Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

Effects of cAMP and cGMP elevating agents on HL-60 cell differentiation.

Previous studies have demonstrated low percentage of HL-60 cell differentiation with theophylline. The present study demonstrate that millimolar concentrations of the non-selective phosphodiesterase inhibitors theophylline, caffeine and isobutyl-methylxanthine all inhibit growth, induce substantial differentiation and elevation of both cAMP and cGMP in HL-60 cells. Selective inhibition of cAMP hydrolysis by Ro20-1724 was without effect. The guanylate cyclase stimulator sodium nitroprusside, which increased cGMP only poorly and also increased cAMP, produced growth inhibition but no differentiation. We put forward the hypothesis that elevation of both cAMP and cGMP above a critical level is necessary for significant cyclic nucleotide induced HL-60 cell differentiation.

Epinephrine induces beta-adrenergic desensitization and differentiation of HL-60 cells.

The HL-60 cell line was cultured in a serum-free medium and exposed to various concentrations of EPI. The effects on cell growth, differentiation and beta-adrenergic response were followed during the culture period of 72 h. Short-term exposure (3 min) to EPI (1 nM-1 mM) in the presence of theophylline (4 mM) caused a dose-dependent increase of cAMP levels with a maximum of 1500% above basal levels. When the cells were exposed to EPI (1 nM-10 microM) for 72 h, a dose-dependent increase of cAMP levels with a maximum of 60% above basal levels. Sustained exposure to EPI generated a dose-dependent desensitization of the beta-adrenergic signal system. After EPI treatment for 72 h, IPR (10 microM for 3 min) in the presence of theophylline (4 mM) increased cAMP-levels by only 80% above baseline level (cAMP levels after maintained exposure to EPI), compared to 1080% above unstimulated level in control cells. The alpha-adrenergic receptor blocker PHENT (10 microM) did not affect baseline cAMP level or IPR-dependent cAMP response, but a mixture of EPI and PHENT increased the response to IPR. The HL-60 cell growth was not influenced by EPI. However, after repeated exposure to EPI for 72 h a concentration-dependent increase of HL-60 differentiation was demonstrated. Differentiation was not influenced by PHENT. These results suggest a differentiation induction due to a beta-adrenergic-induced cAMP elevation.

Elevation of cyclic AMP levels in HL-60 cells accumulated in G1 or G2 by transmethylation inhibitors.

Effects of the transmethylation inhibitors 3-deazaadenosine (c3Ado) and 3-deaza-(+/-)-aristeromycin (c3Ari) on cell cycle and cyclic AMP (cAMP) concentrations in human promyelocytic leukemia cells (HL-60) were studied by flow cytometry and radioimmunoassay techniques. Previously described cell cycle accumulations, after incubation with drugs (25 microM) for two cell doublings (36 hr), were localized to G1 and G2 after incubation with c3Ado and c3Ari, respectively. cAMP levels were elevated in cells treated with c3Ado (35%) and c3Ari (92%) for 36 hr. Addition of the phosphodiesterase (PDE) inhibitor theophylline, increased cAMP levels further, while cAMP responsiveness to the beta-adrenergic stimulator isoproterenol was attenuated after c3Ado and c3Ari incubation. Homocysteine thiolactone (Hcy) alone reduced cell growth slightly (5%) and increased cAMP levels (17%). Hcy increased the growth inhibitory effects of c3Ado, while no modulating effect was seen in combination with c3Ari, nor did Hcy counteract the effects on the cell cycle perturbations. The results suggest that c3Ado- and c3Ari-induced cell cycle accumulation is, at least in part, mediated through cAMP elevation, possibly due to PDE inhibition secondary to S-adenosyl-homocysteine hydrolase inhibition and S-adenosyl-homocysteine build-up.

Physiological cyclic AMP stimulation and oncogene mRNA levels in HL-60 cells.

Altered gene expression of the proto-oncogenes c-fos and c-myc is associated with differentiation of the human promyelocytic leukemia (HL-60) cells. We studied changes of cyclic AMP levels and c-fos and c-myc mRNA levels after stimulation with theophylline and theophylline plus isoproterenol. Reduced c-fos and c-myc mRNA levels were detected, but the reduction could not, however, be related to the observed alternations in cyclic AMP concentrations. Expression of c-jun and c-Ha-ras was not affected under these conditions.

Growth, differentiation and the beta-adrenergic signal system of HL-60 cells. Characterization in a medium with insulin as the only added protein.

The purpose of the present investigation was to define experimental conditions for studies on growth, differentiation and the beta-adrenergic signal system of HL-60 cells. The cell medium was completely devoid of added proteins and hormones other than insulin. The HL-60 cell was able to grow and differentiate in this medium. The spontaneous differentiation along the granulocytic pathway after 72 hr, as assessed by the Nitro Blue tetrazolium test, increased by 400% compared to the serum supplemented medium, but the response to 1 microM retinoic acid was equal in the two media. Induction of monocytic differentiation by 0.16 microM phorbol-13-acetate-12-myristate, as determined by surface adherence after 24 hr, was lower in the absence than in the presence of serum. cAMP levels were elevated in response to (-)-isoproterenol. The EC50 was 0.36 +/- 0.01 microM (mean +/- SE, N = 3). The beta-adrenergic ligand 3H-CGP 12177 was specifically bound to 1 single class of binding sites (Kd: 0.15 +/- 0.04 nM, Bmax: 2220 +/- 150, mean +/- SE, N = 3). These data are comparable to our previously reported findings in serum supplemented medium. The present data show that HL-60 cells are able to grow and differentiate in the absence of serum proteins and hormones other than insulin. Under the present experimental conditions, these cells possessed functional beta-adrenergic receptors.

The human promyelocytic leukemia cell (HL-60 cell) beta-adrenergic receptor.

We have characterized the beta-adrenergic receptor binding site and the beta-adrenergic cAMP response of the HL-60 cell. The hydrophilic ligand [3H]-(-)-CGP-12177 was specifically and reversibly bound to one single class of binding sites (Kd 220 pM and Bmax 1,970 sites/cell). The adrenergic agonists inhibited the specific radioligand binding. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. The beta-2 selective antagonist ICI 118551 had a binding affinity 3 orders of potency higher than the beta-1 selective antagonist, atenolol. The adrenergic agonists elevated the cAMP accumulation in a concentration-dependent mode. The order of potency was isoproterenol greater than epinephrine greater than norepinephrine. Both the binding and the functional studies revealed stereospecificity and reversibility. The present data show that HL-60 cells possess beta-2 adrenergic receptors functionally coupled to adenylate cyclase.

Analysis of Campylobacter jejuni antigens with monoclonal antibodies.

To develop monoclonal reagents for antigenic analysis and serotyping of Campylobacter spp., hybridoma cell lines were produced by fusion of mouse myeloma cells and spleen cells from mice immunized with Formalin-treated Campylobacter jejuni organisms. An enzyme immunoassay was used for preliminary screening of the cell culture supernatants and ascites. Twenty-nine clones which reacted with the immunogen were obtained. Seven of these clones were positive in passive hemagglutination tests with sheep erythrocytes coated with boiled saline extract of whole bacteria; four of these reacted with the purified polysaccharide preparation and with the autoclaved saline extract, but not with lipopolysaccharide prepared from the immunogen strain. Two of the antipolysaccharide clones agglutinated live bacteria in slide tests. Four additional clones gave positive slide agglutination tests with live bacteria, but in tube testing no clones agglutinated Formalin-treated bacteria. No cross-reactions with unrelated bacteria were seen, but several clones reacted in the enzyme immunoassay with many of the 24 Campylobacter strains studied. The clone which gave the highest mean enzyme immunoassay values with Campylobacter coli and C. jejuni strains also reacted with Campylobacter fetus subsp. veneralis and C. fetus subsp. fetus strains. This clone also gave the highest enzyme immunoassay value with an acid glycine extract of the immunogen, which indicates the presence of common antigens in the extract. The results suggest that monoclonal antibodies may be used to devise serotyping schemes for Campylobacter spp.

Studies of monoclonal and polyclonal anti-digoxin antibodies for serum digoxin radioimmunoassay.

We compared the advantages of monoclonal antibodies (produced by plasma cell-myeloma cell hybrid lines) and those of conventional antibodies in the radioimmunoassay of digoxin. It was found that antibodies produced by some hybrid cell lines (hybridomas) were highly specific for the digoxin structure; this way the cross-reactions to related structures (e.g. spironolactone) could be avoided. When the hybridoma lines were grown in ascites, the resulting fluid could have as high or higher titre than the serum of a hyperimmunized rabbit. The high titre, the specificity and the permanent growth of the hybridoma lines make them an optimal source of the specific antibody in clinical radioimmunoassays for the measurement of drug or hormone levels.

Thymus cells suppress the in vitro cytotoxic response against trinitrophenyl (TNP) modified syngeneic cells.

The regulatory role of adult thymocytes in the in vitro cytotoxic cell formation against TNP-coupled syngeneic spleen cells was studied. Thymocytes are known to amplify the cytoxic response of lymph node cells against allogeneic cells. This kind of synergism was not found in the response against TNP-coupled cells; on the contrary, thymocytes had a clear suppressive effect. Thymocytes inducing this suppression must be present already at the beginning of the in vitro response. Mitotically blocked (mitomycin-C treated) thymocytes but not heat killed or lysed cells were also capable to suppress anti-TNP cytotoxicity.

Generation of H-2-restricted cytotoxic T cells by ultraviolet light-treated trinitrophenyl-modified syngeneic cells: increased requirement for adherent cells.

Metabolically inactive (ultraviolet light- [UV] irradiated) cells are incapable of serving as allogeneic stimulators in a mixed lymphocyte reaction (MLR). The reason for the requirement of metabolic activity is not known. Now we have used UV-irradiated, trinitrophenyl- (TNP) coupled syngeneic spleen cells as stimulators in vitro to generate TNP-specific cytotoxic T cells (CTL). It was found that UV-irradiated cells were stimulatory only if adherent cells (nylon wool [NW] and carbonyl iron-adherent Thy-1-) were present in the responder cell population. Nonadherent allogeneic cells were also able to augment the CTL response to UV-irradiated stimulators, suggesting that the requirement for adherent cells can be replaced by the nonspecific stimulatory effects of a MLR. When spleen cells from mice primed in vivo with TNP-coupled syngeneic cells were used in vitro, it was noticed that UV-irradiated stimulators were able to induce as strong a secondary CTL response as metabolically active stimulators but this response was also entirely dependent on the presence of adherent cells in the responder cell population. As a summary, these results suggest that metabolically inactive haptenated stimulators do not present the necessary proliferative (?) stimulus to the CTL precursor cells, but this 'signal' can be mediated via the adherent cell population, thus offering an explanation for the significance of this cell type in CTL responses in vitro.