Several of the new 4-quinolones significantly increase the incorporation of [3HJthymidine into the DNA of mitogen-stimulated human lymphocytes. This study suggests that ciprofloxacin inhibits de novo pyrimidine biosynthesis, thereby resulting in a compensatory increase in the uptake of pyrimidine precursors through salvage pathways, and that additional effects may affect eucaryotic cell growth. Incorporation of deoxyuridine, uridine, and orotic acid as well as thymidine was increased in the presence of ciprofloxacin, one of the antibacterially most active of the new 4-quinolones. In contrast, the uptake was decreased in very high concentrations of the drug. Culture in HAT (hypoxanthine, aminopterine, thymidine) medium, which blocks de novo thymidylate synthesis, abrogated the increase in [3H]thymidine incorporation induced by ciprofloxacin.Ciprofloxacin also failed to increase the uptake of [14C]hypoxanthine or leucine, indicating a selective effect on pyrimidine and not on purine nucleotide biosynthesis. N-(Phosphonacetyl)-L-aspartate, an inhibitor of pyrimidine nucleotide biosynthesis, also increased [3H]thymidine incorporation in phytohemagglutininstimulated lymphocytes in a fashion similar to ciprofloxacin. The growth of several cell lines was partially inhibited by ciprofloxacin at 20 ,ig/ml and completely inhibited at 80 to 160 jug/mi. Growth inhibition by ciprofloxacin could not be restored by the addition of uridine to the medium. Chromosome breaks, gene amplification, or other genetic alterations could not be detected in human lymphocytes incubated with up to 25 jig of ciprofloxacin per ml.Many of the new 4-quinolones have a broader spectrum and are more potent than their structurally related predecessors (7,12). The antibacterial activity of nalidixic acid is caused by inhibition of DNA synthesis, resulting from inhibition of DNA gyrase (23,28). Although the new 4-quinolones have not been studied as extensively as nalidixic acid, they have been shown to inhibit gyrase function (2,10,21,22), possibly through DNA binding (19). However, a recent study demonstrated a lack of correlation between antibacterial activity and inhibition of gyrase activity, thereby suggesting that other factors are responsible for the potency of these drugs (29).We have found that in eucaryotic cells, many of the new quinolones cause a significant (11 to 231%) increase in the uptake of [3H]thymidine into trichloroacetic acid (TCA)-precipitable material in mitogen-stimulated human lymphocytes at drug concentrations of 1.6 to 50 ,ug/ml (8; A. Forsgren, S. F. Schlossman, and T. F. Tedder, submitted for publication). However, cell growth and progression through the cell cycle were inhibited by 20 jig or more of ciprofloxacin per ml. In addition, ciprofloxacin at a clinically achievable concentration of 5 ,ug/ml caused a 50% decrease in immunogloblin production by mitogen-stimulated lymphocytes in vitro.In this study, we investigated the reasons for the increased [3H]thymidine uptake and the decreased function of lymphocytes cultured...

Protein D, a surface-exposed 42-kDa membrane lipoprotein, is well conserved among both type b and nontypeable Haemophilus influenzae strains, and it is considered a vaccine against H. influenzae infections. Here, we report the large-scale purification of a nonacylated form of protein D (PDm) from the periplasmic space of Escherichia coli overexpressing PDm. Screening of human sera for levels of antibodies to PDm demonstrated that the immunoglobulin G (IgG) antibody level is above background levels in infants less than 6 months of age. Following a drop to background values in the age group 6 months to 1 year, IgG antibody levels start to increase, together with IgA antibody levels, after 1 year of age. The first appearance of serum IgM antibodies is in 6-month-to 1-year-old infants whose IgG antibody levels have dropped to the postnatal background level. Affinity-purified antibodies from humans and from PDm-immunized rats detected epitopes of protein D which are normally exposed on the bacterial surface. Affinity-isolated human anti-PDm antibodies eluted in acidic buffer were not bactericidal against H. influenzae. Loss of bactericidal activity may occur in this buffer, as was demonstrated in pooled human sera with high bactericidal activity after incubation in the same buffer. Hyperimmunization of rats with PDm induced high levels of serum IgG and IgA antibodies against PDm and significant bactericidal activity against homologous and heterologous H. influenzae strains.

The cytotoxic quinolone CP-115,953 specifically exerts its inhibitory effect upon eukaryotic topoisomerase II. CP-115,953 stimulates DNA cleavage mediated by topoisomerase II with a potency approximately 600 times greater than that of ciprofloxacin, a quinolone antibacterial agent that currently is in clinical use. Because ciprofloxacin has been reported to strongly enhance interleukin-2 production, we considered it important to study the effect of CP-115,953 on interleukin-2 and gamma interferon (IFN-␥) mRNA and protein expression in mitogen-stimulated human peripheral blood lymphocytes. For comparison, novobiocin and the antineoplastic drug etoposide were also included in the study. CP-115,953 (25 M) enhanced interleukin-2 mRNA levels up to 8-fold and IFN-␥ mRNA concentrations up to 6.5-fold. In contrast, ciprofloxacin (282 M) induced mRNAs for interleukin-2 and IFN-␥ up to 20-fold and 7.8-fold, respectively. However, CP-115,953 showed more prolonged kinetics of IFN-␥ mRNA production than ciprofloxacin. At high concentrations (Ն141 M), ciprofloxacin was a greater inducer of interleukin-2 production and exhibited a higher level of stimulatory action than CP-115,953 on IFN-␥ synthesis. At low concentrations, however, CP-115,953 (Յ25 M) was more potent than ciprofloxacin in inducing interleukin-2 and IFN-␥ synthesis. Etoposide or novobiocin did not influence cytokine mRNA expression. Thus, among the topoisomerase II inhibitors tested, fluoroquinolones are unique in stimulating cytokine synthesis in lymphocyte cultures.DNA topoisomerases are a class of enzymes that primarily alter DNA conformation through a concerted breaking and rejoining of DNA strands, thereby controlling the topological state of DNA (3, 4, 49). Topoisomerase II is not only crucially involved in DNA replication but also participates in transcription, DNA repair, and recombination (18). Nucleotide sequencing of genes encoding the topoisomerase enzymes from eukaryotic and prokaryotic cells shows all type II topoisomerases to be structurally and phylogenetically related. Eukaryotic DNA topoisomerase II corresponds to the bacterial DNA gyrase and is a ubiquitous ATP-dependent type II topoisomerase (4). Eukaryotic topoisomerase II is the primary cellular target for several clinically important antitumor agents (e.g., etoposide) (7, 46), while the prokaryotic counterpart is the target for fluoroquinolone antibacterial drugs (24).The lethal target of fluoroquinolone antibiotics in bacteria is the association between DNA and DNA gyrase (24, 34). Fluoroquinolones in clinical use have, in general, been found to be only weak inhibitors of the eukaryotic topoisomerase II in in vitro assays exploring relaxation or catenation of supercoiled double-stranded DNA (19,25,26). However, a number of new fluoroquinolone derivatives with enhanced activity against eukaryotic topoisomerase II exhibited by their strong inhibitory effects on eukaryotic DNA replication have been characterized, and their structure-activity relationships have been determined (3,9,12,13,17,1...

We searched for antibodies against Moraxella (Branhamella) catarrhalis proteins in the sera of patients with lower respiratory tract infection. Sera from 48 patients with M. catarrhalis and 39 patients without M. catarrhalis in their lower respiratory tract specimens were studied by a gel electrophoresis-immunoperoxidase technique; sera from 23 healthy adult blood donors were also included. Immunoglobulin G (IgG) antibodies against a 28-kDa protein were found significantly more frequently in patients with M. catarrhalis in lower respiratory tract specimens (71%) than in patients without M. catarrhalis in lower respiratory tract specimens (28%) or healthy adult blood donors (22%). Seroconversion, from the acute to the convalescent stages, occurred in at least eight patients with M. catarrhalis and in one patient without detectable M. catarrhalis. IgG antibodies against other M. catarrhalis proteins were found in most sera, including those obtained from blood donors. By adsorption experiments the 28-kDa protein was demonstrated to be surface exposed. IgM antibodies against an 85-kDa protein were found in serum from one patient from whom M. catarrhalis and Streptococcus pneumoniae were isolated from the lower respiratory tract, while IgA antibodies against M. catarrhalis proteins could not be detected in any serum specimen.