High-yield expression and purification of human interferon alpha-1 in Pichia pastoris.

For several years, interferon alpha-1, also known as interferon alpha-D, has been studied for treatment of various viral diseases, such as hepatic fibrosis caused by hepatitis B, herpes simplex virus keratitis, and bovine respiratory diseases in calves. Currently, recombinant human interferon alpha-D (rHuIFNalphaD) is expressed intracellularly in Escherichia coli or secreted by Bacillus subtilis and Saccharomyces cerevisiae. In this report, we describe the process of obtaining a relatively high-yield secretion of biologically active recombinant rHuIFNalphaD using the Pichia pastoris system. The process produced as high as 0.7 mg of purified protein per 20 ml of shake culture of rHuIFNalphaD with better bioactivity than the commercially available rHuIFNalphaD molecule produced in E. coli.

Functional characterization of monoclonal antibodies to interferon-tau.

Interferon tau (IFNtau) produces an array of biological effects, including antiluteolytic, antiviral, antiproliferative and immunomodulatory activities, without the consequent cytotoxicity associated with other type I IFNs. Four anti-IFNtau monoclonal antibodies (MAbs) have been characterized by determining regional epitopes and observation of their effects on IFNtau binding, antiviral and antiproliferative activity. Using an enzyme-linked immunoadsorbent assay (ELISA) developed against six overlapping synthetic peptides representing the entire linear sequence of IFNtau, three antibodies, HL-98, HL-100 and HL-127, were found to react with the carboxy terminal peptide, while HL-129 bound the penultimate amino terminal peptide. Binding studies indicated that MAbs directed against either region could effectively inhibit the binding of alkaline phosphatase labeled IFNtau to cells expressing type I IFN receptors. While only two of the MAbs significantly reversed IFNtau-induced growth inhibition, the antiviral activity of IFNtau was significantly inhibited by MAbs that bound the amino and carboxy termini, confirming the functional importance of these domains in the binding and subsequent activity of IFNtau.

Nitric oxide regulation of interleukin-8 gene expression.

While the regulation of nitric oxide (NO) by inflammatory cytokines or lipopolysaccharide (LPS) has received considerable attention, NO modulation of cytokine expression has yet to be fully explored. The NO synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME), inhibited interleukin (IL)-8 and IL-6 production in LPS-stimulated human whole blood in a dose-dependent manner. In the presence of 1 microgram/mL LPS, L-NAME blocked IL-8 release (72 +/- 4% inhibition at 20 mM (mean +/- SEM, p < .05)) 24 h post-LPS without affecting cellular viability. IL-6 production was significantly inhibited only with the highest dose of L-NAME used. L-NAME inhibition of IL-8 production was also observed at the mRNA level. Conversely, direct exposure of whole blood to NO with the spontaneous NO liberator DETA NONOate caused a dose-dependent stimulation of IL-8, but had no effect on IL-6 release. IL-8 concentrations rose from 8.3 +/- 1.9 ng/mL at 24 h to 31.7 +/- 7.6 ng/mL at 72 h with a single stimulation of 10 mM DETA NONOate. The hydroxyl radical scavenger dimethyl sulfoxide (DMSO) prevented the DETA NONOate induction of IL-8, suggesting the participation of the hydroxyl radical in the NO-induced IL-8 production. These results provide important evidence substantiating a role for NO as a regulator of cytokine expression.

Transcriptional and post-transcriptional regulation of interleukin-8.

Steady-state mRNA for interleukin (IL) 8 persists significantly longer than mRNA for tumor necrosis factor (TNF)-alpha in lipopolysaccharide (LPS) stimulated human whole blood. Nuclear run-ons demonstrated consistent levels of transcriptional activity of the IL-8 gene at 2 and 26 hours after LPS stimulation when compared with the rapid induction and arrest of the TNF-alpha gene. Inhibition of cellular transcription with actinomycin D added at 2 hours after LPS resulted in the substantial decrease of both IL-8 and TNF-alpha mRNAs, demonstrating half-lives of 4.6 and 2.3 hours, respectively. In contrast, inhibition of transcription at 23 hours after LPS revealed extremely stable IL-8 mRNA with a half-life of > 10 hours. The half-life of beta-actin in the same actinomycin-D-treated samples did not vary significantly from the half-life calculated at the 2-hour time point (5.5 hours versus 5.6 hours), indicating that the observed IL-8 mRNA stability was not an artifact of the system. Both IL-8 and TNF-alpha protein levels decreased when actinomycin D was added 2 hours after LPS stimulation. However, no effect in IL-8 protein levels was evident when actinomycin D was added at 23 hours after LPS. These results demonstrate that IL-8 mRNA stability is controlled at both the transcriptional and posttranscriptional levels of gene regulation.

Nitric oxide regulation of IL-8 expression in human endothelial cells.

The regulatory signals required to induce the production of IL-8, an important neutrophil chemoattractant and activator, have yet to be clearly defined. We examined the role of nitric oxide (NO) in IL-8 regulation. The NO synthase inhibitor, (L)-NG-nitroarginine methyl ester (L-NAME), inhibited the TNF-stimulated IL-8 production in the human endothelial cell line, ECV304, in a dose-dependent manner without affecting cellular viability (TNF alone, 5.5 +/- 0.9 ng/ml; TNF + 5 mM L-NAME, 2.4 +/- 0.5 ng/ml). Moreover, exogenously added NO produced by the spontaneous NO generating compounds, S-Nitroso-N-acetyl-D,L-pennicillamine (SNAP) and Ethanamine, 2,2'-(hydroxynitrosohydrazono)bis- (DETA NONOate), induced a dose-dependent release of IL-8 from these cells. Maximal stimulation of IL-8 was found to be 1.2 +/- 0.1 ng/ml with the 1 mM concentration of SNAP and 1.6 +/- 0.1 ng/ml with the 2 mM concentration of DETA NONOate. These results provide key evidence substantiating a regulatory role of NO in IL-8 expression.