Quantitative PCR identifies a minimal deleted region of 120 kb extending from the Philadelphia chromosome ABL translocation breakpoint in chronic myeloid leukemia with poor outcome.

Fluorescence in situ hybridization (FISH) analysis has shown previously that 10-15% of chronic myeloid leukemias (CML) have hemizygous deletions of variable sizes affecting regions that flank the ABL and BCR translocation breakpoints on the derivative chromosome 9, and these patients have a poor outcome. FISH studies using large commercial genomic probes have previously suggested that haploinsufficiency of sequences flanking either ABL or BCR modify the disease process of CML and lead to an unfavorable prognosis. In this present study, real-time quantitative PCR (Q-PCR) analysis was used to identify and map much smaller hemizygous microdeletions in a subset of CML patients that were not deleted using large genomic FISH probes. Microdeletions were identified by Q-PCR in 25 of 71 patients selected based on less favorable outcome (chronic phase duration of less than 96 months and a survival time of less than 84 months). In contrast, no microdeletion was detected in any of 18 CML samples selected from a group with a more favorable outcome. Detailed mapping of the 25 Q-PCR microdeletions showed that the minimal deleted region extended approximately 120 kb from the 5' end of the ABL gene in the centromeric direction on the derivative chromosome 9, and the region 3' to BCR on chromosome 22 was excluded. Of the four ESTs and/or genes that map to the 120 kb region, the putative tumor suppressor PRDM12 is the strongest candidate gene. The potential role for each sequence in modifying the clinical behavior of CML is presented.

In vitro suppression of anti-DNA antibody and immunoglobulin synthesis in systemic lupus erythematosus patients by human gamma interferon.

The immunoregulator human gamma interferon (IFN-gamma) suppressed the spontaneous in vitro synthesis and secretion of anti-DNA antibodies by peripheral blood mononuclear cells of patients with systemic lupus erythematosus (SLE-PBMC). Comparable level of suppression were observed with both natural human IFN-gamma and recombinant derived human IFN-gamma. In addition, the inhibitory effects of human IFN-gamma were completely neutralized by a monoclonal antibody directed against it. Human IFN-gamma also inhibited the antigen induced production of anti-DNA synthesis by SLE-PBMC. Based on the kinetics of inhibition, human IFN-gamma appeared to be acting directly on the B cell. Lastly, human IFN-gamma also suppressed the spontaneous production of IgG and IgM by SLE-PBMC. Our findings support the conclusion that SLE Ig production can be regulated and that human IFN-gamma may be clinically useful in the treatment of SLE as well as other immune complex diseases.

MIF-like activity of natural and recombinant human interferon-gamma and their neutralization by monoclonal antibody.

By utilizing elutriation-purified human monocytes, we found that human interferon (IFN) inhibits monocyte migration in a manner similar to migration inhibitory factor (MIF) and does it without demonstrable cytotoxicity. We observed that human IFN-gamma is 10 to 300 times more potent in its MIF activity than is IFN-alpha and that monoclonal antibodies (MoAb) can be used to distinguish between them. Studies with recombinant IFN-gamma indicate that the migration inhibition seen with natural IFN-gamma is due to IFN-gamma itself and is not due to co-purification of another lymphokine with the natural IFN-gamma. Although interferons exhibit MIF activities, there are apparently other cytokines, without antiviral activity, that also have MIF activities. MIF from the lymphoblastoid cell line RPMI 1788 was not neutralized by MoAb to IFN. However, MIF activity in supernatant fluid from human peripheral blood lymphocyte cultures stimulated with Con A-Sepharose was completely neutralized with MoAb anti-IFN-gamma. These data indicate that MIF is really a family of cytokines that inhibit macrophage/monocyte migration and that the major portion of MIF activity associated with crude supernatant of mitogen-stimulated lymphocytes is due to IFN-gamma.

Purification of human gamma-interferon to essential homogeneity and its biochemical characterization.

A multistep procedure has been developed which enables human gamma-interferon (HuIFN-gamma) to be purified to essential homogeneity. The procedure takes advantage of a modification of a previously described sequential chromatographic technique [Braude, I.A. (1983) Prep. Biochem. 13, 177-190] and the high isoelectric point of HuIFN-gamma (pH 9.5-9.8). The steps include Controlled Pore Glass adsorption chromatography, concanavalin A-Sepharose and heparin-Sepharose affinity chromatography, cation-exchange chromatography, and gel filtration chromatography. The purified HuIFN-gamma had a specific activity of 5.9 X 10(7) units/mg. This represents a purification of more than 70 000-fold and a 33% recovery. In addition, one gel filtration fraction had a specific activity of 2.5 X 10(8) units/mg. This represents a purification of greater than 300 000-fold and a recovery of greater than 17%. This fraction, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was shown to be composed of one major 26-kilodalton (kDa) species and four minor species of 74, 67, 56, and 22 kDa. Analysis of this material with anti-HuIFN-gamma monoclonal antibody immunoabsorbent columns indicates that both the 26- and the 22-kDa species are HuIFN-gamma. Thus, the final product is essentially homogeneous (90-92% HuIFN-gamma), and the specific activity of pure HuIFN-gamma is approximately (2.7-2.8) X 10(8) units/mg of protein. Finally, the 26- and 22-kDa moieties are shown to be similar, if not identical, proteins as judged by amino acid and sequence analyses.

A simple and efficient method for the production of human gamma interferon.

A method for the production of human gamma interferon (HuIFN-gamma), which is both simple and efficient, has been developed. The 2 co-inducers, A-23187 and mezerein, stimulate human peripheral blood lymphocytes to produce high levels of HuIFN-gamma in both stationary cultures and spinner cultures. The compound 2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione (FOD) has also proven to be a useful co-inducer. The production of HuIFN-gamma appears to occur in at least 2 phases.

A simple and efficient method for the purification of human gamma interferon.

A preparative, sequential chromatographic procedure has been developed for the purification of human gamma interferon (HuIFN-gamma). The four steps in the procedure are Controlled Pore Glass-adsorption chromatography, Concanavalin-A affinity chromatography, Heparin-Sepharose affinity chromatography and gel-filtration. By virtue of the development of a coordinated effluent-affluent buffer scheme, eluants can also serve as loading buffers for the succeeding column. Consequently, crude HuIFN-gamma preparations can be purified rapidly (approximately one week), easily, and is amenable to a semi-automated process. The procedure has also been shown to be efficient. Here, as an example, it is reported that an overall purification of greater than 75,000-fold can be achieved, yielding a specific activity of 5.2 X 10(7) units/mg, and a recovery of 95.5%. In addition, the peak fraction, representing 37.8% of the applied activity, had a specific activity of 1.0 X 10(8) units/mg protein and represents a purification of more than 145,000-fold. An SDS-PAGE analysis of one such fraction indicated that approximately 40% of the final material was HuIFN-gamma.

Further heterogeneity of human alpha interferon mRNA species.

Translationally active (in Xenopus oocytes) human alpha interferon (IFN) messenger RNA's (mRNA's) derived from Sendai virus--induced leukocyte cultures display a bimodal distribution of RNA lengths on electrophoresis through agarose-CH3HgOH gels. The major population (alpha s) consists of mRNA of length 0.7 to 1.4 kilobases, while the minor population (alpha L) consists of RNA of length 1.6 to 3.5 kilobases. Induction of human leukocytes in the presence of 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB; 100 micromolar) appears to inhibit the accumulation of IFN-alpha s and to enhance that of IFN-alpha L mRNA's (average length about 1.8 kilobases in preparations from DRB-treated cells). Interferons derived from the alpha s mRNA's represent the group of previously recognized alpha interferons while the alpha L interferons are distinguishable from this group by their lower heterospecific activity on bovine cells compared to human cells, their apparent slower mobility in sodium dodecyl sulfate--polyacrylamide gels, and their apparent heteroclitic response toward an antiserum to IFN-alpha.

Size characteristics of human leucocyte interferon under reducing and non-reducing conditions.

Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis was used to characterize human leucocyte interferon (HuIFN-alpha) under reducing and non-reducing conditions. Under non-reducing conditions HuIFN-alpha possesses two size forms, but under reducing conditions (r-HuIFN-alpha) only one is observed. The apparent molecular weight of this one form varies with the concentration of 2-mercaptoethanol used. When r-HuIFN-alpha is permitted to reoxidize the bimodal configuration of HuIFN-alpha is restored. The size heterogeneity of native HuIFN-alpha can be eliminated by mild treatment with NaIO4 [HuIFN-alpha/IO4; Stewart II, Lin, Wiranowska-Stewart & Cantell (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 4200-4204]. The size of the HuIFN-alpha/IO4 increases after treatment with 2-mercaptoethanol (r-HuIFN-alpha/IO4) and the apparent molecular weight of this component also varies with the concentration of 2-mercaptoethanol used. In the case of r-HuIFN-alpha the single peak observed apparently originates from both the higher- and lower-molecular-weight components.

Isolation of a Biologically active fragment of human alpha interferon.

When treated with pepsin under limited digestion conditions, human leukocyte-derived alpha interferon (HulFN-alpha) is partially inactivated. While the initial HulFN-alpha preparation is about equally active on human and on bovine cells, the residual antiviral activity of HulFN-alpha after pepsin treatment is significantly more active on bovine cells than on human cells. Furthermore, pepsin-treated HulFN-alpha, when assayed on bovine cells, yields an active production which is smaller and antigenically distinguishable from the native forms of HulFN-alpha.

Mechanism of binding of mouse interferon to controlled pore glass.

Many proteins bind to controlled pore glass; they are either acid elutable or alkali elutable. Mouse interferon is an acid-elutable protein. Since poly(L-lysine) and, to some extent, poly(L-arginine) are also eluted from controlled pore glass under acidic conditions, one may postulate that mouse interferon binds to controlled pore glass via some of the protein's epsilon-amino groups (of lysine) and/or guanidinium groups (of arginine) and the beads' silanol (hydroxyl groups). The necessity of lysine in the binding of interferon to controlled pore glass is further substantiated by the fact that citraconylated interferon does not bind to controlled pore glass. A requirement for Lewis acid-base interaction between the beads' B2O3 groups and the amide groups of arginine is unlikely in view of the results obtained with the alternative system, ZrOH, which, being devoid of B2O3, did bind interferon. Since a substantial amount of interferon could be eluted from controlled pore glass with ethylene glycol and high salt, one may assume that some hydrophobicity is involved in the binding of interferon to controlled pore glass.

Purification of mouse interferon by sequential chromatography.

Two schemes for the purification of mouse interferon are described, based on the concerted application of various physicochemical and affinity/adsorption column chromatographic techniques. Mouse interferon was purified to a final specific activity of 1.0--8.0 x 10(8) units/mg when first precipitated with ammonium sulphate and further processed by hydrophobic chromatography and adsorption chromatography on AFFI-Gel 202 and Controlled Pore Glass. It was purified to a final specific activity of 2.5--3.7 x 10(8) units/mg when first precipitated with ammonium sulphate and further processed by gel filtration with Ultrogel AcA 54, ion-exchange chromatography with Carboxymethyl Boi-Gel Agarose, hydrophobic chromatography with AFFI-Gel 202 and adsorption chromatography with Controlled Pore Glass.