Use of predicted risk of mortality to evaluate the efficacy of anticytokine therapy in sepsis. The rhIL-1ra Phase III Sepsis Syndrome Study Group.

OBJECTIVES: To investigate a novel anticytokine therapy in patients with sepsis syndrome, and the relationship between a patient's baseline mortality risk and survival benefit. DESIGN: Data from a recent phase III, double-blind, placebo-controlled, multicenter clinical trial with patients randomized to three treatment arms: an intravenous loading dose of recombinant human interleukin-1-receptor antagonist (rhIL-1ra) or placebo, followed by a continuous infusion of rhIL-1ra (1.0 mg/kg/hr, or 2.0 mg/kg/hr), or placebo for 72 hrs. SETTING: Sixty-three investigative centers in eight countries. PATIENTS: The study population consisted of 893 patients: 302 placebo patients; 298 patients treated with 1.0 mg/kg/hr of rhIL-1ra; and 293 patients treated with 2.0 mg/kg/hr of rhIL-1ra. MEASUREMENTS AND MAIN RESULTS: An independent, sepsis-specific, log-normal regression model that predicts the risk of mortality over 28 days was applied to all patients enrolled into the rhIL-1ra sepsis study. The ability of the Predicted Risk of Mortality model to predict 28-day mortality in the placebo patients was determined and the relationship between mortality risk and efficacy of rhIL-1ra was investigated. The trial data were also analyzed using two other risk-assessment models for comparison with Predicted Risk of Mortality. A significant increase in survival time was demonstrated for all patients treated with rhIL-1ra (n = 893, p < .02 Predicted Risk of Mortality log-normal), but patients with a Predicted Risk of Mortality of < 24% derived little benefit. Retrospective examination of time-to-death data demonstrated that rhIL-1ra reduced risk of death in the first 2 days for patients with > or = 24% Predicted Risk of Mortality (n = 580, p < .005 Predicted Risk of Mortality log-normal). This same effect was not present in patients with a Predicted Risk of Mortality of < 24% on entry into the study. The Predicted Risk of Mortality model predicted a 28-day mortality rate of 35% for placebo patients compared with 34% observed and accurately stratified patients along the full range of risks. There was a wide distribution of individual patient risks for 28-day mortality for all patients, as well as within categorical subgroups, such as shock and organ system dysfunction. Two alternate risk models were assessed and the Acute Physiology Score of Acute Physiology and Chronic Health Evaluation III also demonstrated a statistically significant survival benefit for rhIL-1ra (p = .04 Predicted Risk of Mortality log-normal) for all patients treated. CONCLUSIONS: Using an appropriate analytic model, a statistically significant increase in survival time from rhIL-1ra was measured. A direct relationship was found between a patient's Predicted Risk of Mortality at study entry to efficacy of rhIL-1ra. Individual risk or severity assessment may be a useful tool for evaluating the clinical benefit of new therapeutic approaches to sepsis and for monitoring outcomes at the bedside.

TRNA2Gln Su+2 mutants that increase amber suppression.

We selected mutants of lambda pSu+2 which had an increased ability to suppress on Escherichia coli trp B9601 amber mutation on translationally stringent rpsL594 streptomycin-resistant ribosomes. tRNA2Gin Su+2 molecules produced from eight independent mutants were purified, and their ribonucleic acid sequences were determined. Two types of mutations were mapped to the tRNA2Gin Su+2(glnV) gene by this method. Both altered the pseudouridine at position 37 of the tRNA anticodon loop. Seven of the isolates were transitions (pseudouridine to cytosine), and one was a transversion (pseudouridine to adenine). These mutations resulted in Su+ transfer ribonucleic acid molecules that exhibited higher transmission coefficients than their parent Su+2 transfer ribonucleic acids. As judged by their suppressor spectra on T4 amber mutants, which were almost identical to that of Su+2, the two mutant Su+ transfer ribonucleic acids inserted glutamine at amber sites.

Isolation and characterization of lambda b221poriCasnA, a plaque-forming specialized transducing phage carrying the origin of replication of the Escherichia coli chromosome.

A specialized transducing phage lambda b221poriCasnA has been isolated carrying oriC the origin of chromosomal replication of Escherichia coli. All phage genes required for lytic growth are retained, thus the phage is capable of lytic growth. The presence of the oriC locus confers upon infecting phage DNA the ability to replicate as a plasmid using only host DNA replication functions. The presence of both oriC and ansA markers has allowed the development of a plaque assay for origin function which can be used to identify mutants at these loci. Comparison of restriction endonuclease cleavage sites present on lambda b221proiCasnA DNA to those on its parent, lambda b221 rex::Tn10 suggests the steps involved in the formation of the transducing phage.

Pseudovirulent mutants of lambda b221poriCasnA resulting from mutations in or near oriC, the E. coli origin of DNA replication.

Mutants of the specialized transducing phage lambda b221poriCasnA have been isolated which form plaques on lambda lysogens. Genetic and physical evidence is presented to show that the mutations responsible for the pseudovirulent phenotype map in or near oriC, the origin of chromosomal replication in Escherichia coli.

The purification and sequence of a temperature-sensitive tryptophan tRNA.

Escherichia coli can be temperature-sensitive due to a lesion in the gene for tRNATrp (Yanofsky, C., and Soll, L. (1977) J. Mol. Biol. 113, 663-677). Purification of tRNATrp from this strain (temperature-sensitive tRNATrp) was achieved by one of two methods. Either a combination of benzoylated DEAE-cellulose column chromatography and two-dimensional polyacrylamide gel electrophoresis, or hybridization to plasmid DNA covalently bound to cellulose (this is a recombinant plasmid carrying the gene for tRNATrp) and electrophoresis of the eluted material on a 10% polyacrylamide gel, produced isotopically pure tRNA. The sequence of the temperature-sensitive tRNATrp was determined by standard methods. We find that the sequence differs from that of wild type tRNATrp by a single residue; G in position 7 (G7) in wild type tRNATrp is A7 in temperature-sensitive tRNATrp. This base difference results in one less base pair in the CCA stem of the temperature-sensitive species. The effect of this base change in the in vitro and in vivo properties of tRNATrp (presented elsewhere (S.P. Eisenberg and M. Yarus, manuscript in preparation.)) are discussed.

The structure of the DNA containing the E. coli tRNATry gene.

Using the pMB9 recombinant plasmid pMY3, which contains a functional gene for the tRNATry mutant Su+7, the EcoRI fragment containing the tRNATry gene is mapped and oriented with respect to the HindIII site in the tetracycline region of pMB9. Complete HpaII and HaeIII maps of the EcoRI fragment are derived. The Su+7 tRNA gene is placed by hybridization to these fragments, and the tRNA gene is oriented by using the restriction sites for HinfI, TaqI, and HpaII in the tRNA gene itself. A tRNAAsp gene is shown to lie adjacent to tRNATry, and is also placed and oriented in the map. The RI fragment itself originates in a locus adjacent to, and transcribed in the same direction as, the ribosomal RNA genes of phi 80d3. The implications of the structure of the cloned DNA for its previously measured regulatory and tRNA gene activities are discussed. In particular, the effect on the regulation of RNA synthesis is attributable to an E. coli DNA sequence, but cannot be due to the presence of a normal tRNA promoter on the plasmid.

Recessive lethal amber suppressors in yeast.

Recessive lethal amber suppressor mutations have been isolated in a diploid strain of Saccharomyces cerevisiae. Diploids carrying these suppressors upon sporulation yield asci with only two live spores, both lacking the suppressor. At least two classes of recessive lethal suppressors exist. Aneuploid strains carrying one wild type and one suppressor locus have been isolated and used in mapping studies; one suppressor maps on chromosome III, the other does not.

Genetic characterization of mutations which affect catabolite-sensitive operons in Escherichia coli, including deletions of the gene for adenyl cyclase.

Sixty-two spontaneous mutations have been characterized which reduce the level of expression of catabolite-sensitive operons. These mutations appear to affect either the crp (catabolite gene activator protein) or cya (adenyl cyclase) loci. No new loci have been discovered. Deletions of the cya gene do not remove an essential function. phi80 transducing phage for the cya gene have been used to do recombination and complementation studies on cya mutants.

Isolation and partial characterization of a mutant of Escherichia coli deficient in DNA polymerase II.

A mutant of Escherichia coli deficient in DNA polymerase II has been isolated from E. coli polA1 by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and assay of polymerase activity in extracts of survivors. The polA1 mutation was suppressed during mutagenesis by introduction of the suppressor, su7(+), into the parental strain. The mutant, HMS83 polA1 polB1, contains less than 0.5% of the normal levels of DNA polymerase II. The only polymerase activity detected in the mutant is DNA polymerase III. E. coli HMS83 grows normally at both 25 and 42 degrees , and supports the growth of bacteriophages T4, T7, lambda, varphiX174, 186, P2, and fd. The polB mutation does not affect sensitivity to ultraviolet irradiation or recombination frequencies.

Recessive lethals: a new class of nonsense suppressors in Escherichia coli.

Two new nonsense suppressors in Escherichia coli were found in partial diploids carrying F'14 and were shown to be on the episome. These suppressors can exist only in cells which also contain the su(-) allele, i.e., su(+)/su(-) heterozygotes. Presumably the mutations cause an alteration of an essential cellular component, the complete loss of which is lethal. Su7, an amber suppressor, has an efficiency of 76 per cent and su8, an ochre suppressor, an efficiency of 4 per cent.