Tiazofurin-induced autosecretion of IL-6 and hemoglobin production in K562 human leukemia cells.

Previous reports have established the synthesis of interleukin-6 (IL-6) and IL-6 receptors (IL-6R) in several human leukemia cells and found that IL-6 and the IL-6R could be expressed in cell lines with erythroid/megakaryocytic features. IL-6 is a pleiotropic cytokine involved in megakaryocytic differentiation. The finding that endogenous IL-6 levels in serum increased after 5-fluorouracil (5-FU) treatment suggests that IL-6 may play some role in the recovery of hematopoietic systems. This observation may assist the understanding of erythroid regeneration caused by antineoplastic agents such as tiazofurin. Tiazofurin inhibits the activity of IMP dehydrogenase. Its exposure to K562 cells at 10 microM tiazofurin stimulates erythroid differentiation. Stimulation of cells with tiazofurin gave a significant increase in IL-6 production. Its levels were quadrupled after 2 days of culture. Tiazofurin also caused a trivial reduction in the percentage of cells with the IL-6R. This evidence implies that tiazofurin produced no significant effect on the IL-6R. Tiazofurin also increased the percentage of benzidine-positive cells representing hemoglobin production, confirmed by GpA expression. We concluded that IL-6 is rate limiting in regard to hemoglobin production and that IL-3 could be used for clinical benefit to stimulate erythropoiesis and synergize with tiazofurin.

Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes.

A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExATM immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

Enzyme immunoassay to determine heavy metals using antibodies to specific metal-EDTA complexes: optimization and validation of an immunoassay for soluble indium.

An immunoassay that measures soluble indium at concentrations from 0.005 ppb to 320 ppm is described. The assay utilized a monoclonal antibody that binds specifically to indium-EDTA complexes in an antigen-inhibition format. The sensitivity of the assay could be modulated by changing the nature of the soluble inhibiting antigen. The range of the assay was from 0.6 to 320 ppm, 0.1 to 120 ppm, or 0.005 to 2000 ppb when indium-EDTA, indium-(p-nitrobenzyl)-EDTA, or indium-EDTA-bovine serum albumin, respectively was used as the soluble inhibiting antigen. The assay reliably monitored indium concentration in the presence of a 100-fold excess of manganese, magnesium, or copper ions and the quantitation of indium by immunoassay correlated closely with the values obtained using atomic absorption spectroscopy. This technology could be employed in immunoassays for other metals that are priority pollutants.

Multiple DNA-dependent RNA polymerases in Trypanosoma cruzi.

DNA-dependant RNA polymerase solubilized from cultured Trypanosoma cruzi epimastigotes was chromatographed on A-25 Sephadex and gave a single peak of activity. Subsequent phosphocellulose chromatography revealed two peaks of RNA polymerase activity. These peaks have different sensitivities to the toxin alpha-amanitin. The first peak is 50% inhibited by 17.8 micrograms ml-1 amanitin while the second peak is 50% inhibited by an amanitin concentration of 44.6 micrograms ml-1. The activity of both peaks is blocked by actinomycin D, but is unaffected by rifampicin. Each peak is stimulated by Mn2+, and is optimal with single stranded DNA as a template.

Interaction between murine natural killer cells and trypanosomes of different species.

The involvement of natural killer (NK) cells in the immunological resistance of mice to murine-specific Trypanosoma musculi was evaluated. Murine NK cells were found to be unable to kill or inhibit T. musculi or to protect recipients from infection. In addition, the ability of spleen cells from normal mice and from mice on day 3 of T. musculi infection, at the time of maximum NK augmentation, to kill Trypanosoma cruzi and Trypanosoma lewisi was evaluated. Spleen cells from normal mice displayed significant killing of both T. cruzi and T. lewisi. Furthermore, augmented spleen cells from T. musculi-infected mice were considerably more effective than normal spleen cells in killing both T. cruzi and T. lewisi. The activity of NK cells toward YAC-1 tumor target cells was inhibited in a dose-dependent fashion by either live T. musculi or extracts of T. musculi, but not by extracts of rat-specific T. lewisi. The results suggest that well-adapted protozoan parasites may be nonsusceptible to the natural cell-mediated resistance mechanisms of their hosts. Their nonsusceptibility could result from the ability to elaborate substances that either inactivate NK cells or block NK cell interaction with complementary sites on the parasite surface.

Destruction of Trypanosoma cruzi by Natural killer cells.

Mice infected with trypanosoma cruzi or stimulated with poly(inosinic.cytodylic acid) were found to possess splenic and peritoneal exudate cells with enhanced cytotoxic activity against epimastigote and trypomastigote forms of Trypanosoma cruzi. By use of specific alloantiserums it was determined that the effector cells responsible for this cytotoxic activity were typical natural killer cells.

Increased natural killer cell activity in experimental American trypanosomiasis.

These studies showed that increased natural killer (NK) cell activity develops during experimental American Trypanosomiasis in mice. Increased cytotoxicity against YAC target cells by peritoneal cavity cells (PEC) was detected as early as 1 day into infection in both C3H/He and C57BL/6 mice. Peak activity occurred 2 days into infection, with significant but variable increases detected in the spleen cells (SC) from these mice. The response subsided in PEC and to a lesser extent in SC by 4 days post-infection. Similar responses were not detected in infected C57BL/6 bgJ/bgJ (beige mutant) mice until the fourth day of infection and peaked at significantly lower levels than the peak on day 2 in beige hybrid mice ([C57BL/6 +/+ X C57BL/6 bgJ/bgJ]F1). The effector cell from infected mice was sensitive to pretreatment with anti-NK 1.2 serum plus complement (C), partially sensitive to anti-Thy 1.2 + C, and insensitive to polyvalent anti-mouse immunoglobulin + C. The cytotoxic activity induced in cells from mice infected for 2 days was recovered in subpopulations nonadherent to plastic or Sephadex G-10. Thus, the anti-YAC effector cell found very early in mice infected with T. cruzi possessed many of the characteristics of natural killer (NK) cells found in normal mice. Other experiments demonstrated that injection of heat-killed preparations of blood-form trypomastigotes (BFT) induced increases in NK activity. The presence of augmented NK activity against YAC tumor cells in both resistant and susceptible strains of mice, together with the presentation of the resistance phenotype in beige mutant mice, which had a lower NK response to infection, is not indicative of a direct role for host protection during infection with T. cruzi by NK cells.

Spontaneous lytic activity against allogeneic tumor cells and depression of specific cytotoxic responses in mice infected with Trypanosoma cruzi.

The ability of cells from mice infected with Trypanosoma cruzi to generate specific as well as nonspecific cell-mediated cytotoxic responses to allogeneic tumor cells was investigated. The capacity of spleen cells from either C3H/He (C3H) or C57BL/6 (B6) mice to develop cytotoxic responses in vitro against P-815 tumor cells was depressed after the 2nd wk of acute infection initiated with 10(3) parasites. However, spleen cell responses in B6 mice sensitized against P-815 were depressed only in those mice infected for 16 to 22 days and were significantly enhanced in mice infected for shorter or longer periods of time. In subsequent experiments, significant cytotoxic activity against P-815 target cells was observed in cells from infected B6 and, to a lesser degree, C3H mice without prior sensitization with the tumor cells. With the use of both P-815 and YAC tumor cell lines in cytotoxicity assays, two phases of apparently nonspecific T. cruzi-induced lytic activity (TILA) were observed after infection: a 1st phase detected within 2 days against YAC target cells, and a subsequent phase detected against both YAC and P-815 target cells at a time dependent upon the number of parasites injected. Other results demonstrated increased cytotoxic activity against P-815 target cells in 2.5-mo-old C3H mice infected with greater numbers of T. cruzi or when older (4.5 to 6.5 mo) mice were infected with 10(3) parasites. Antibodies cross-reactive to tumor cells were not detected in sera from infected mice, suggesting that TILA is an antibody-independent phenomenon. Possible relationships between the phases of TILA and the modification of specific responses generated in infected mice are discussed.