Detection of poly(I):poly(C12U), mismatched double-stranded RNA, by rapid solution hybridization: blood values after intravenous infusion.

A rapid solution hybridization technique has been developed for estimating blood concentrations of poly(I):poly(C12U), a high molecular weight bioactive double-stranded RNA. Samples were prepared by mixing 100 microL of blood with, 165 microL of 6 M guanidine thiocyanate (GuSCN) and 0.16 M EDTA pH 8.0, and freezing. Hybridizations were carried out with a [3H]poly(C) probe in 3 M GuSCN for 10 min at 37 degrees C. Ribonuclease-resistant hybrids were collected by precipitation with trichloroacetic acid and filtration. Validation studies demonstrated minor interassay variance; the assay was accurate in the range 0.1 to 10 ng poly(I):poly(C12U). Thirty-one blood samples from 15 patients were collected and prepared before and immediately after an average 35 min intravenous infusion of 40-500 mg poly(I):poly(C12U). Postadministration values averaged 48% (s.d. 23%) of the theoretical maximum (range 20-102%). These results confirm previous observations of rapid elimination kinetics of poly(I):poly(C12U) in patients.

Nucleic acid hybridization in plasma: method for the quantitation of poly(I).poly(C12, U) in plasma of cancer patients.

Using a new method for the direct measurement of the double-stranded RNA (dsRNA) molecule poly(I).poly(C12, U) in plasma, levels of 100 X 10(-9) g of drug were routinely quantified. The samples were digested by proteinase K in a buffered solution containing 0.1% of Brij-35 and deoxycholate detergents. The digestions were terminated after 1 h by the addition of Brij-58 and boiling saturated NaI (1.67 g/ml). Serially diluted samples were filtered onto nitrocellulose and the filters washed and hybridized. Levels of the hybridized-radioactive probe, synthesized de novo in an RNA dependent DNA transcription system, were determined by liquid scintillation spectrophotometry and quantified by comparison to a standard curve. The efficiency of hybridization declined when the plasma concentration in the reaction fell below 1.0 mg/ml. Incubation and denaturation temperatures significantly altered the amount of radioactive probe hybridized; results varied in the extent of hybridization and in the concentration range of dsRNA showing a linear response. Elevated temperature during proteinase K digestion showed reduced hybridization efficiencies: 100% at 25, 80% at 37, 35% at 45, and 25% at 55 degrees C. Incubation at elevated temperatures, prior to the addition of NaI, caused a decline in the amount of radioactivity hybridized, but did not have an effect during hybridization.

Human serum digests poly(rI) X poly(rC) differently from other mammalian sera.

Using a new spectrophotometric assay, we found that human serum digests predominately (rC)n of (rI)n X (rC)n, in agreement with results of Nordlund et al. (Proc. Soc. Exp. Biol. Med., 1970, 133, 439-444) and DeClercq (Eur. J. Biochem., 1979, 93, 165-172) while rhesus monkey, cat, and rabbit sera digest primarily (rI)n. The results of Nordlund et al. also show (in agreement with our results) that human serum digests more of the (rC)n strand of (rI)n X (rC)n than rabbit serum. However, their results differ from ours, since they found that rabbit serum digests nearly equal, but small amounts of (rI)n and (rC)n. Results are discussed in relation to interferon (IFN) induction and toxicity following administration of (rI)n X (rC)n.

Inhibition of antigodies binding polyinosinic-polycytidylic acid in human and mouse lupus sera by viral and synthetic ribonucleic acids.

The specificities of anti-RNA antibodies of diverse origin were studied by inhibition of the binding of radioative polyinosinic polycytidylic acid. The antibodies were from human patients with systemic lupus erythematosus (SLE), older NZB/NZW F(1) mice who have SLE, and young NZB/NZW F(1) mice immunized with either synthetic or viral double-stranded (ds) RNA. The inhibitors were two viral ds and two synthetic ds RNAs, ribosomal RNA and transfer RNA. The human sera were more heterogeneous than the mouse lupus sera, and had greatest specificity for reovirus RNA. The mouse lupus sera were more homogeneous and, in general, were inhibited efficiently by all four ds RNAs. Sera from mice immunized with synthetic RNA reacted poorly with viral RNA, whereas sera from mice immunized with viral RNA reacted with all four ds RNAs and resembled the lupus sera. These results suggest a role for viruses in the induction of anti-RNA antibodies, and are compatible with the concept that virus infection as well as excessive antibody responses are involved in the pathogenesis of SLE.