Serological characterization of HTLV-III infection in AIDS and related disorders.

Current efforts to test blood donors and other persons for exposure to the human T cell lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immunodeficiency syndrome (AIDS), are based on the measurement of serum antibodies to viral antigens. We studied presence of serum antibodies to HTLV-III-related antigens from 767 individuals with AIDS or AIDS-related complex (ARC) or asymptomatic persons at risk for AIDS by using ELISA and immunoblot techniques. Of the 280 specimens from AIDS and ARC subjects that were tested, 99% were ELISA reactive and 96% were immunoblot reactive. Greater than 96% of the seropositive subjects manifested antibodies to the p24 core antigen, whereas only 88% had antibodies to the gp41 envelope-related glycoprotein. Contrary to previous reports, a short incubation time in the immunoblot assay failed to detect low-titer or low-affinity antibodies that were detected by overnight incubation. There was no apparent difference in pattern of antibodies to HTLV-III-related antigens in symptomatic vs. asymptomatic seropositive individuals.

Human leukocytic pyrogen test for detection of pyrogenic material in growth hormone produced by recombinant Escherichia coli.

Human growth hormone is biosynthetically produced in recombinant strains of Escherichia coli as methionyl human growth hormone (met-hGH). When purified from the bacterial culture, met-hGH is biologically active in established assays for growth hormone. Therefore, a phase I trial of met-hGH was carried out in healthy human adults; during the first trial, however, signs, symptoms, and clinical laboratory tests characteristic of an acute-phase response to pyrogenic agents was observed. Prior testing of the met-hGH preparation used in the phase I trial did not reveal evidence of toxicity, and the U.S. Pharmacopeial Convention rabbit pyrogen test, as well as the Limulus amoebocyte lysate (LAL) test, had not detected significant levels of exogenous pyrogens or endotoxin. In addition, standard inhibition studies with added endotoxin showed no inhibition by the LAL test. When this preparation of met-hGH was incubated with human blood mononuclear cells, leukocytic pyrogen (LP) was released into the supernatant medium, suggesting that the preparation contained pyrogenic material. Various lots of met-hGH based on different purification and formulating methods were tested by the human LP assay for contaminating pyrogens. The results of these tests aided in the identification of procedures for met-hGH preparations which did not induce LP in vitro. Thus, subsequent lots of met-hGH which had passed the LP test were used in repeat clinical studies, and no inflammatory or pyrogenic reactions were observed. When the LP test was used, experiments revealed that the original lot of met-hGH was contaminated with endotoxin which had not been detected in the LAL or rabbit pyrogen tests. Lyophilization in glycine-phosphate buffer had resulted in a 10- to 20-fold reduction of endotoxin reactivity in the LAL test and the U.S. Pharmacopeial Convention rabbit pyrogen test. These data provide a probable explanation for the negative result from the LAL and rabbit pyrogen test in the initial lot of met-hGH which induced acute-phase reactions. In addition, these studies demonstrate that the release of LP from human cells is a reliable indicator of the presence of materials that are pyrogenic for humans.

[Sensitivity of bacteria to antibiotics. Evaluation of a rapid automatic method (author's transl)]. / Sensibilité des bactéries aux antibiotiques. Evaluation d'une méthode rapide automatisée.

A rapid automated sensitivity testing system is evaluated in this report. The results have been compared with the standard disk diffusion method (ICS). Three hundred strains recently isolated from clinical cases : Enterobacteriaceae, Staphylococcus aureus, Streptococci D, Pseudomonas aeruginosa, have been tested. The general agreement between the two techniques was 89 p. cent.

Bacteriophage T4 inhibits colicin E2-induced degradation of Escherichia coli deoxyribonucleic acid. II. Inhibition by T4 ghosts and by T4 in the absence of protein synthesis.

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we showed previously that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with phage T4 and that at least two modes of inhibition can be differentiated on the basis of their sensitivity to chloramphenicol (CM). This report deals exclusively with the inhibition of E2 produced by T4, or T4 ghosts, in the absence of protein synthesis. The following observations are described. (i) The stage of T4 infection that inhibits E2 occurs after reversible adsorption of the phage to the bacterial surface, but probably prior to injection of T4 DNA into the cell's interior. (ii) The extent of inhibition increases as the T4 multiplicity is increased; however, the fraction of bacterial DNA that eventually is solubilized is virtually independent of the phage multiplicity. (iii) Phage ghosts (DNA-less phage particles) possess an approximately 15-fold greater inhibitory capacity toward E2 than do intact phage; however, because highly purified T4 (completely freed of ghost contamination) still inhibit E2, we discount the possibility that preparations of "intact phage" inhibit exclusively by virtue of contaminating ghosts. (iv) T4 infection does not liberate an extracellular inactivator of E2. In fact, infection with sufficiently high multiplicities of T4 produces a supernatant factor that protects E2 from nonspecific inactivation at 37 C. This protective factor does not interfere with the colicin's ability to induce DNA solubilization. (v) Inhibition of E2 occurs even when phage are added well after initiation of DNA solubilization by E2, suggesting that a late stage of E2 action is the target of inhibition by T4 infection. (vi) Increasing the CM concentration from 50 mug/ml to 200 mug/ml appears to reduce the inhibition appreciably; however, this can be attributed to an enhancement by CM of the rate of E2-induced DNA solubilization. (vii) The same degree of inhibition of E2 by T4 seen in CM is observed when CM is replaced by puromycin or rifampin. (viii) Others have shown that raising the multiplicity of E2 increases the rate of DNA solubilization. We find that the fractional inhibition (i), [i = (1 - y(i)/y(o)), where y(i) and y(o) represent the inhibited and uninhibited rates of solubilization of DNA, respectively], produced by a given T4 multiplicity is independent of the multiplicity of E2 and hence is independent of the rate of DNA solubilization induced by E2.

Bacteriophage T4 inhibits colicin E2-induced degradation of Escherichia coli deoxyribonucleic acid. I. Protein synthesis-dependent inhibition.

The deoxyribonucleic acid (DNA) of Escherichia coli B is converted by colicin E2 to products soluble in cold trichloroacetic acid; we show that this DNA degradation (hereafter termed solubilization) is subject to inhibition by infection with bacteriophage T4. At least two modes of inhibition may be differentiated on the basis of their sensitivity to chloramphenicol. The following observations on the inhibition of E2 by phage T4 in the absence of chloramphenicol are described: (i) Simultaneous addition to E. coli B of E2 and a phage mutated in genes 42, 46, and 47 results in a virtually complete block of the DNA solubilization normally induced by E2; the mutation in gene 42 prevents phage DNA synthesis, and the mutations in genes 46 and 47 block a late stage of phage-induced solubilization of host DNA. (ii) This triple mutant inhibits equally well when added at any time during the E2-induced solubilization. (iii) Simultaneous addition to E. coli B of E2 and a phage mutated only in gene 42 results in extensive DNA solubilization, but the amount of residual acid-insoluble DNA (20 to 25%) is more characteristic of phage infection than of E2 addition (5% or less). (iv) denA mutants of phage T4 are blocked in an early stage (endonuclease II) of degradation of host DNA; when E2 and a phage mutated in both genes 42 and denA are added to E. coli B, extensive solubilization of DNA occurs with a pattern identical to that observed upon simultaneous addition of E2 and the gene 42 mutant. (v) However, delaying E2 addition for 10 min after infection by this double mutant allows the phage to develop considerable inhibition of E2. (vi) Adsorption of E2 to E. coli B is not impaired by infection with phage mutated in genes 42, 46, and 47. In the presence of chloramphenicol, the inhibition of E2 by the triple-mutant (genes 42, 46, and 47) still occurs, but to a lesser extent.