Preclinical development of AMG 139, a human antibody specifically targeting IL-23.

BACKGROUND AND PURPOSE: AMG 139 is a human anti-IL-23 antibody currently in a phase II trial for treating Crohn's disease. To support its clinical development in humans, in vitro assays and in vivo studies were conducted in cynomolgus monkeys to determine the pharmacology, preclinical characteristics and safety of this monoclonal antibody. EXPERIMENTAL APPROACH: The in vitro pharmacology, pharmacokinetics (PK), pharmacodynamics and toxicology of AMG 139, after single or weekly i.v. or s.c. administration for up to 26 weeks, were evaluated in cynomolgus monkeys. KEY RESULTS: AMG 139 bound with high affinity to both human and cynomolgus monkey IL-23 and specifically neutralized the biological activity of IL-23 without binding or blocking IL-12. After a single dose, linear PK with s.c. bioavailability of 81% and mean half-life of 8.4-13 days were observed. After weekly s.c. dosing for 3 or 6 months, AMG 139 exposure increased approximately dose-proportionally from 30 to 300 mg·kg(-1) and mean accumulation between the first and last dose ranged from 2- to 3.5-fold. Peripheral blood immunophenotyping, T-cell-dependent antigen responses and bone formation markers were not different between AMG 139 and vehicle treatment. No adverse clinical signs, effects on body weight, vital signs, ophthalmic parameters, clinical pathology, ECG, organ weights or histopathology were observed in the monkeys with the highest dose of AMG 139 tested (300 mg·kg(-1) s.c. or i.v.). CONCLUSIONS AND IMPLICATIONS: The in vitro pharmacology, PK, immunogenicity and safety characteristics of AMG 139 in cynomolgus monkeys support its continued clinical development for the treatment of various inflammatory diseases.

Pharmacology of AMG 181, a human anti-α4 ß7 antibody that specifically alters trafficking of gut-homing T cells.

BACKGROUND AND PURPOSE: AMG 181 is a human anti-α4 ß7 antibody currently in phase 1 and 2 trials in subjects with inflammatory bowel diseases. AMG 181 specifically targets the α4 ß7 integrin heterodimer, blocking its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the principal ligand that mediates α4 ß7 T cell gut-homing. EXPERIMENTAL APPROACH: We studied the in vitro pharmacology of AMG 181, and the pharmacokinetics and pharmacodynamics of AMG 181 after single or weekly i.v. or s.c. administration in cynomolgus monkeys for up to 13 weeks. KEY RESULTS: AMG 181 bound to α4 ß7 , but not α4 ß1 or αE ß7 , and potently inhibited α4 ß7 binding to MAdCAM-1 (but not vascular cell adhesion molecule-1) and thus inhibited T cell adhesion. Following single i.v. administration, AMG 181 Cmax was dose proportional from 0.01 to 80 mg·kg(-1) , while AUC increased more than dose proportionally. Following s.c. administration, dose-proportional exposure was observed with single dose ranging from 5 to 80 mg·kg(-1) and after 13 weekly doses at levels between 20 and 80 mg·kg(-1) . AMG 181 accumulated two- to threefold after 13 weekly 80 mg·kg(-1) i.v. or s.c. doses. AMG 181 had an s.c. bioavailability of 80%. The linear elimination half-life was 12 days, with a volume of distribution close to the intravascular plasma space. The mean trend for the magnitude and duration of AMG 181 exposure, immunogenicity, α4 ß7 receptor occupancy and elevation in gut-homing CD4+ central memory T cell count displayed apparent correlations. CONCLUSIONS AND IMPLICATIONS: AMG 181 has in vitro pharmacology, and pharmacokinetic/pharmacodynamic and safety characteristics in cynomolgus monkeys that are suitable for further investigation in humans.

Responsibility for truth in research.

For over half a century, cell cultures derived from animals and humans have served researchers in various fields. To this day, cross-contamination of cultures has plagued many researchers, often leading to mistaken results, retractions of results, cover-ups and some out-and-out falsification of data and results following inadvertent use of the wrong cells. Also, during years of examining cultures for purity we learned that many virologists were not too concerned about the specificity of the cultures they used to propagate the particular virus under study as long as the substrate (whatever it might have been) gave optimal virus yield. Polio virus propagates in primate cells, and much research has involved cells from man and various species of primates. In the 1950s a large number of chimpanzees were held in captivity in Africa for extensive studies of the efficacy of polio vaccine in production at the Wistar Institute in Philadelphia and elsewhere. Chimpanzee tissues, particularly kidneys, were thus readily available and could have also provided substrates for polio virus production, since little was known about the purity of substrates and little attention was paid to their specificity at that time.

T cells compete for access to antigen-bearing antigen-presenting cells.

These studies tested whether antigenic competition between T cells occurs. We generated CD8(+) T cell responses in H-2(b) mice against the dominant ovalbumin epitope SIINFEKL (ova8) and subdominant epitope KRVVFDKL, using either vaccinia virus expressing ovalbumin (VV-ova) or peptide-pulsed dendritic cells. CD8(+) T cell responses were visualized by major histocompatibility complex class I-peptide tetrameric molecules. Transfer of transgenic T cells with high affinity for ova8 (OT1 T cells) completely inhibited the response of host antigen-specific T cells to either antigen, demonstrating that T cells can directly compete with each other for response to antigen. OT1 cells also inhibited CD8(+) T cell responses to an unrelated peptide, SIYRYGGL, providing it was presented on the same dendritic cells as ova8. These inhibitions were not due to a more rapid clearance of virus or antigen-presenting cells (APCs) by the OT1 cells. Rather, the inhibition was caused by competition for antigen and antigen-bearing cells, since it could be overcome by the injection of large numbers of antigen-pulsed dendritic cells. These results imply that common properties of T cell responses, such as epitope dominance and secondary response affinity maturation, are the result of competitive interactions between antigen-bearing APC and T cell subsets.

Pharmacokinetics and therapeutic efficacy of rimantadine in horses experimentally infected with influenza virus A2.

OBJECTIVE: To determine pharmacokinetics of single and multiple doses of rimantadine hydrochloride in horses and to evaluate prophylactic efficacy of rimantadine in influenza virus-infected horses. ANIMALS: 5 clinically normal horses and 8 horses seronegative to influenza A. PROCEDURE: Horses were given rimantadine (7 mg/kg of body weight, i.v., once; 15 mg/kg, p.o., once; 30 mg/kg, p.o., once; and 30 mg/kg, p.o., q 12 h for 4 days) to determine disposition kinetics. Efficacy in induced infections was determined in horses seronegative to influenza virus A2. Rimantadine was administered (30 mg/kg, p.o., q 12 h for 7 days) beginning 12 hours before challenge-exposure to the virus. RESULTS: Estimated mean peak plasma concentration of rimantadine after i.v. administration was 2.0 micrograms/ml, volume of distribution (mean +/- SD) at steady-state (Vdss) was 7.1 +/- 1.7 L/kg, plasma clearance after i.v. administration was 51 +/- 7 ml/min/kg, and beta-phase half-life was 2.0 +/- 0.4 hours. Oral administration of 15 mg of rimantadine/kg yielded peak plasma concentrations of < 50 ng/ml after 3 hours; a single oral administration of 30 mg/kg yielded mean peak plasma concentrations of 500 ng/ml with mean bioavailability (F) of 25%, beta-phase half-life of 2.2 +/- 0.3 hours, and clearance of 340 +/- 255 ml/min/kg. Multiple doses of rimantadine provided steady-state concentrations in plasma with peak and trough concentrations (mean +/- SEM) of 811 +/- 97 and 161 +/- 12 ng/ml, respectively. Rimantadine used prophylactically for induced influenza virus A2 infection was associated with significant decreases in rectal temperature and lung sounds. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of rimantadine to horses can safely ameliorate clinical signs of influenza virus infection.

Mepivacaine: its pharmacological effects and their relationship to analytical findings in the horse.

Mepivacaine is a local anaesthetic drug that is widely used in equine medicine and is classified by the Association of Racing Commissioners International (ARCI) as a Class 2 foreign substance that may cause regulators to impose significant penalties if residues are identified in post-race urine samples. Therefore, an analytical/pharmacological database was developed for this agent and its metabolites. Using an abaxial sesamoid local anaesthetic model, it was determined that the highest no-effect dose (HNED) for its local anaesthetic effect was 2 mg. Using enzyme-linked immunosorbent assay (ELISA) screening, it was determined that subcutaneous (s.c.) administration of the HNED of mepivacaine to eight horses yielded a peak urinary concentration of apparent mepivacaine of 63 ng/mL 2 h after injection. The major identified metabolite recovered from equine urine after dosing with mepivacaine is 3-hydroxymepivacaine. Therefore, 3-hydroxymepivacaine was synthesized, purified and characterized, and a quantitative mass spectrometric method was developed for this metabolite as isolated from horse urine. Following subcutaneous injection of the HNED of mepivacaine, the concentration of 3-hydroxymepivacaine recovered from horse urine reached a peak of about 64.6 ng/mL at 4 h after administration as measured by GC/MS. The concentration of mepivacaine or its metabolites after administration of a HNED dose are detectable by mass spectral techniques. Within the limits of this research, the study suggests that recovery of concentrations less than about 65 ng/mL of 3-hydroxymepivacaine from post-race urine samples may not be associated with a recent LA effect of mepivacaine.

Lidocaine in the horse: its pharmacological effects and their relationship to analytical findings.

Lidocaine is a local anaesthetic agent that is widely used in equine medicine. It is also an Association of Racing Commissioners International (ARCI) Class 2 foreign substance that may cause regulators to impose substantial penalties if residues are identified in post race urine samples. Therefore, an analytical/pharmacological database was developed for this drug. Using our abaxial sesamoid local anaesthetic model, the highest no-effect dose (HNED) for the local anaesthetic effect of lidocaine was determined to be 4 mg. Using enzyme-linked immunosorbent assay (ELISA) screening, administration of the HNED of lidocaine to eight horses yielded peak serum and urine concentrations of apparent lidocaine of 0.84 ng/mL at 30 min and 72.8 ng/mL at 60 min after injection, respectively. These concentrations of apparent lidocaine are readily detectable by routine ELISA screening tests (LIDOCAINE ELISA, Neogen, Lexington, KY). ELISA screening does not specifically identify lidocaine or its metabolites, which include 3-hydroxylidocaine, dimethylaniline, 4-hydroxydimethylaniline, monoethylglycinexylidine, 3-hydroxymonoethylglycinexylidine, and glycinexylidine. As 3-hydroxylidocaine is the major metabolite recovered from equine urine, it was synthesized, purified and characterized, and a quantitative mass spectrometric method was developed for 3-hydroxylidocaine as recovered from horse urine. Following subcutaneous (s.c.) injection of the HNED of lidocaine, the concentration of 3-hydroxylidocaine recovered from urine reached a peak of about 315 ng/mL at 1 h after administration. The mean pH of the 1 h post dosing urine samples was 7. 7, and there was no apparent effect of pH on the amount of 3-hydroxylidocaine recovered. Within the context of these experiments, the data suggests that recovery of less than 315 ng/mL of 3-hydroxylidocaine from a post race urine sample is unlikely to be associated with a recent local anaesthetic effect of lidocaine. Therefore these data may be of assistance to industry professionals in evaluating the significance of small concentrations of lidocaine or its metabolites in postrace urine samples. It should be noted that the quantitative data are based on analytical methods developed specifically for this study, and that methods used by other laboratories may yield different recoveries of urine 3-hydroxylidocaine.

Amantadine and equine influenza: pharmacology, pharmacokinetics and neurological effects in the horse.

Amantadine is an antiviral agent effective against influenza A viruses. We investigated 1) the antiviral efficacy, 2) analytical detection, 3) bioavailability and disposition, 4) pharmacokinetic modelling and 5) adverse reactions of amantadine in the horse. In vitro, amantadine and its derivative rimantadine suppressed the replication of recent isolates of equine-2 influenza virus with effective doses (EDs) of less than 30 ng/ml. Rimantadine was more effective than amantadine against most viral isolates; we suggest a minimum plasma concentration of 300 ng/ml of amantadine for therapeutic efficacy. In vivo an i.v. dose of amantadine 15 mg/kg bwt produced mild, transient CNS signs which were no longer apparent after 30 min. Amantadine administered at a dose of 15 mg/kg bwt was established as the maximum safe single i.v. dose. However, if repeated i.v. administration of amantadine is required no more than 10 mg/kg bwt t.i.d. should be used. The maximal safe plasma concentration of amantadine was not evaluated but is probably greater than 2000 ng/ml and possibly greater than 4000 ng/ml. On the other hand, horses with lower seizure thresholds, or those on medications that lower seizure thresholds, may be at increased risk of amantadine-induced seizures, which show few premonitory signs and are rapidly fatal. After i.v. administration of amantadine 10 mg/kg bwt, the disposition kinetics were well fitted by a 2-compartment open model. The estimated peak plasma concentration after this dose was about 4500 ng/ml, the volume of distribution at steady-state (Vdss) was (mean +/- s.d.) 4.9 +/- 1.9 l/kg bwt and the beta phase half-life was 1.83 +/- 0.87 h. Computer projections of plasma amantadine concentrations after i.v. administration of amantadine at a dose of 10 mg/kg bwt t.i.d. at 8 h intervals suggest peak plasma concentrations of 4000-5000 ng/ml and troughs of less than 300 ng/ml will be achieved. Amantadine administered orally at 10 mg/kg bwt and 20 mg/kg bwt showed mean oral bioavailability of about 40-60% and a plasma half life of 3.4 +/- 1.4 h; however, there was substantial inter-animal variation in bioavailability. Projections based on the kinetics observed in individual animals suggest that some animals readily maintain effective plasma concentrations of amantadine after oral administration of 20 mg/kg bwt t.i.d. On the other hand, animals in which amantadine is poorly bioavailable may require up to a 6-fold (120 mg/kg bwt) increase in the oral dose to achieve effective blood concentrations. Withholding food for 15 h did not reduce these inter-animal differences in bioavailability. Our results showed that simple dosing with oral amantadine will not yield effective plasma concentrations in all animals. While i.v. administration yielded more reproducible plasma concentrations, care should be taken to see that the seizure threshold is not exceeded. In acute situations, i.v. administration (5 mg/kg bwt) every 4 h should maintain safe and effective plasma and respiratory tract concentrations of amantadine.

Complexes of N antitermination protein of phage lambda with specific and nonspecific RNA target sites on the nascent transcript.

The mechanisms that control N protein dependent antitermination in phage lambda have counterparts in many eukaryotic systems, including specific regulatory interactions of the antitermination protein with the nascent RNA transcript. Here we describe the specific and nonspecific RNA binding modes of antitermination protein N. These modes differ markedly in RNA binding affinity and in structure. N protein, either free in solution or as a complex with nonspecific RNA, lacks observable secondary and tertiary structure and binds RNA sequences indiscriminately with a dissociation constant (Kd) of approximately 10(-6) M. In contrast N becomes partially folded with at least 16-18 amino acids of ordered alpha-helical structure and binds much more tightly (Kd approximately 10(-9) M) on forming a highly specific 1:1 complex with its cognate boxB RNA hairpin. These observations and others are used to help define a bipartite model of N-dependent antitermination in which these specific and nonspecific interactions control the binding of N to the nascent transcript. Finally the role of RNA looping in delivering the bound N to the transcription complex and determining the stability (and thus the terminator specificity) of the resulting antitermination interaction of N with the RNA polymerase is considered in quantitative terms.

Regulation of the elongation-termination decision at intrinsic terminators by antitermination protein N of phage lambda.

The mechanisms that control N-protein-dependent antitermination in the phage lambda life cycle have counterparts in the regulatory systems of other organisms. Here we examine N-dependent antitermination at the intrinsic tR' terminator of lambda to elucidate the regulatory principles involved. The tR' terminator consists of a sequence of six base-pairs along the template at which the transcription complex is sufficiently destabilized to make RNA release possible. Within this "zone of opportunity" for termination the termination efficiency (TE) at each template position is determined by a kinetic competition between alternative reaction pathways that lead either to elongation or to termination. TE values at each position within tR' have been mapped as a function of NTP concentration, and it is shown that N protein (in the presence of NusA and a nut site; the minimal system for N-dependent antitermination) can offset increases in TE that are induced by limiting the concentrations of each of the next required NTPs. By limiting NTP concentrations or working at low temperature we show that a significant effect of N within the minimal system is to increase the rate of transcript elongation three- to fivefold at most positions along the template. Assuming that a comparable increase in elongation rate applies at template positions within the terminator, we show that an increase of this magnitude is not sufficient to account for the antitermination efficiency observed and that an approximately 100-fold stabilization of the transcription complex at intrinsic termination sites as a consequence of binding the N-containing antitermination sub-assembly must be invoked as well. A general method for partitioning TE effects in antitermination between changes in elongation rate and termination complex stability is demonstrated, based on competing free energy of activation barriers for the elongation and termination reactions. The analysis and utility of such mixed modes of transcriptional regulation are considered in general terms.

Specificity mechanisms in the control of transcription.

In this overview we analyze and illustrate the principles underlying some of the specificity mechanisms that control the initiation, elongation, and termination phases of transcription. Thermodynamic mechanisms dominate in the first steps of initiation, where promoters at various levels of activation can be considered to be in competition for a limiting supply of core RNA polymerase. In the later stages of initiation, as well as in elongation and termination, the regulatory mechanisms that control specificity are largely kinetic, involving rate competition between branching reaction pathways where the outcome depends on the rates (and equilibria) of reaction and interconversion of different forms of the transcription complex. Elongation complexes are very stable at most positions along the DNA template, meaning that only RNA chain elongation (and editing) can occur at these positions. However, the stability of transcription complexes decreases abruptly when termination sequences are encountered, and here the outcome can be easily switched between elongation and termination (RNA release) by minor changes in the relative rates of these competing processes. Cis effectors, defined as sites at which regulatory proteins bind to upstream activation loci on either the DNA or the nascent RNA, play important roles in the control of both initiation and of the elongation-termination decision. Examples, drawn from studies of phage lambda N-dependent antitermination and E. coli rho-dependent termination processes, illustrate the flexibility and additivity of regulatory components within control mechanisms in transcription that involve multiple determinants. The generality of such regulatory principles are stressed.

Biophysical studies of T-cell receptors and their ligands.

Recently developed methodologies for the production of the soluble extracellular domains of alpha beta TCRs have allowed several biophysical characterizations. The thermodynamic and kinetic parameters associated with specific ligand interactions between the TCR and MHC-peptide complexes, as well as superantigens, are now being established. Crystallographic studies of isolated TCR fragments have yielded the structures of a V alpha domain and the two extracellular domains of a beta-chain. These investigations are beginning to allow a new visualization of antigen recognition and T-cell activation processes.

Bacteriophage lambda N protein alone can induce transcription antitermination in vitro.

Specific and processive antitermination by bacteriophage lambda N protein in vivo and in vitro requires the participation of a large number of Escherichia coli proteins (Nus factors), as well as an RNA hairpin (boxB) within the nut site of the nascent transcript. In this study we show that efficient, though nonprocessive, antitermination can be induced by large concentrations of N alone, even in the absence of a nut site. By adding back individual components of the system, we also show that N with nut+ nascent RNA is much more effective in antitermination than is N alone. This effect is abolished if N is competed away from the nut+ RNA by adding, in trans, an excess of boxB RNA. The addition of NusA makes antitermination by the N-nut+ complex yet more effective. This NusA-dependent increase in antitermination is lost when delta nut transcripts are used. These results suggest the formation of a specific boxB RNA-N-NusA complex within the transcription complex. By assuming an equilibrium model, we estimate a binding constant of 5 x 10(6) M-1 for the interaction of N alone with the transcription complex. This value can be used to estimate a characteristic dissociation time of N from the complex that is comparable to the dwell time of the complex at an average template position, thus explaining the nonprocessivity of the antitermination effect induced by N alone. On this basis, the effective dissociation rate of N should be approximately 1000-fold slower from the minimally processive (100-600 bp) N-NusA-nut+ transcription complex and approximately 10(5)-fold slower from the maximally processive (thousands of base pairs) complex containing all of the components of the in vivo N-dependent antitermination system.

Determination of highest no effect dose (HNED) for local anaesthetic responses to procaine, cocaine, bupivacaine and benzocaine.

The highest no effect doses (HNEDs) for the local anaesthetic (LA) effects of procaine, cocaine, bupivacaine and benzocaine were determined using the heat lamp/hoof withdrawal model of Kamerling et al. (1985b) and the abaxial sesamoid block model of local anaesthesia. The heat lamp rapidly (4 or 5 s) increased the temperature of the superficial skin layers of the pastern to about 90 degrees C, at which point the animal sharply withdrew its hoof. Effective LA blockade precluded this response and superficial skin temperatures exceeded 120 degrees C. Thermal stimulus experiments were routinely terminated after 10 s of exposure to prevent undue tissue damage. Following abaxial sesamoid block with bupivacaine, the HNED for that drug was about 0.25 mg/site. Increasing the dose to 2 mg/site apparently produced complete and prolonged LA blockade. Analogous work showed that the HNED for procaine was about 2.5 mg/site. Similarly, the dose response curve for procaine was parallel with that of bupivacaine but was shifted 10-fold to the right. The duration of the LA response following procaine injection was less than for bupivacaine with the statistically significant response following 40 mg/site injection lasting less than 45 min. Cocaine was less potent than procaine, showing a shallower dose response curve. The HNED for cocaine was less than 5 mg/site, although at this dose the duration of action was extremely short (< 7.5 min). Benzocaine had no significant LA action when a dose of 800 mg was applied topically as a 5% preparation. These results show that the HNEDs for bupivacaine and procaine are remarkably low, that cocaine is somewhat less potent as a LA than might be expected, and that 5% topical benzocaine has no significant pharmacology. The small doses of bupivacaine and procaine producing effective local anaesthesia suggests that developing plasma thresholds for these agents is likely to be very challenging.

RNA interactions in the regulation of transcription.

In this overview we describe interactions between RNA, DNA, and proteins that are involved in the regulation of the various functional cycles of E. coli transcription. The role of the RNA terminator hairpin in destabilizing the transcription elongation complex and controlling the efficiency of termination at various template positions near intrinsic terminators are discussed, as are the mechanisms whereby rho protein triggers RNA release at rho-dependent terminators. Mechanisms used by phage 1-coded antitermination factor N in "fine-tuning" termination efficiencies are also considered, as are the roles of kinetically different elongation complex conformations in controlling transcriptional fidelity. General principles of the control of transcription by such RNA-protein interaction mechanisms are discussed.

Structural and thermodynamic characteristics of the binding of the lambda N protein to a RNA hairpin.

The specific binding of the lambda N protein to a 15 nucleotide RNA oligomer that forms a hairpin structure has been investigated by biophysical methods. Using fluorescence spectroscopy and equilibrium ultra-centrifugation, it was found that the N protein binds specifically to this RNA hairpin as a monomer. Circular dichroism experiments show that both the N protein and the RNA hairpin undergo structural change upon association of the complex.

Evidence of DNA bending in transcription complexes imaged by scanning force microscopy.

Complexes of Escherichia coli RNA polymerase with DNA containing the lambda PL promoter have been deposited on mica and imaged in air with a scanning force microscope. The topographic images reveal the gross spatial relations of the polymerase relative to the DNA template. The DNA appears bent in open promoter complexes containing RNA polymerase bound to the promoter and appears more severely bent in elongation complexes in which RNA polymerase has synthesized a 15-nucleotide transcript. This difference could be related to the conformational changes that accompany the maturation of open promoter complexes into elongation complexes and suggests that formation of the elongation complex involves a considerable modification of the spatial relations between the polymerase and the DNA template.

Betaine can eliminate the base pair composition dependence of DNA melting.

We show that the amino acid analogue betaine shares with small tetraalkylammonium ions [Melchior, W. B., Jr., & von Hippel, P. H. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 298-302] the ability to reduce or even eliminate the base pair composition dependence of DNA thermal melting transitions. The "isostabilizing" concentration of betaine (at which AT and GC base pairs are equally stable) is approximately 5.2 M. Betaine exerts its isostabilizing effect without appreciably altering the conformation of double-stranded DNA from the B form. The presence of > 5 M betaine also does not greatly change the behavior of DNA as a polyelectrolyte; this lack of effect on electrostatic interactions is expected because betaine exists as a zwitterion near neutral pH. Study of DNA melting transitions in high concentrations of betaine thus allows the experimental separation of compositional and polyelectrolyte effects on DNA melting. As a consequence, betaine solutions can also be used to investigate DNA-protein interactions under isostabilizing (or close to isostabilizing) conditions, which has not been possible using isostabilizing salts. This potential is illustrated by examining the highly salt concentration-dependent interaction of ribonuclease A with DNA in concentrated betaine solutions.

Biological characteristics and viral susceptibility of a stable dog kidney cell line.

A cell line derived in 1956 from normal dog kidney is described. The cells are epithelial, contact-inhibited, and can be maintained in the same culture vessels for period of more than 2.5 yr. Karyologically, the cells are hypodiploid with a modal number of 72 as opposed to the diploid number of 78. The karyotype indicates male origin of the cells and clonal derivation of extant cultures due to the presence of two marker chromosomes in all metaphases observed. At the 159th passage the dog kidney (DK) cells did not produce tumors in athymic rats. At least 13 viruses of various types produced transmissible cytopathogenic effects in the DK cells, including all of the human influenza viruses investigated.