Identification of alpha interferon-induced genes associated with antiviral activity in Daudi cells and characterization of IFIT3 as a novel antiviral gene.

A novel assay was developed for Daudi cells in which the antiviral (AV) and antiproliferative (AP) activities of interferon (IFN) can be measured simultaneously. Using this novel assay, conditions allowing IFN AV protection but no growth inhibition were identified and selected. Daudi cells were treated under these conditions, and gene expression microarray analyses were performed. The results of the analysis identified 25 genes associated with IFN-α AV activity. Upregulation of 23 IFN-induced genes was confirmed by using reverse transcription-PCR. Of 25 gene products, 17 were detected by Western blotting at 24 h. Of the 25 genes, 10 have not been previously linked to AV activity of IFN-α. The most upregulated gene was IFIT3 (for IFN-induced protein with tetratricopeptide repeats 3). The results from antibody neutralizing experiments suggested an association of the identified genes with IFN-α AV activity. This association was strengthened by results from IFIT3-small interfering RNA transfection experiments showing decreased expression of IFIT3 and a reduction in the AV activity induced by IFN-α. Overexpression of IFIT3 resulted in a decrease of virus titer. Transcription of AV genes after the treatment of cells with higher concentrations of IFN having an AP effect on Daudi cells suggested pleiotropic functions of identified gene products.

Human IFN-alpha protein engineering: the amino acid residues at positions 86 and 90 are important for antiproliferative activity.

Human IFN-alpha is a family of structurally related proteins that exhibit a wide range of antiproliferative activities. To understand the structural basis for these different antiproliferative activities, eight recombinant human IFN-alpha hybrids (HY) of alpha21a/alpha2c (HY-4, HY-5) and mutants (site-directed mutagenesis (SDM)-1, 2 and cassette mutagenesis (CM)-1, 2, 3, and 4) have been expressed, purified, and characterized. The data showed that the amino acid region 81-95 is important for antiproliferative activity. Site-directed mutagenesis and cassette mutagenesis studies showed that if serine (S) 86 and asparagine (N) 90 were replaced by tyrosine (Y), the antiproliferative activity was increased. We have also observed that if Y86 was replaced by isoleucine (I), the antiproliferative activity was comparable. However, if Y86 was replaced by aspartic acid (D), lysine (K), or alanine (A), the antiproliferative activity was substantially decreased. Our results indicate that Y and/or I at position 86 and Y at position 90 are very important in antiproliferative activity of human IFN-alpha. Circular dichroism spectra showed that the amino acid replacements at position 86 did not change the secondary structure. Thus the biological activity changes among those mutants do not appear to be due to conformational changes. The results also suggest that hydrophobic residue(s) at position 86 may be important for the interaction of the molecule with its receptor. The competitive binding data correlated with the antiproliferative activity. The N-terminal region of the molecule and the hydrophobic residues (including Y and I) on the C-helix region at positions 86 and/or 90 are important for binding and antiproliferative activities of human IFN-alphas.

Significance of anti-interferon-alpha2 and sICAM-1 activities in the sera of viral hepatitis B and C patients treated with human recombinant interferon-alpha2.

In this study the presence of an IFN-binding activity in the sera of patients with chronic viral hepatitis B or C treated with rIFN-alpha2 was screened by a radioimmune assay (RIA) using radiolabeled rIFN-alpha2. Incidence of an anti-IFN activitywas compared with hepatitis B virus (HBV) or hepatitis C virus (HCV) serum markers as hepatitis B s antigen (HBsAg), hepatitis B e antigen (HBeAg), antibodies to HBsAg (anti-HBsAg), antibodies to HBeAg (anti-HBeAg), seroconversion, HBV DNA, HCV RNA, and serum soluble intracellular adhesion molecule I (sICAM). Injections (intramuscular) of rIFN-alpha2 caused an anti-rIFN activity formation in 8 (27.6%) of 29 patients with chronic active hepatitis B (CAH-B) and in 8 (30.8%) of 26 patients with chronic active hepatitis C (CAH-C). The presence of the anti-rIFN activity in CAH-B patients correlated frequently with the persistence of HBsAg, HBeAg and HBV-DNA, while its absence was often accompanied by the anti-HBeAg and anti-HBsAg seroconversion, respectively, and HBV-DNA negativity. In two CAH-C patients who became HCV RNA-negative no anti-IFN activity was found. Levels of serum sICAM-1 in CAH-B patients responding to the IFN treatment were higher than those in non-responders or in which the anti-IFN activity was present. The anti-IFN activity may negatively influence the effect of the IFN therapy of CAH-B or CAH-C patients at early stages of the therapy. The appearance of the anti-IFN activity at the end of a long-term IFN therapy does not seem to influence the outcome of the therapy. sICAM-1 may be involved in the process of CAH-B reactivation and IFN-triggered cytotoxicity during the IFN therapy.

Radioiodination of human interferon-alpha2 interferes with binding of C-terminal specific antibodies.

Radioimmunoassays based on reactivity between a monoclonal antibody (mAb) and human 125I-interferon (IFN)-alpha2 are frequently exploited in interferon research. In general, epitopes of antibodies specific for human IFN-alpha2 are located on the two immunodominant structures formed in the N- and C-terminal domains, respectively. We found that labelling of IFN-alpha2 with Na(125)I by the chloramine-T method did not affect the binding of antibodies recognising the N-terminal region 30-53. In contrast, radioiodination of IFN was associated with a dramatic decrease in IFN reactivity with mAbs specific for the C-terminus (residues approximately 120-145 approximately ). We suggest that steric hindrance araising from the incorporation of 125I into the tyrosine residues at positions 123, 130 and 136 may be responsible for the change in immunoreactivity. The adverse effect of radioiodination of IFN-alpha2 on the binding potency of C-terminal specific mAbs must be taken into consideration in experiments based on the interaction of such antibodies (i.e. NK2) with the labelled antigen.

Stereological analysis in scanning electron microscopy.

The traditional field of stereological analysis based on quantitative image analysis data is expanded to include scanning electron microscopy (SEM). Two major methodological advances enable useful results to be obtained: 1) Image recording of backscattered electrons using an annular semi-conductor detector; 2) Direct link between SEM and image analyser by means of an on-line coupling providing synchronisation of both sub-systems.

[Coronary angiographic findings in 308 patients with the clinical diagnosis of intramural myocardial infarction (author's transl)]. / Der nicht transmurale Herzinfarkt--ein diagnostisches Problem. Koronarangiographische Befunde bei 308 Patienten, bei denen klinisch di Diagnose eines nicht transmuralen Herzinfarktes gestellt worden war.

308 patients with the clinical diagnosis of intramural myocardial infarction made elsewhere were re-investigated more than eight weeks after the acute event. ECGs and pulmonary "wedge" pressures were recorded at rest and during exercise and coronary angiography performed (Sones' or Judkin's technique). In the first group (1973/74) of 77 patients, 35 (45.5%) had a normal coronary angiogram, compared with 0.7% in a control group with transmural myocardial infarction. A normal coronary angiogram was found in 85% of the 40 patients who had no angina during exercise. In a second group (1974/77) of 231 patients, there was a steady decrease in the number of patients without angina pectoris during exercise, in parallel with a decrease in the number of those with normal coronary angiograms. This change in pattern was apparently due to improved diagnosis in the referring hospitals. Of the 37 patients (first group) with angina during exercise, all but one were subsequently found to have significant coronary arteriosclerosis. It is suggested that most patients with a normal coronary angiogram had sustained a myocarditis and (or) pericarditis which produced the symptoms and the altered ECGs, leading to the misinterpretation of "intramural myocardial infarction".