Transduction of primitive human hematopoietic cells with recombinant adenovirus vectors

We have examined the ability of recombinant adenoviral vectors to transduce human hematopoietic cells. Our findings indicate that adenovirus readily infects a large proportion of CD34+ cells. Using adenovirus vectors that transduce either a lacZ or an alkaline phosphatase reporter gene, we observed up to 45% of total CD34+ cells infected. Upon more detailed analysis, we observed comparable levels of transduction for CD34+/CD38- cells and for CD34+ cells in G(zero) phase of the cell cycle. Importantly, exposure to adenovirus resulted in negligible levels of toxicity as assayed by propidium iodide staining and colony-forming ability. Using adenovirus vectors, we also describe a model system for regulated gene expression in early hematopoietic tissues. CD34+ cells were simultaneously infected with two viruses, one carrying a TetR/VP16 transactivator (tTA) and the second carrying a tTA- dependent lacZ reporter gene. Using this approach, beta-gal expression was only observed upon coinfection with the transactivator vector. In addition, as shown previously (Gossen and Bujard, Proc Natl Acad Sci USA 89:5547, 1992), tetracycline was able to inhibit tTA mediated induction, thereby providing an effective means to regulate expression of the reporter gene. We conclude that recombinant adenovirus is an effective vehicle for transiently expressing genes in primitive human hematopoietic cells.     

The myeloid blood cell differentiation-inducing protein MGI-2A is interleukin-6

The mouse myeloid blood cell differentiation-inducing protein, macrophage and granulocyte inducer, type 2A (MGI-2A), was purified, and the amino acid sequence of a CNBr cleavage peptide (22 residues) was determined. This amino acid sequence is identical to the sequence found in positions 73 to 94 of mouse interleukin-6 (IL-6). Recombinant mouse IL-6 protein induces differentiation of mouse myeloid leukemic cells that are induced to differentiation by MGI-2, and monoclonal antimouse- MGI-2 antibody, which neutralizes MGI-2, also completely neutralizes this IL-6-induced differentiation. These results show that the major type of mouse myeloid differentiation-inducing protein (MGI-2A) and IL- 6 are very similar and most likely identical proteins. Recombinant human IL-6 (also called interferon-beta 2 or B-cell differentiation factor), which shows only a 41% similarity to mouse IL-6, has 11 identical amino acid residues out of the 22 in the mouse MGI-2A peptide and also induces differentiation of the same myeloid leukemic cells.     

Purification of common acute lymphoblastic leukemia antigen positive cells from normal human bone marrow

Mononuclear cells expressing the common acute lymphoblastic leukemia antigen (CALLA) were purified from normal adult human bone marrow, where they constitute a small fraction of the total population. This was accomplished by a two-step purification from Ficoll-Hypaque- isolated mononuclear cells. Isolated mononuclear cells were first labeled with a mixture of monoclonal antibodies (MoAb) specific for myeloid and erythroid precursor cells, and immune rosettes were then formed with sheep erythrocytes coated with rabbit anti-mouse antibodies (R/M-SRBC). Sedimentation through Ficoll-Hypaque then eliminated the majority of mature myeloid cells. The second step consisted of labeling the remaining rosette-negative cells with CALLA-specific MoAb and purifying CALLA+ cells by fluorescence activated cell sorting. Alternatively, CALLA+ cells were purified in a second R/M-SRBC rosette sedimentation step. The purified CALLA+ cells, which morphologically were medium to large lymphoid cells, were subsequently studied using dual fluorescence techniques to identify surface markers as well as intracytoplasmic staining to detect terminal deoxynucleotidyl transferase enzyme (TdT) and intracytoplasmic mu. While the CALLA+ cell suspensions contained very few mature myeloid cells or T lymphocytes, the finding that 5% to 11% of them were cyto-mu+ and 13% to 22% expressed the B1 differentiation antigen clearly indicated that at least some of these cells were B cell precursors. Because 48% to 63% of the cells were TdT+ and practically all of them expressed Ia antigen, it appears that these cells are a mixture of very early lymphoid precursor cells as well as more differentiated pre-B cells. The phenotype of these normal cells is very similar to that of common ALL cells. Differences in the surface marker phenotypes between adult and fetal CALLA+ cells that have previously been purified were also identified.     

Induction of proliferation of purified human myeloid progenitor cells: a rapid assay for granulocyte colony-stimulating factors

The proliferation and differentiation of granulocyte and monocyte progenitor cells (CFU-C) in vitro is dependent on the presence of a group of closely related glycoproteins termed colony-stimulating factors (CSF). In order to investigate the interaction of these factors with CFU-C, we purified CFU-C from the peripheral blood of chronic myeloid leukemia patients with an immune rosette technique using specific monoclonal antibodies (mean 74-fold enrichment, 45% cloning efficiency). Colony formation by purified CFU-C demonstrated an absolute dependence on an exogenous source of CSF. Liquid culture of small aliquots of enriched CFU-C with CSF-containing medium resulted in a rapid, time- and concentration-dependent induction of DNA synthesis as measured by 3H-thymidine incorporation. This specific CSF induction of DNA synthesis by enriched CFU-C was used to develop a microassay system for CSF activity. CSF activity could be reproducibly quantitated in 24-48 hr. The proliferating cells in this assay system were shown to be myeloid progenitor cells by examining the morphology of their progeny and by determining the surface antigen phenotype of the responding cells (Ia+, T3-, B1-, Mo1-). This microassay provides a quantitative assessment of CSF activity that may be useful in the purification of human CSF and in the generation of monoclonal antibodies to CFU-C surface structures.     

Analysis of antigenic determinants on human monocytes and macrophages

Mo1, 2, 3, and 4, and Plt-1 are a series of five distinct antigens detected on the surface of human peripheral blood monocytes by mouse monoclonal antibodies. Mo2 and 3 are restricted to the monocyte- macrophage series, while Mo1, as previously reported, is also expressed by human granulocytes and null cells. Mo3, as distinguished from Mo1 and Mo2, is weakly expressed by virgin peripheral blood monocytes but becomes well expressed if monocytes are cultured overnight at 37 degrees C. Mo4 is coexpressed by monocytes and platelets, while Plt-1 appears to be a platelet-specific antigen whose detection on monocytes reflects adherence of platelets to monocyte membranes. That Mo2-4 are true monocyte antigens is demonstrated by their resynthesis following protease treatment of monocytes (Mol expression is resistant to proteolytic digestion). During myeloid-monocyte differentiation, the Mo antigens are infrequently expressed by immature myeloid cells but are found at higher frequency on leukemic monocytic forms. Macrophages from cultured peripheral blood monocytes and HL-60 cells exposed to lymphokines or phorbol diester express Mo1-4, but noncirculating peritoneal macrophages lack Mo3. The Mo antigens are differentiation markers whose expression reflects membrane heterogeneity during myeloid- monocyte-macrophage maturation.     

Isolation of myeloid progenitor cells from peripheral blood of chronic myelogenous leukemia patients

Myeloid progenitor cells (colony- and cluster-forming cells in semisolid medium, CFU-GM) were purified from the peripheral blood of chronic myelogenous leukemia (CML) patients. Lymphocytes, monocytes, and most immature myeloid cells were simultaneously depleted with specific monoclonal antibodies using an erythrocyte rosette technique for cell separation. Cells expressing Ia-like antigen were then selected from the residual cell population. Day 7 CFU-GM were enriched 44--116-fold in the IA+ cell fraction, when compared to the unseparated cells, and up to 47% of the cells could form a myeloid colony or cluster in culture. This cell fraction contained up to 92% undifferentiated blasts, with the remainder mostly promyelocytes. The enriched CFU-GM cells were dependent on an exogenous supply of colony- stimulating factor for growth, and colony formation was linear with cell concentration over a large range (10(4)-10(1) cells/ml). This technique of rosette depletion and enrichment with specific monoclonal antibodies provides a unique method for purifying a homogenous population of myeloid precursor cells with defined surface antigen characteristics.     

Occult hepatitis B virus infection: diagnosis, implications and management?

Abstract   Occult hepatitis B virus (HBV) infection is generally defined as the detection of HBV-DNA in the serum or liver tissue of patients who test negative for hepatitis B surface antigen. In most cases, occult HBV infection is related to low level HBV infection with subdetectable levels of HBsAg and not infection with HBV variants that cannot express S proteins or produce S proteins with aberrant epitopes that are not detected by conventional serological assays. Prevalence of occult HBV infection is related to the overall prevalence of HBV infection in that country, being more common in persons with prior exposure to HBV. Occult HBV infection has been found in a substantial proportion of patients with cirrhosis and hepatocellular carcinoma but other causes of liver disease are frequently present. Future studies should focus on delineating the pathogenic role of occult HBV infection and the basis for failure to detect circulating hepatitis B surface antigen.     

A carboxyl terminal truncation mutant of CD36 is secreted and binds thrombospondin: evidence for a single transmembrane domain

CD36 has been implicated in several intracellular signalling events, including platelet and monocyte activation, and receptor-mediated internalization of bound ligands such as oxidized low-density lipoprotein and apoptotic neutrophils. These processes are presumably mediated by the intracytoplasmic domain(s) of the molecule. By analysis of hydrophobicity plots and by analogy to rat LIMPII, which has a 60% homology to CD36, a two-transmembrane domain model has been proposed. To characterize the structure-function relationships of CD36 involved in transducing the signal, we have defined the number of transmembrane and intracellular domains experimentally using a mutagenesis approach. A truncated CD36 cDNA was constructed that encodes a protein that terminates just proximal to the putative C-terminal transmembrane domain. This mutant was cloned into eukaryotic expression plasmid vectors to generate short-term and stable transfected cells. Our results indicate that the truncated mutant is secreted by the transfectants into the postculture medium, indicating that there is only one transmembrane domain in CD36, which is present at the C- terminal end. The soluble secreted protein from all of these cells is functional as indicated by its binding to thrombospondin.