Purification and characterization of prokaryotically expressed human interferon-λ2

A system for the production of soluble interferon (IFN)-λ2 was developed by fusing the IFN-λ2, NusA protein, polyhistidine and S peptide genes and then expressing the fused product (Nus-His-S-tagged IFN-λ2) in Escherichia coli. The expressed fusion protein was purified by Ni-NTA affinity chromatography. The fusion tag was removed from recombinant IFN-λ2 by cleavage with enterokinase. N-Terminal sequencing confirmed the identity of the purified protein. When compared with commercial IFN-α2b, IFN-λ2 had similar antiviral activity. The production and characterization of biologically active IFN-λ2 will be beneficial for its potential role in clinical applications.     

Signal peptide of Arabinosidase enhances secretion of interferon-α2b protein by <Emphasis Type="Italic">Bifidobacteria longum</Emphasis>

Bifidobacteria can potentially be used for gene therapy. Here, we reported that 65% of the total hIFN-α2b produced from Bifidobacteria longum transformed with pBAD-SPIFN plasmids encoding a fusion protein of the arabinosidase signal peptide and human IFN-α2b (hIFN-α2b), was secreted. For B. longum transformed with pBAD-IFN plasmids (hIFN-α2b without the signal peptide), only 15% of the total IFN-α2b was secreted and western blotting and N-terminal amino-acid sequence analysis revealed cleavage of the arabinosidase signal peptide from the secreted hIFN-α2b. Moreover, the active level of the secreted hIFN-α2b in the supernatant of B. longum transformed with pBAD-SPIFN plasmids was over 1,000 IU/ml commercial rhIFN-α2b. Hence, the arabinosidase signal peptide can enhance the secretion efficiency of IFN-α2b from B. longum. Q. Deng, W. Zeng and Z. Yu contributed equally to this work.     

Synonymous codon usage bias and overexpression of a synthetic gene encoding Interferon α2b in yeast

To achieve higher level expression of Interferon α2b (IFN-α2b) in methylotrophic yeast (Pichia pastoris), a cDNA fragment coding for the mature IFN-α2b was designed and synthesized based on the synonymous codon bias of P. pastoris and optimized G+C content. The synthetic IFN-α2b was inserted into the secreted expression vector pPICZαA, and then integrated into P. pastoris GS115 genome by electroporation. Multi-copy integrants in the Mut⁺ recombinant P. pastoris strain were screened by high concentrations of Zeocin. 120 hours culturing allowed expression of the IFN-α2b transformant up to 810 mg/L as detected by SDS-PAGE and quantitative methods. In addition, Western blot analysis showed that the recombinant proteins had immunogenicity. The significant antiviral activity of the recombinant IFN-α2b protein was verified by WISH/ VSV system, which was 3.3×10⁵ IU/mL. Foundation items: The National ‘973’ Basic Research Program (2002CB111302); The National Natural Science Foundation of China (30370807)     

PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes

 The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-γ (IFN-γ)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 α- and β-subunits. The deduced amino acid sequences of the α- and β-subunits were 49% identical. We also determined the primary structure of the mouse PA28 γ-subunit (Ki antigen), a protein of unknown function structurally related to the α- and β-subunits. The amino acid sequence identity of the γ-subunit to the α- and β-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the α- and β-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-γ-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the γ-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a γ-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-γ-inducible α- and β-subunits emerged by gene duplication from a γ-subunit-like precursor. Received: 11 March 1997     

Designing of hybrid human interferon alfa-2 strain-producers and the use of enteropeptidase for obtaining N-terminal methionine-free interferons

A system for production of human interferon-alpha2a (IFN-alpha2a) and IFN-alpha2b lacking N-terminal methionine has been developed. Plasmids containing genes of hybrid IFN-alpha2 under the control of different promoters were constructed; a sequence encoding the enteropeptidase hydrolysis site being introduced in proximal part of the genes. As the result, 4 strains of Escherichia coli producing hybrid IFN-alpha2 have been obtained. The methodology for IFN-alpha2 renaturation, hydrolysis of its N-terminal part, chromatographic purification of N-terminal methionine-free IFN-alpha2 has been developed.     

Interferon-γ- and lipopolysaccharide-induced tumor necrosis factor-α is required for nitric oxide production: Tumor necrosis factor-α and nitric oxide are independently involved in the killing ofMycobacterium microti in interferon-γ-and lipopolysaccharide-treated J774A.1 cells

A comparative study was done using J774A.1 and J774A. 1-derived transfected cells (J774A.1 C.1) containing antisense tumor necrosis factor α (TNF-α) plasmid to determine the role of endogenous TNF-α on nitric oxide production as well as on the growth ofMycobacterium microti in interferon γ (IFN-γ)- and lipopolysaccharide (LPS)-treated cells. On stimulation with IFN-γ and LPS a higher level of NO was observed in J774A.1 cells compared to J774A.1 C.1 which indicated that endogenous TNF-α is required for the production of NO. Comparing the effect of IFN-γ and LPS on the intracellular growth ofM. microti, the growth-reducing activity was higher in J774A.1 cells than in J774A.1 C.1 cells and was not completely abrogated in the presence of the nitric oxide inhibitorN ^G-methyl-l-arginine (l-NMA). J774A.1 C.1 cells infected withM. microti produced a significant amount of NO when exogenous TNF-α was added along with IFN-γ and LPS and the concentration of intracellular bacteria decreased almost to that in IFN-γ and LPS treated parental J774A.1 cells. Addition of exogenous TNF-α even in the presence ofl-NMA in J774.1 C.1 cells could also partially restore intracellular growth inhibition ofM. microti caused by IFN-γ and LPS. TNF-α is probably required for the production of NO in J774A.1 cells by IFN-γ and LPS but TNF-α and NO are independently involved in the killing of intracellularM. microti with IFN-γ and LPS.     

Comparison of expression systems for the production of human interferon-α2b

The production of human interferon alpha2b (IFN-α2b) in two expression systems, tobacco (Nicotiana tabaccum) and Escherichia coli, was compared in various aspects such as safety, yield, quality of product and productivity. In the E. coli system, IFN-α2b was expressed under a pelB signal sequence and a T7lac promoter in a pET 26b(+) vector. The same gene was also cloned in expression plant vector (pCAMBIA1304) between cauliflower mosaic virus promoter (CaMV35S) and poly A termination region (Nos) and expressed in transgenic tobacco plants. The expression of protein in both systems was confirmed by western immunoblotting and the quantity of the protein was determined by immunoassay. The amount of periplasmic expression in E. coli was 60 μg/L of culture, while the amount of nuclear expression in the plant was 4.46 μg/kg of fresh leaves. The result of this study demonstrated that IFN-α2b was successfully expressed in periplasm of bacterial and plant systems. The limitations on the production of IFN-α2b by both systems are addressed and discussed to form the basis for the selection of the appropriate expression platform.     

Different physiology of interferon-α/-γ in models of liver regeneration in the rat

Liver regeneration may take place after liver injury through replication of hepatocytes or hepatic progenitor cells called oval cells. Interferons (IFN) are natural cytokines with pleiotrophic effects including antiviral and antiproliferative actions. No data are yet available on the physiology and cellular source of natural IFNs during liver regeneration. To address this issue, we have analyzed the levels and biologic activities of IFN-α/IFN-γ in two models of partial hepatectomy. After 2/3rd partial hepatectomy (PH), hepatic levels of IFN-α and IFN-γ declined transiently in contrast to a transient increase of the IFN-γ serum level. After administration of 2-acetylaminofluorene and partial hepatectomy (AAF/PH model), however, both IFN-α and IFN-γ expression were up-regulated in regenerating livers. Again, the IFN-γ serum level was transiently increased. Whereas hepatic IFN-γ was up-regulated early (day 1–5), but not significantly, in the AAF/PH model, IFN-α was significantly up-regulated at later time points in parallel to the peak of oval cell proliferation (days 7–9). Biological activity of IFN-α was shown by activation of IFN-α-specific signal transduction and induction of IFN-α specific-gene expression. We found a significant infiltration of the liver with inflammatory monocyte-like mononuclear phagocytes (MNP) concomitant to the frequency of oval cells. We localized IFN-α production only in MNPs, but not in oval cells. These events were not observed in normal liver regeneration after standard PH. We conclude that IFN-γ functions as an acute-phase cytokine in both models of liver regeneration and may constitute a systemic component of liver regeneration. IFN-α was increased only in the AAF/PH model, and was associated with proliferation of oval cells. However, oval cells seem not to be the source of IFN-α. Instead, inflammatory MNP infiltrating AAF/PH-treated livers produce IFN-α. These inflammatory MNPs may be involved in the regulation of the oval cell compartment through local expression of cytokines, including IFN-α.     

Treatment with TNF-α and IFN-γ alters the activation of SER/THR protein kinases and the metabolic response to IGF-I in mouse c2c12 myogenic cells

The aim of this study was to compare the effects of TNF-α, IL-1β and IFN-γ on the activation of protein kinase B (PKB), p70^S6k, mitogen-activated protein kinase (MAPK) and p90 ^rsk , and on IGF-I-stimulated glucose uptake and protein synthesis in mouse C2C12 myotubes. 100 nmol/l IGF-I stimulated glucose uptake in C2C12 myotubes by 198.1% and 10 ng/ml TNF-α abolished this effect. Glucose uptake in cells differentiated in the presence of 10 ng/ml IFN-γ increased by 167.2% but did not undergo significant further modification upon the addition of IGF-I. IGF-I increased the rate of protein synthesis by 249.8%. Neither TNF-α nor IFN-γ influenced basal protein synthesis, but both cytokines prevented the IGF-I effect. 10 ng/ml IL-1β did not modify either the basal or IGF-I-dependent glucose uptake and protein synthesis. With the exception of TNF-α causing an 18% decrease in the level of PKB protein, the cellular levels of PKB, p70^S6k, p42^MAPK, p44^MAPK and p90 ^rsk were not affected by the cytokines. IGF-I caused the phosphorylation of PKB (an approximate 8-fold increase above the basal value after 40 min of IGF-I treatment), p42^MAPK (a 2.81-fold increase after 50 min), and the activation of p70^S6k and p90 ^rsk , manifesting as gel mobility retardation. In cells differentiated in the presence of TNF-α or IFN-γ, this IGF-I-mediated PKB and p70^S6k phosphorylation was significantly diminished, and the increase in p42^MAPK and p90 ^rsk phosphorylation was prevented. The basal p42^MAPK phosphorylation in C2C12 cells treated with IFN-γ was high and comparable with the activation of this kinase by IGF-I. Pretreatment of myogenic cells with IL-1β did not modify the IGF-I-stimulated phosphorylation of PKB, p70^S6k, p42^MAPK and p90 ^rsk . In conclusion: i) TNF-α and IFN-γ, but not IL-1β, if present in the extracellular environment during C2C12 myoblast differentiation, prevent the stimulatory action of IGF-I on protein synthesis. ii) TNF-α- and IFN-γ-induced IGF-I resistance of protein synthesis could be associated with the decreased phosphorylation of PKB and p70^S6k. iii) The activation of glucose uptake in C2C12 myogenic cells treated with IFN-γ is PKB independent. iv) The similar effects of TNF-α and IFN-γ on the signalling and action of IGF-I on protein synthesis in myogenic cells could suggest the involvement of both of these cytokines in protein loss in skeletal muscle.     

Effects of interferon-alpha subtypes on the Th1/Th2 balance in peripheral blood mononuclear cells from patients with hepatitis virus infection-associated liver disorders

Summary Interferon-alpha (IFN-α) has recently been shown to modulate in vitro T helper (Th) 1-driven responses in the peripheral blood mononuclear cells (PBMC) of patients with hepatitis B virus or C virus infection. In this study, we examined the in vitro effects of IFN-α subtypes (IFN-α1, −α2, −α5, −α8, and −α10) on the Th1/Th2 balance in PBMC obtained from patients with hepatitis virus infection-associated liver disorders and chronic hepatitis (CH), in comparison with the effect on healthy control volunteer PBMC. The Th1-type cell percentages and Th1/Th2 ratios were significantly higher in the PBMC of patients when compared with controls both before and after cultivation in vitro, with the IFN-α subtypes. The IFNα-5 induced an increase in the Th2-type cell percentages in both control and patient PBMC, resulting in that IFN-α5 lowered the Th1/Th2 ratio in patients with CH. Furthermore, statistical analysis revealed that IFN-α8 significantly promoted an increase in the Th1/Th2 ratios of PBMC from patients with CH and liver cirrhosis (LC) but not that of PBMC from patients with LC-hepatocellular carcinoma (HCC) and HCC. These findings imply that hepatitis virus infection and its disease status modify the effects of IFN-α subtypes on Th1 and Th2 immune balance in patients. Our findings should help to elucidate the mechanisms underlying successful IFN therapy for hepatitis virus infection and prevention of hepatocellular carcinogenesis.     

Functional genomics of PPAR-γ in human immunomodulatory cells

Keeping in view the fact that peroxisome-proliferators activated receptors-PPARs (α,γ) play a crucial role in atherogenic inflammation, the present study was addressed to explore as to how selective and specific PPAR-γ gene silencing within human mononuclear cells affects genes involved in lipid metabolism and innate immune process. Such a study revealed that with respect to control cells, the PPAR-γ knock-out cells exhibited significant reduction in the expression of genes coding for PPAR- α, CD-36, LDL-R as well as significant increase in the expression of genes coding for IL-4, IL-8, IFN-γ, CX3CR1, hTERT. However, the expression of genes coding for LXR-α and Receptor-C _k could not be detected in PPAR-γ knock-out cells. Based on these results, we propose that PPAR-γ gene has the inherent capacity to influence the lipid mediated inflammation process in atherosclerotic lesions.     

NMR Based Metabonomics Study of DAG Treatment in a C2C12 Mouse Skeletal Muscle Cell Line Myotube Model of Burn-Injury

After severe burn injury, proinflammatory cytokine levels are elevated in serum and skeletal muscle, which in turn increases protein breakdown and decreases protein synthesis. In this study, C2C12 mouse skeletal muscle cell line myotubes were exposed to proinflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) as an in vitro cell-line model of catabolic response to burn injury and then treated with des-acyl ghrelin (DAG), a 28 amino acid polypeptide hormone thought to inhibit protein breakdown and increase protein synthesis, to assess its therapeutic potential. Nuclear magnetic resonance-based metabonomics was used to monitor metabolic activity of C2C12 myotubes under four treatment conditions: (1) control, (2) TNF-α/IFN-γ (TI), (3) DAG (DA), and (4) TNF-α/IFN-γ followed by DAG (TIDA) to assess the effect of DAG treatment on cellular metabolic response during basal or catabolic conditions. Twelve metabolites showed significant changes in concentrations following treatments in the hydrophilic cell extracts. Lactate (P < 10⁻⁴) and citrulline (P < 10⁻⁹) increased with TNF-α/IFN-γ treatment, indicating increased protein degradation, and returned to control levels in the TIDA group. Adenosine nucleotide levels had decreased trends in TI myotubes that returned to baseline levels after DAG treatment (P < 10⁻⁴). Guanidinoacetate and pantothenate, metabolites involved in protein synthesis and cell proliferation, had increased concentration trends following DAG treatment in both the DA and TIDA groups. Our metabonomics analysis provides further evidence that DAG counteracts the catabolic response caused by elevated muscle TNF-α/IFN-γ cytokine levels following severe burns and can play a potential therapeutic role in treatment of burn injury.     

Recombinant bacillus Calmette-Guérin (BCG) expressing interferon-alpha 2B enhances human mononuclear cell cytotoxicity against bladder cancer cell lines in vitro

Purpose  The proper induction of cellular immunity is required for effective bacillus Calmette-Guérin (BCG) immunotherapy of bladder cancer. It has been known that BCG stimulation of human peripheral blood mononuclear cells (PBMC) leads to the generation of effector cells cytotoxic to bladder cancer cells in vitro. To improve BCG therapy, we previously developed human interferon (IFN)-α 2B secreting recombinant (r) BCG (rBCG-IFN-α). We demonstrated that rBCG-IFN-α augmented T helper type 1 (Th1) cytokine IFN-γ production by PBMC. In this study, we further investigated whether rBCG-IFN-α could also enhance PBMC cytotoxicity toward bladder cancer cells. Materials and methods  PBMC were prepared from healthy individuals, left alone or stimulated with rBCG-IFN-α or control MV261 BCG, and used as effector cells in ⁵¹Cr-release assays. Human bladder cancer cell lines T24, J82, 5637, TCCSUP, and UMUC-3 were used as target cells. To determine the role of secreted rIFN-α as well as endogenously expressed IFN-γ and IL-2 in inducing the cytotoxicity, PBMC were stimulated with rBCG-IFN-α in the presence of neutralizing antibodies to IFN-α, IFN-γ or IL-2. To determine the role of natural killer (NK) and CD8⁺ T cells in inducing the cytotoxicity, both cell types were isolated after BCG stimulation of PBMC and used as effector cells in ⁵¹Cr-release assays. Results  Non-stimulated PBMC showed basal levels of cytotoxicity against all target cell lines tested. MV261 BCG increased the PBMC cytotoxicity by 1.8- to 4.2-fold. rBCG-IFN-α further increased the PBMC cytotoxicity by up to 2-fold. Elevated production of IFN-γ and IL-2 by PBMC was observed after rBCG-IFN-α stimulation. Blockage of IFN-α, IFN-γ or IL-2 by neutralizing antibodies during rBCG-IFN-α stimulation reduced or abolished the induction of PBMC cytotoxicity. Both NK and CD8⁺ T cells were found to be responsible for the enhanced PBMC cytotoxicity induced by rBCG-IFN-α with the former cell type being more predominant. Conclusions  rBCG-IFN-α is an improved BCG agent that induces enhanced PBMC cytotoxicity against bladder cancer cells in vitro. This rBCG strain may serve as an alternative to BCG for the treatment of superficial bladder cancer.     

Production of interferons and change of the lymphocyte subpopulation phenotype in peripheral blood at cervical papillomavirus infection

IFN-γ and IFN-α productionin vitro by peripheral blood cells activated by phytohemagglutinin or the Newcastle disease virus was impaired in patients with a benign process, cervical intraepithelial neoplasm and cancerin situ associated with human papillomavirus infection. In case of IFN-γ and IFN-α production impairment following cervical papillomavirus infection, the increased severity of disease was accompanied by remarkable IFN system suppression. The lower synthesis of both IFN correlated with changes of some lymphocyte-subpopulation phenotype in peripheral blood. Lower CD4⁺ and CD3⁺ DR⁺ T cell concentrations were observed in papillomavirus-infected patients with impaired IFN production; impaired IFN-γ production was accompanied by lower CD4/CD8 index.     

Preliminary study of combination therapy with interferon-α and zinc in chronic hepatitis C patients with genotype 1b

We have evaluated the efficacy of interferon-α (IFN-α) plus zinc therapy in hepatitis C patients with genotype 1b, poor responders for IFN alone. Ten patients were injected with 10 MU of IFN-α every day for 4 wk, followed by three times a week for 20 wk (control group). Nine patients took 300 mg of zinc sulfate a day orally during IFN-α therapy (zinc sulfate group), and 15 patients took IFN-α and 150 mg of polaprezinc (polaprezinc group). On the d 8 of IFN therapy, circadian zinc levels in serum elevated significantly in the polaprezinc group compared to the zinc sulfate group or control group. Serum ALT levels normalized in 73.3% of the polaprezinc group, 55.6% of the zinc sulfate group, and 40.0% of the control group at 6 mo after the end of IFN therapy. Sustained eradication for the hepatitis C virus RNA judged at the end of the 6-mo follow-up period was higher in the polaprezinc group than in the zinc sulfate group (53.3% vs 11.1%, p<0.05) or the control group (20.0%). No clinical side effects of zinc were observed at the dose used. The data suggest that polaprezinc is expected to increase the therapeutic response of IFN-α for chronic hepatitis C with genotype 1b.