Influence of poly(I:C) variability on thermoregulation, immune responses and pregnancy outcomes in mouse models of maternal immune activation.

Maternal immune activation (MIA) models that are based on administration of the viral mimetic, poly(I:C), are widely used as experimental tools to study neuronal and behavioral dysfunctions in relation to immune-mediated neurodevelopmental disorders and mental illnesses. Evidence from investigations in non-pregnant rodents suggests that different poly(I:C) products can vary in terms of their immunogenicity, even if they are obtained from the same vendor. The present study aimed at extending these findings to pregnant mice, while also controlling various poly(I:C) products for potential contamination with lipopolysaccharide (LPS). We found significant variability between different batches of poly(I:C) potassium salt obtained from the same vendor (Sigma-Aldrich) in terms of the relative amount of dsRNA fragments in the high molecular weight range (1000-6000 nucleotides long) and with regards to their effects on maternal thermoregulation and immune responses in maternal plasma, placenta and fetal brain. Batches of poly(I:C) potassium salt containing larger amounts of high molecular weight fragments induced more extensive effects on thermoregulation and immune responses compared to batches with minimal amounts of high molecular weight fragments. Consistent with these findings, poly(I:C) enriched for high molecular weight dsRNA (HMW) caused larger maternal and placental immune responses compared to low molecular weight (LMW) poly(I:C). These variable effects were unrelated to possible LPS contamination. Finally, we found marked variability between different batches of the poly(I:C) potassium salt in terms of their effects on spontaneous abortion rates. This batch-to-batch variability was confirmed by three independent research groups using distinct poly(I:C) administration protocols in mice. Taken together, the present data confirm that different poly(I:C) products can induce varying immune responses and can differentially affect maternal physiology and pregnancy outcomes. It is therefore pivotal that researchers working with poly(I:C)-based MIA models ascertain and consider the precise molecular composition and immunogenicity of the product in use. We recommend the establishment of reference databases that combine phenotype data with empirically acquired quality information, which can aid the design, implementation and interpretation of poly(I:C)-based MIA models.

Quantitative fluorescence method for continuous measurement of DNA hybridization kinetics using a fluorescent intercalator.

We present a quantitative fluorescence method for continuous measurement of DNA or RNA hybridization (including renaturation) kinetics using a fluorescent DNA intercalator. The method has high sensitivity and can be used with reaction volumes as small as 1 microliter and amounts of DNA around 1 ng. The method is based on the observations that (i) for the usual hybridization conditions, intercalators such as ethidium bromide bind (intercalate) to double-stranded DNA (dsDNA) but not single-stranded DNA or RNA and (ii) there is a large increase in fluorescence intensity when intercalators such as ethidium bromide bind to dsDNA. In this application, the intercalator can be considered as a quantitative indicator of dsDNA concentration. When a small amount of intercalator is added to a hybridizing solution, the fluorescence intensity of the intercalators increases with increase in dsDNA. The hybridization reaction can thus be monitored by continuously recording fluorescence intensity vs time. Because the amount of intercalator bound to dsDNA is not necessarily proportional to dsDNA concentration, the time-dependent fluorescence intensity graph is not identical to the kinetic graph [dsDNA] vs t. However, the fluorescence intensity vs time graph can easily be converted to the true [dsDNA] vs t graph by means of an experimental calibration graph of fluorescence intensity vs [dsDNA]. This calibration graph is obtained in a separate experiment using samples containing known amounts of dsDNA in the ethidium bromide buffer used in the kinetic measurement. We present results of experimental tests of the intercalator technique using ethidium bromide as an intercalator and DNA from Escherichia coli and lambda-phage and Poly(I)-Poly(C) RNA hybrids. These DNA and RNA samples have Cot1/2 values that cover a range of 10(6). Our experimental results show that (i) the kinetics of hybridization are not significantly perturbed by the intercalator at concentrations where no more than 10% of the binding sites on DNA or RNA hybrids are occupied, (ii) the kinetic graphs obtained by the intercalator fluorescence method and corrected with the calibration graph agree with kinetic graphs obtained by optical absorbance measurements at 260 nm, and (iii) the intercalator technique can be used in the different salt environments often used to increase the velocity of the hybridization reaction and at the hybridization temperatures (35-75 degrees C) normally used to minimize nonspecific hybridization.

Centrifugal counter-current partition chromatography with helical coil rotor. Simplified counter-current chromatography with a rotating face-seal.

A centrifugal counter-current partition chromatograph has been developed and tested in order to simplify earlier counter-current chromatographic (CCC) procedures. It includes a helical coil rotor and a rotating face-seal. The rotor is designed to be adapted in an ordinary laboratory centrifuge for toroidal coil CCC. Twisting between the inlet and outlet tubing is avoided by using the rotating seal in the rotor. The seal is placed between the rotor, on which helical coils are mounted, and a newly designed centrifuge lid. This chromatographic rotor, rotating simply around its own axis, has simplified a previous CCC device in which a coil planet mechanism is used to avoid tube twisting whilst retaining the capability for chromatographic separations. Results for separations of nystatin, dinitrophenylamino acids and Poly I:C were comparable to those obtained by liquid chromatography and the previous CCC procedure.

Poly(I).poly(C), a potential drug carrier for the antitumor agent mitoxantrone: in vitro drug binding study.

Coupling of mitoxantrone, a new antitumor agent, to a macromolecular carrier system may improve the drug's selectivity of action and pharmacokinetic properties. We have studied in vitro binding of mitoxantrone to poly(I).poly(C), a macromolecular, double-stranded homoribopolymer, by equilibrium dialysis and high-performance liquid chromatography (HPLC). Results showed high binding affinity for mitoxantrone to poly(I).poly(C) (Kd = 1.05 X 10(-6) M), the calculated number of mitoxantrone-binding sites is 60 per molecule poly(I).poly(C). In view of the good tolerance in clinical studies, poly(I).poly(C) may thus be a useful drug carrier for mitoxantrone. A mitoxantrone:poly(I).poly(C) ratio of 1:30 (w/w) is recommended for therapeutic studies.

Differential effect of poly rI.rC and Newcastle disease virus on the expression of interferon and cellular genes in mouse cells.

The expression of type I murine interferon (MuIFN) genes and several other cellular genes was examined in poly rI.rC induced and Newcastle disease virus (NDV) infected mouse cells. Northern analysis of RNA from induced L cells revealed that the MuIFN-alpha s are expressed efficiently in NDV infected cells but only at low levels in poly rI.rC induced cells. MuIFN-beta 1, however, is expressed equally well in cells treated with poly rI.rC or infected with NDV. As shown by the use of a probe specific for poly rI.rC, interferon induction correlates with the cellular uptake of poly rI.rC into the cells. The relative levels of alpha and beta 1 mRNAs in the cells reached a maximum at 10 hr after the induction which indicates coordinate expression of alpha and beta 1 interferon genes. The effect of viral infection on the expression of two murine genes coinduced with interferon (pMIF20/11 and pMIF3/10) and several cellular genes was also examined. While pMIF20/11 is an inducible gene, the pMIF3/10 gene is expressed constitutively in mouse L cells. Viral infection, but not poly rI.rC treatment, enhanced the expression of the pMIF3/10 gene, as well as two other cellular genes; H-2 and c-myc, however, the expression of beta-actin gene was unaltered. These data indicate that enhancement of gene expression in virus infected cells in not limited to the interferon system.

[Study of poly(C) and poly(I)-poly(C) complexes with model phospholipid membranes by infrared spectroscopy]. / Izuchenie kompleksov poly(C) i poly(I)-poly(C) s model'nymi fosfolipidnymi membranami metodom IK-spektroskopii.

The temperature dependence of poly(C) is shown by the infrared spectroscopy to be different for the free polynucleotide and for the polynucleotide in complexes with membranes. The intensity of stretching vibrations of C = 0 bond of poly(C) in the complex appears to be sensitive to the temperature. The intensity of this band is sharply decreased by increasing the temperature. This effect depends upon concentration of Mg2+-cations. Adsorption of poly(I)-poly(C) on the surface of vesicles from phosphatidylcholine results in the increase of the double helix.

Positive Limulus amoebocyte lysate reactions with polyriboinosinic acid x polyribocytidylic acid.

The annealed copolymer polyriboinosinic acid x polyriboinosinic acid reacted with Limulus amoebocyte lysate to cause gelation at a concentration approximately 2,000-fold or greater than bacterial endotoxins. This copolymer was pyrogenic in rabbits and demonstrated hypochromicity, but no significant correlation was noted among Limulus amoebocyte lysate reactivity, progenicity, and hypochromicity. Like endotoxin, polyriboinosinic acid x polyribocytidylic acid did not react with purified Limulus coagulogen. Similar concentrations of the homopolymers polyriboinosinic acid and polyribocytidylic acid were negative or significantly below the Limulus amoebocyte lysate reactivity of the copolymear and essentially nonpyrogenic. Thus, polyriboinosinic acid x polyribocytidylic acid is a compound in addition to endotoxin that effects a positive Limulus amoebocyte lysate test.