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In the present study, a pre-B cell leukemia L1210-C7, representing a very early stage of the B lineage, was used to characterize the molecular mechanisms exploited by IFN-gamma to modulate B cell activity. A cDNA library was prepared with poly (A)+ RNA from cells stimulated with IFN-gamma and three cDNAs clones complementary to IFN-gamma inducible mRNAs were isolated by differential screening. Of these, the 9.5 cDNA hybridized to a 2.4 kb mRNA not homologous with previously cloned IFN-gamma inducible mRNAs. Furthermore, when compared with RNAs obtained from cells of different origins (fibroblasts and T cells) the 9.5 mRNA appeared to be increased only in cells belonging to the B lineage. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the expression of a gene that could be employed as specific cell activation marker.

When treated with IFN-alpha, NIH-3T3 cells express after a few hours high levels of the mouse 202 gene mRNA. This activation takes place at the transcriptional level as shown by nuclear "run on" assay. For this purpose a fragment of 806 base-pairs (the b fragment), spanning the 5'-flanking region of the 202 gene, was linked to the reporter CAT gene and transiently transfected into mouse NIH 3T3. The data suggest that the b fragment is sufficient to confer transcriptional inducibility upon IFN stimulation and can account in large part for the response of the 202 gene. Binding assays, using a 40-bp probe derived from the IFN-stimulated response element (ISRE) comprised in the b fragment, demonstrated the presence of two DNA-binding proteins. One of these, defined as complex A, was inducible upon IFN treatment, whereas the other, defined as complex B, was constitutively present regardless of IFN treatment. The IFN-alpha-induced complex A appears to have the necessary characteristics to be the transcriptional activator of the 202 gene: it requires the same nucleotides for binding as are required for IFN-dependent gene activation and is dependent on IFN-alpha treatment.

High serum levels of antibodies to interferon-gamma (IFN-gamma) have been found in patients infected with human immunodeficiency virus (HIV). A radioimmunoassay (RIA) with a recombinant IFN-gamma protein or an affinity purified IFN-gamma preparation as antigens, was developed to detect the specific anti-IFN-gamma antibodies. Reactivity of sera to IFN-gamma was confirmed by Western blot analysis. These antibodies, however, do not seem to recognize the active site of the molecule, since they do not neutralize the antiviral IFN-gamma activity in a biological assay. These results enforce the hypothesis of the role of autoimmunization during the course of the disease.

A 17-aminoacid peptide corresponding to the C terminal of the smaller form of human 2'-5' oligoadenylate synthetase was coupled to keyole lymphet haemocyanin (KLH) and used as immunogen in rabbits. After a cycle of four immunizations two animals produced immunoglobulins able to recognize the 17-aminoacid peptide as evaluated in ELISA assays. The specific Ig were purified by an immunoadsorbent with the peptide immobilized on Sepharose CL-4B and used in Western blot employing either protein A iodinated or conjugated with peroxidase as indicator system. The results obtained using extracts from HeLa or WISH cells treated for 15 hr with HuIFN-alfa as antigen demonstrate that the anti-peptide antibodies recognize the 40 kDa form of the 2'-5' oligoadenylate synthetase enzyme complex. These antibodies therefore represent a useful tool for monitoring the induction of the above enzyme.

We compared the activity of human recombinant alpha and gamma interferons (IFNs) on normal T lymphocytes and various T cell lines. IFN gamma, unlike IFN alpha, did not promote the antiviral state in these cells, or induce the activity of 2'-5' oligoadenylate synthetase. The lack of antiviral effect was observed using an RNA virus (VSV) and a DNA virus (HSV, type 1) as challenger viruses.