Simultaneous measurement of antibodies to three HIV-1 antigens in newborn dried blood-spot specimens using a multiplexed microsphere-based immunoassay.

We developed a fluorescent immunoassay to simultaneously measure antibodies to three HIV-1 antigens from newborn dried blood-spot specimens. The multiplexed assay uses fluorescent microspheres and a flow analyzer. The procedure is sensitive, precise and accurate, and can be expanded to simultaneously measure additional multiple analytes from a single specimen.

Cell separation based on the reversible interaction between calmodulin and a calmodulin-binding peptide.

A cell separation system based on the calcium-dependent interaction of calmodulin (CM) with a calmodulin-binding peptide (CBP) has been developed. The prototype of this system utilizes an indirect method to label the target cell population. Cells are first labeled with a primary monoclonal antibody directed to a specific cell surface antigen, then with a secondary affinity reagent, consisting of a polyclonal goat anti-mouse IgG (GAM-IgG) that has been cross-linked to a CBP derived from the sequence of the rabbit skeletal muscle myosin light chain kinase. In the presence of Ca2+, the CBP on the cells labeled with GAM-IgG-CBP binds to biotinylated calmodulin (CM-Biotin) with high affinity. The target cells are then captured with a solid-phase streptavidin. The unbound non-target cells are washed away and the immobilized target cells are released by chelating Ca2+ with EGTA. The specificity of the GAM-IgG-CBP and CM-Biotin and the feasibility of using this system to separate cells was demonstrated using the KG-1 human acute myelogenous leukemia cell line. KG-1 cells were fractionated on the basis of cell surface expression of HLA-DR. The cell selection reagents and the cell separation process did not affect KG-1 cell viability while cells selected by this procedure were 90% pure with a yield of 75%. This cell separation system also was used for rare cell isolation from normal human peripheral blood mononuclear cells. T cells expressing the Vbeta5 T cell receptor, which represent < 5% of the unfractionated cells, were isolated with 89% viability, 72% purity, 80% yield, and retained the ability to respond to activation signals as measured by blast transformation. The results from this study show that a cell selection system based on the reversible interaction between CM and a CBP can be applied to gently and efficiently isolate cells from a heterogeneous starting population that are free of the solid matrix without exposure to the stresses of mechanical or enzymatic release.

Hydroquinone resistance in a murine myeloblastic leukemia cell line. Involvement of quinone reductase and glutathione-dependent detoxification in nonclassical multidrug resistance.

A hydroquinone-resistant derivative of the M1 cell line, designated M1HQ, was generated and used to evaluate the biochemical mechanism responsible for resistance to oxidative stress-inducing agents. The hydroquinone concentrations that were cytotoxic to 50 and 90% of the parental M1 cell line in 48 hr were 25 and 90 microM, respectively, whereas exposure to 500 microM hydroquinone did not decrease M1HQ viability significantly. M1HQ cells grew slower than M1 cells and exhibited significantly higher resistance to colchicine, doxorubicin, hydrogen peroxide, 4-hydroperoxycyclophosphamide, and 1,3-bis (2-chloroethyl)-1-nitrosourea but not to benzoquinone, vinblastine, or gamma-radiation. M1HQ cells possessed significantly higher levels of total thiols, glutathione, glutathione peroxidase, glutathione reductase, quinone reductase, and gamma-glutamyl transpeptidase than the parental M1 cell line. Steady-state gamma-glutamylcysteine synthetase mRNA expression also was 1.6-fold higher in M1HQ cells. P-glycoprotein transcripts were detectable in both M1 and M1HQ cells, but were 2-fold higher in M1HQ. Multidrug resistance-associated protein transcripts were not detectable in either M1 or M1HQ. Hydroquinone resistance in M1HQ cells was partially reversible with a combination of inhibitors of quinone reductase, gamma-glutamylcysteine synthetase, glutathione peroxidase, and the multidrug resistance-associated protein, but not with inhibitors of P-glycoprotein, gamma-glutamyl transpeptidase, or glutathione-S-transferase. When treated with [14C]hydroquinone, M1HQ cells did not generate significant hydroquinone-protein adducts but did release an adduct similar to N-acetylcysteinyl-benzoquinone. In contrast, numerous [14C]hydroquinone-protein adducts were produced in M1 cells, while the N-acetylcysteinyl-benzoquinone-like molecule was undetectable. Thus, hydroquinone resistance in M1HQ cells appeared to result from a glutathione-dependent detoxification and export mechanism.

Influence of oxygen partial pressure on human and mouse myeloid cell line characteristics.

Mouse and human cells have been reported to have different thiol characteristics (J. M. Messina and D. A. Lawrence, 1992, Int. J. Immunopharmacol. 14, 1221-1234). In addition, when cells are undergoing active growth, they usually have an increased thiol content. In an attempt to determine whether a mouse and a human cell line with similar characteristics can be induced to change their phenotype simply by being cultured at a lower oxygen partial pressure (pO2), the human KG-1 and mouse M1 myeloid cell lines were cultured at 5 and 20% oxygen. It is important to note that 5% O2 is close to the physiological pO2, whereas the percentage of O2 usually employed for most in vitro methods is atmospheric pO2. After long-term culturing at 5% (L cell lines) versus 20% O2 (H cell lines), the thiol content of the original (H) cell lines significantly changed. The amount of total and surface thiols was lower in both L cell lines, but only significantly different on the M1 lines, whereas the glutathione content was significantly lower in the L cell lines of KG-1 and M1. The mouse lines showed the greatest sensitivity to pO2 changes; however, the mouse cell lines were not more sensitive to hydrogen peroxide than the human cell lines even though they possess significantly less total thiols and glutathione. Interestingly, cell lines maintained at lower pO2 (physiological pO2) were more sensitive to hydrogen peroxide than their parental counterparts that were maintained at atmospheric oxygen levels.

Protection of Purified Human Hematopoietic Progenitor Cells by Interleukin-1ß or Tumor Necrosis Factor-α.

Hematopoiesis is the process of blood cell formation in the adult human, the bone marrow (BM) is the primary hematopoietic organ. Each day, the BM produces billions of leukocytes, erythrocytes, and thrombocytes, which enter the circulation. Production of such enormous numbers of mature blood cells results from the exponential expansion and differentiation of pluripotent hematopoietic stem cells through a series of increasingly more differentiated hematopoietic progenitor cells (HPCs) Regulation of hematopoiesis is accomplished by the transduction of signals that follow interactions of the HPCs and developing blood cells with BM stromal cells, cytokines, and the extracellular matrix.

The effects of interleukin-1 beta and tumor necrosis factor-alpha on in vitro colony formation by human hematopoietic progenitor cells exposed to doxorubicin or hydroquinone.

Previous studies have shown that treatment of bone marrow (BM) cells with interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha) can protect hematopoietic progenitor cells (HPC) from the toxic effects of 4-hydroperoxycyclophosphamide (4HC) or gamma-irradiation. Since doxorubicin (DX) and hydroquinone (HQ) may inhibit hematopoiesis through mechanisms similar to 4HC and gamma-irradiation, it was of interest to determine whether IL-1 beta or TNF-alpha could protect HPC from DX and HQ as well. Bone marrow mononuclear cells (BMMNC) or purified HPC (pHPC) were exposed to 50 ng/mL IL-1 beta or 25 ng/mL TNF-alpha alone or in combination with DX or HQ for 22 hours at physiological O2 partial pressure and temperature. The cells were washed free of the cytokines and toxicants and plated in cytokine-containing semisolid medium. Under these concurrent cytokine +/- toxicant treatment conditions, neither IL-1 beta nor TNF-alpha significantly affected progenitor cell frequencies (assessed as CFU-C) or lineage commitment compared with the medium-treated controls. Treatment with either 100 nM DX or 30 microM HQ, however, reduced CFU-C frequencies by approximately 70%. When BMMNC were used, treatment with neither IL-1 beta nor TNF-alpha consistently protected CFU-C from either DX or HQ. In contrast, using pHPC, IL-1 beta or TNF-alpha treatment conferred nearly two-fold protection of CFU-C from DX in all donors tested. TNF-alpha protected CFU-C from HQ using pHPC from all but one donor, while IL-1 beta did not protect CFU-C from HQ. Using phPC, maximum protection of CFU-C from DX was reached at IL-1 beta or TNF-alpha concentrations above 10 ng/mL or 1 ng/mL, respectively. Treatment of pHPC with TNF-alpha for at least 8 hours was necessary before significant protection from DX could be detected. Therefore, we conclude that IL-1 beta and TNF-alpha can act directly on human HPC to protect them from the inhibitory effects of DX and that, to a lesser extent, TNF-alpha can directly protect HPC from HQ.

In vitro effects of hydroquinone, benzoquinone, and doxorubicin on mouse and human bone marrow cells at physiological oxygen partial pressure.

A comparative study was undertaken in order to assess the hematotoxic effects of hydroquinone (HQ), 1,4-benzoquinone (BQ), and doxorubicin (DX) on mouse and human bone marrow (BM) cells. Initial experiments indicated that the inhibitory effects of near-ambient pO2 and HQ on granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation were additive. Thus, subsequent experiments were done under conditions of continuous toxicant exposure in complete medium at physiological temperature and O2 partial pressure. Viability was measured 24 hr after exposure, and at the concentrations tested, HQ was less cytotoxic than BQ. DX did not exhibit significant cytotoxicity at the concentrations used. Both HQ and BQ were slightly more cytotoxic to mouse BM cells than to human BM cells. Dose-response analyses of HQ, BQ, or DX inhibition of GM-CSF-induced proliferative and colony-forming responses indicated that murine GM progenitors were significantly less sensitive to HQ than to the majority of myeloid BM cells that proliferated in response to GM-CSF. This preferential resistance of GM progenitors to HQ was not observed when human BM cells were used. HQ was somewhat more inhibitory to human than to mouse GM-CSF responses. Inhibition of GM-CSF-induced responses by BQ correlated closely with cytotoxicity, and DX was 1000-fold more inhibitory to GM-CSF-induced proliferative and colony-forming responses than either HQ or BQ. Again, DX appeared to be slightly more inhibitory to human BM cells than to mouse BM cells. Purified human hematopoietic progenitor cells (HPCs) were also used in the dose-response analyses of HQ, BQ, or DX inhibition of GM-CSF-induced proliferative and colony-forming responses. Inhibition of GM-CSF-induced HPC responses by HQ, BQ, and DX was very similar to that obtained when BM mononuclear cells were used, suggesting that the human HPC is a target for the direct effects of HQ, BQ, and DX.

Extrachromosomal recombination in vaccinia-infected cells requires a functional DNA polymerase participating at a level other than DNA replication.

Homologous recombination was measured in vaccinia-infected cells cotransfected with two plasmid recombination substrates. One plasmid contains a vaccinia protein lacZ coding region bearing a 1.1 kb 3' terminal deletion while the other plasmid contains a non-promoted lacZ coding region bearing a 1.1 kb 5' terminal deletion. Homologous recombination occurring between the 825 bp of lacZ common to both plasmids regenerates a functional lacZ gene from which B-galactosidase expression was measured. The entire 3 kb lacZ gene was used as a positive control. A panel of thermosensitive mutants was screened in cells either transfected with the positive control plasmid or cotransfected with the recombination substrates. A DNA - mutant, ts42, known to map to the viral DNA polymerase gene was found to be defective in recombination. Significantly, other DNA - mutants, ts17 or ts25, or other DNA polymerase mutants did not exhibit a defect in recombination similar to ts42. Inhibitors of viral DNA synthesis did not uniformly affect recombination. Cytosine arabinoside and aphidicolin inhibited B-galactosidase expression from the recombination substrates but not from the positive control plasmid, whereas hydroxyurea enhanced expression from both. Marker rescue with the cloned wildtype DNA polymerase gene repaired the defect in ts42. Southern and western analyses demonstrated that B-galactosidase activity was consistent with a recombined lacZ gene and unit size 116 kDa protein. Measurement of plasmid and viral DNA replication in cells infected with the different DNA - mutants indicated that recombination was independent of plasmid and viral DNA replication. Together these results suggest that the vaccinia DNA polymerase participates in homologous recombination at a level other than that of DNA replication.

A DNA ligase gene in the Copenhagen strain of vaccinia virus is nonessential for viral replication and recombination.

Biochemical and genetic analyses have been conducted to determine whether a vaccinia virus open reading frame (orf) with extensive homology to the Saccharomyces cerevisiae DNA ligase gene encodes a functional ligase activity. This orf in HindIII A, designated A50R, is capable of encoding a 552-amino-acid, 63.4-kDa polypeptide. Full-length A50R mRNA produced in vitro directed the synthesis of a polypeptide with an apparent molecular weight of 57 kDa. Significantly, translation reactions programmed with A50R mRNA were capable of ligating a 3-kb Notl restriction fragment into multimers. DNA ligase activity was not detectable when either truncated sense or full-length antisense mRNA was translated in vitro. In extracts prepared from cells infected with wt vaccinia virus, DNA ligase activity was detected as assayed by the formation of a 57 kDa ligase-AMP adduct which was expressed early in the viral replication cycle. In cells infected with a DNA ligase deletion mutant no equivalent AMP-labeled adduct was detected. Relative to wt virus, the DNA ligase deletion mutant exhibited no significant differences in homologous recombination. These results indicate that the vaccinia orf A50R encodes a functional DNA ligase expressed early in infection, but this DNA ligase is nonessential for either recombination or viral replication.

Reaction of reduced metronidazole with guanosine to form an unstable adduct.

Metronidazole, which is known to react with DNA under certain conditions, forms an adduct with guanosine in the presence of the reducing agent, sodium dithionite. This product has been purified by HPLC, characterized by UV and fast atom bombardment mass spectrometry, and tentatively identified as 1-(2-hydroxyethyl)-2-methyl-5-(N2-guanosinylamino)imidazole. This adduct is very unstable and decomposes to a variety of products including guanosine. Formation of this and similar DNA adducts from metronidazole in vivo, and the decomposition of these products, are probably both strongly dependent on details of intracellular metabolism.

Ribonuclease activity associated with the 60S ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin.

All purified preparations of the ribosome-inactivating proteins ricin A, phytolaccin and Shiga toxin were shown to exhibit ribonuclease activity with 5S or 5.8S rRNA substrates. These toxin species generated reproducible patterns of RNA fragments distinct for each toxin species while multiple preparations of a single toxin species yielded similar RNA fragment patterns. The heat inactivation profile of Shiga toxin was identical for its RNase and protein synthesis inhibitory activities. These data are the first to indicate that the ribosome-inactivating catalytic toxins, in addition to alpha-sarcin, exhibit RNase activity. These results suggest RNase activity may be responsible for ribosome-inactivation catalyzed by ricin, phytolaccin and Shiga toxin proteins.