cytoplasmic domain, which retained the conserved Box 1 and Box 2 sequence motifs, decreased the ability of hG-CSFR to transduce G-CSF- mediated growth signals without an associated loss in receptor binding affinity. Substitution of proline by alanine at amino acid positions 639 and 641 within Box 1 completely abolished the G-CSF-mediated growth signal. Rapid induction of tyrosine phosphorylation of several cellular proteins, including a 75-kD protein (p75) identified as c-rel, was an early event associated with transduction of proliferative signals by hG- CSFR in Ba/F3 transfectants. Mutant receptors containing Pro-to-Ala substitutions that inactivated the receptor for mitogenic activity also inactivated the receptor for tyrosine-specific phosphorylation of p75. These results show that the conserved Box 1 sequence motif (amino acids 634 to 641) is critical for mitogenesis and activation of cellular tyrosine kinases by hG-CSFR.     

Pulmonary artery obstruction due to giant cell arteritis

Giant cell arteritis is often referred to in the context of polymyalgia rheumatica with temporal artery involvement. There are, however, more malignant forms of presentation of this necrotizing arteritis involving either the great vessels of the aorta or, occasionally, the pulmonary arteries. Our case relates to giant cell arteritis presenting as pulmonary artery obstruction in a patient without polymyalgia rheumatica or extensive aortic or proximal great vessel involvement.     

Immunolocalization of beta 1 integrins in platelets and platelet- derived microvesicles

Human platelets contain several adhesion receptors belonging to the integrin superfamily. At least three beta 1 integrins are present on platelets and have been shown to mediate platelet adhesion to collagen, fibronectin, and laminin. To study the cellular localization of the beta 1 integrins in platelets, we produced a polyclonal antibody by immunization of goat 172 with purified beta 1 subunit from HPB-ALL cells. Antibody 172 (Ab172) specifically immunoblotted a 135-Kd protein in a lysate of whole platelets. The reactivity of Ab172 with platelet membrane proteins was further determined by immunoprecipitation of lysates of surface-radioiodinated platelets. Ab172 immunoprecipitates, resolved by nonreducing/reducing two-dimensional sodium dodecyl sulfate- polyacrylamide gel electrophoresis consisted of three labeled proteins with migrational properties of platelet glycoprotein (GP)Ia, GPIc and GPIIa. Neither GPIIb/IIIa nor the vitronectin receptor were immunoprecipitated by Ab172, confirming a lack of cross-reactivity with the beta 3 integrins in platelets. Immunofluorescence studies using Ab172 were performed to investigate the cellular distribution of beta 1 integrins in platelets. Fluorescent labeling of intact cells demonstrated the presence of beta 1 antigen on the surface of resting cells. Permeabilization of platelets with Triton X-100 showed the presence of an intracellular pool of beta 1 antigen. Double-label experiments using Ab172 and AP-2 (anti-GPIIb/IIIa) showed identical labeling patterns, suggesting a similar subcellular distribution for these integrins. Following thrombin stimulation, permeabilized cells showed a centralized clearing of both beta 1 antigen and GPIIb/IIIa as well as an intensification of surface labeling for beta 1 antigen. These findings suggest the translocation of intracellular beta 1 antigen to the platelet surface as a result of thrombin stimulation. Because platelet-derived microvesicles have been reported to contain GPIIb/IIIa, we investigated the possible distribution of beta 1 integrins in these structures. Microvesicles, produced as a result of platelet activation, were labeled with Ab172, suggesting the distribution of beta 1 integrins in these structures as well as in intact cells.     

Loxoribine induces chronic lymphocytic leukemia B cells to traverse the cell cycle

Leukemic B cells from a majority of patients with chronic lymphocytic leukemia (CLL) enter the cell cycle upon stimulation in vitro with loxoribine, a potent 7,8-disubstituted guanine ribonucleoside immunostimulant. In the absence of added costimulants, a proportion of these cells become activated and undergo DNA synthesis and mitosis accompanied by a marked increase in expression of an array of cell surface activation antigens. The resultant activated B-CLL cells exhibit greatly enhanced sensitivity to cycle-active cytotoxic drugs. This approach may be of potential value in the therapy of CLL.     

Expression of CD33, CD38, and HLA-DR on CD34+ human fetal liver progenitors with a high proliferative potential

High proliferative-potential colony-forming cells (HPP-CFC) have been identified in the bone marrow of mice and adult humans, and have been characterized as a compartment of primitive progenitors possibly including stem cells. In this report we describe the human fetal liver (FL) as a source of HPP-CFC. These FL HPP-CFC develop in clonal cultures in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) within 3 to 4 weeks. The median frequency of HPP-CFC in FL tissues between 16 and 21 weeks of gestational age was 1 in 3,000 total FL cells. After 4 weeks of growth, FL HPP-CFC grew to a median colony size of 8.3 x 10(4) cells/colony. Using cell-sorting techniques FL HPP-CFC were shown to be predominantly contained in the CD34+ CD33+ CD38- fraction of FL cells. FL HPP-CFC were heterogeneous for HLA-DR expression, and no differences in proliferative capacities were observed between HLA-DR+ and HLA-DR- HPP- CFC. The CD34+ CD33-HLA-DR- CD38- population, previously suggested to contain stem cells, was observed to be very rare in the FL, representing approximately 1 in 1.7 x 10(5) light-density FL cells and containing almost no CFC. Therefore, it is possible that stem cells are contained in the CD33+ fraction of FL cells. Phenotypic characterization of CD34+ CD33+ CD38- lin -LDFL cells showed that these cells are also CD13+, predominantly Thy-1+, CD45RA-, CD45RO-, CD71-, and heterogenoeous for c-kit expression. These data suggest that FL HPP- CFC represent a heterogeneous compartment of primitive myeloid progenitors that may include stem cells.     

Modulation of murine pluripotential stem cell proliferation in vivo by lithium carbonate

The effect of administering lithium in vivo on murine pluripotential stem cell proliferation was studied. Lithium was injected i.p. at various meq/liter concentrations (0.5-5.0 meq/liter). Data was obtained establishing that lithium increased the pluripotential stem cell population from normal mice as measured by the Till and McCulloch CFUs assay. Significant increases were demonstrated in: (1) bone marrow CFUs; (2) bone marrow organ cellularity, and (3) peripheral blood WBC. Marrow CFUs increase was maximum at 4 days post-lithium injection and greatest in the 1 meq/liter group (p < 0.001). Further increases in lithium concentration, i.e., 5 meq/liter, effectively reduced CFUs levels below normal, suggesting toxicity of the drug was apparent at this dose level. We conclude lithium may modulate granulopoiesis by increasing the CFUs stem cell compartment, thereby directly channeling differentiation into the granulopoietic pathway. This increases the committed progenitor stem cell (CFUc) population and ultimately peripheral blood end-stage cells.