Use of paper-absorbed fingerstick blood samples for studies of antibody to human immunodeficiency virus type 1 in intravenous drug users

The suitability of paper-absorbed (PA) fingerstick blood specimens for antibody testing of human immunodeficiency virus type 1 (HIV-1) was examined in two populations of intravenous drug users (IVDU): 393 persons from a drop-in counseling and testing clinic and 145 from a methadone treatment clinic. From the first group, the same 66 immunoblot-confirmed enzyme immunoassay (EIA)-positive specimens were identified in sera from venipuncture and parallel fingerstick PA specimens. The latter had slightly higher EIA mean background levels resulting in 10 immunoblot-negative EIA-positive samples versus 6 in the sera group. HIV-1 seroprevalence was 17% of 393 from the drop-in clinic. By category of IVDU, the rates were 34% and 14% for active and recovering IVDU, respectively (P less than .001), and 36% in black and Latino compared with 13% in white IVDU (P less than .002). Of the 145 participants in the methadone program, 39% had antibody to HIV-1: 49% for blacks and Latinos compared with 30% in whites (P less than .01). The data indicate that antibody testing for HIV-1 by PA is equivalent to the serum antibody assay of venipuncture specimens. The fingerstick method appears to have greater use for serosurveys and screening programs because of convenience, safety, and ease of storage, transport, and processing of samples.     

Enhancement of in vitro beta-thalassemic and normal hematopoiesis by a noncytotoxic monoclonal antibody, 9.1C3: evidence for negative regulation of hematopoiesis by monocytes and natural killer cells

The enhancement of in vitro human hematopoiesis by the addition of a noncytotoxic monoclonal antibody, 9.1C3, is described. Enhancement of all aspects of in vitro hematopoiesis was observed on addition of 9.1C3 antibody to cultures of mononuclear cells from normal bone marrow, cord blood, and peripheral blood from beta-thalassemia major patients. In cultures with no exogenous colony-stimulating factor (CSF), the addition of 9.1C3 resulted in a two- to eightfold increase in nonerythroid colony formation. Similarly, for cultures maximally stimulated with CSF, the addition of 9.1C3 antibody resulted in a one- to fourfold increase in colony formation. These effects were abrogated by the removal of either adherent, Leu-M3+ or Leu-7+ cells. Colony- forming cells were shown to be present among the 9.1C3-negative cells when mononuclear cells were sorted by flow cytometry. Media conditioned in the presence of 9.1C3 and mononuclear cells were able to enhance colony formation in vitro for normal nonadherent bone marrow cells beyond that achieved with supramaximal amounts of human placental- conditioned medium and erythropoietin. The data suggest that natural killer cells interact with monocytes to exert a negative regulatory control on in vitro granulopoiesis and erythropoiesis. Consequently, the number of progenitor and multipotential cells in cultures of unfractionated cell populations may be greatly underestimated.     

Functional and immunologic protein S levels are decreased during pregnancy

Protein S, is a natural anticoagulant protein which serves as a cofactor for activated protein C. During pregnancy and in the postpartum period, functional protein S levels are significantly reduced (38% +/- 17.3%, mean +/- 1 SD) when compared to nonpregnant females (97% +/- 31.6%) (P less than 0.001). In plasma an equilibrium exists between functionally active free protein S and protein S complexed with C4b-binding protein, which is functionally inactive. As a result of this equilibrium either a decreased level of total protein S antigen or an elevation of C4b-binding protein could lead to reduced protein S activity. C4b-binding protein levels measured by enzyme- linked immunoassay are not significantly different in pregnant women versus nonpregnant controls (103.5% +/- 21.2% v 100% +/- 16.9%). However, during pregnancy and in the postpartum period, total protein S levels are reduced (68% +/- 10.7%) compared to nonpregnant controls (100% +/- 17.0%). This difference is significant at P less than 0.001. These data demonstrated that the reduction in protein S activity observed during pregnancy is a result of reduced total protein S antigen.     

Terminal deoxynucleotidyl transferase-containing cells in peripheral blood: implications for the surveillance of patients with lymphoblastic leukemia or lymphoma in remission

An indirect immunofluorescence assay was used to quantitate TdT- containing (TdT+) cells in the mononuclear leukocyte fraction of peripheral blood from normal subjects and patients with acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). In normal children (10) and adults (10), 0.036% +/- 0.014% (mean +/- SD) and 0.030% +/- 0.015% TdT+ cells were found. In peripheral bloods from 10 children receiving chemotherapy for tumors other than ALL or LL, 0.040% +/- 0.039% TdT+ cells were found. Serial determinations were performed on 15 patients with ALL or LL who were in clinical remission. Eight of these patients remained in continuous remission and always had fewer than 0.11% TdT+ cells in their peripheral blood. Three patients who developed systemic relapse were found to have progressively rising numbers of TdT+ cells in their peripheral blood prior to clinical evidence of relapse. All 3 of these patients had greater than 0.1% TdT+ cells in their peripheral blood from 3 to 8 wk prior to clinical relapse. In 3 other patients, localized extramedullary relapse developed, but no trend was found on serial TdT determinations. Thus, the indirect immunofluorescence assay for TdT detects a small population of cells in normal peripheral blood. In patients with ALL, progressive increases above this normal level were associated with subsequent bone marrow relapse.     

Localization of the G-CSF gene on chromosome 17 proximal to the breakpoint in the t(15;17) in acute promyelocytic leukemia

The human granulocyte-colony stimulating factor gene (G-CSF) is localized at 17q11.2-q21, the region of one of the breakpoints in the 15;17 chromosome translocation specific for acute promyelocytic leukemia (APL). As G-CSF induces differentiation and loss of tumorigenicity in myeloid leukemic cells or cell lines, it was possible that the translocation in APL involved the DNA of the G-CSF coding region or its regulatory region. In situ hybridization to chromosomes with the t(15;17) from patients with the APL translocation using a G- CSF cDNA clone revealed that the coding region of this gene is proximal to the t(15;17) breakpoint on chromosome 17. Southern analysis of DNA from patients with the APL translocation showed no differences in hybridization between normal and leukemic cells. These results indicate that the G-CSF coding sequence is not disrupted by the chromosomal rearrangement characteristic of APL.     

Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.     

Analysis of hematopoietic stem and progenitor cell populations in cytomegalovirus-infected mice

We have studied the effects of murine cytomegalovirus (MCMV) infection on bone marrow stem and progenitor cell populations to find an explanation for the defects in hematopoiesis that accompany CMV infections in patients. Sublethal MCMV infection of BALB/c mice resulted in a 5- to 10-fold decrease in the numbers of myeloid (colony- forming unit-granulocyte-macrophage [CFU-GM]) and erythroid (burst- forming unit-erythroid [BFU-E]) progenitor cells in the marrow, but not in primitive myeloerythroid progenitor cell (colony-forming unit-spleen [CFU-S]) numbers. In contrast, we observed a 10- to 20-fold reduction in CFU-S as well as CFU-GM and BFU-E in lethally infected mice. Depletion of marrow CFU-GM was less severe in C57BL/10 and C3H/HeJ mice, which are more resistant to the effects of MCMV infection. Treatment of bone marrow cells with MCMV preparations in vitro did not reduce the numbers of CFU-GM, although up to 10% of the cells were productively infected. This finding suggests that CFU-GM were not susceptible to lytic MCMV infection in vitro and are probably not eliminated by lytic infection in vivo. Increases in the frequencies of Sca-1+Lin- marrow cells, a population that includes cells with the characteristics of pluripotential stem cells, were observed in MCMV- infected BALB/c, C57BL/10, and DBA/2J mice. Increases in the frequencies of c-kit+Lin- marrow cells were only seen in DBA/2J mice. MCMV infection did not impair the function of pluripotential stem cells because transplantation of marrow from MCMV-infected donors into irradiated recipient mice resulted in successful reconstitution of the T, B, and myeloid cell lineages.     

Regulation of hematopoiesis-IV: The role of interleukin-3 and bryostatin 1 in the growth of erythropoietic progenitors from normal and anemic W/Wv mice

We and others have established a role for T lymphocytes and their products in the regulation of erythropoiesis. Interleukin-3 (IL-3) is a multipotential lymphokine with burst-promoting activity that is produced by activated T lymphocytes. In the anemic, stem cell-defective W/Wv mouse we have described the absence of a functionally active thymocyte population that in normal animals enhances erythroid progenitor growth and stem cell self-renewal. In studies reported here we find that W/Wv mouse marrow responds to exogenous IL-3 by increased erythroid progenitor cell growth. The BFU-E and CFU-E from anemic donors are more sensitive to IL-3 than are those in +/+ marrow. We have recently observed a stimulatory effect of bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate) on normal erythropoiesis in human bone marrow progenitor assays. To test the effects of this molecule on murine normal and anemic W/Wv cells we grew these cells in the presence of increasing doses of bryostatin 1. Bryostatin mimics the stimulatory action of IL-3 on W/Wv bone marrow. Polyclonal antibody directed against murine IL-3 blocks the stimulatory effect of bryostatin on erythropoiesis. Otherwise inactive thymocytes from W/Wv mice in coculture with W/Wv bone marrow showed stimulation of erythropoiesis in the presence of bryostatin. We believe that bryostatin may in part act by stimulating T lymphocytes to release physiologic concentrations of lymphokines.