Postantibiotic effect and postantibiotic sub-MIC effect of LTX-109 and mupirocin on Staphylococcus aureus blood isolates.

The development of new synthetic antimicrobial peptides like LTX-109 provides a new class of drugs for the treatment of Staphylococcus aureus infections. We evaluated LTX-109 and mupirocin for pharmacodynamic parameters against 10 methicillin-resistant S. aureus isolates. The postantibiotic effect (PAE) is defined as the length of time that bacterial growth is suppressed following a brief exposure to an antibiotic. We also determined the sub-MIC effect (SME) which measures the direct effect of subinhibitory levels on strains that have not previously been exposed to antibiotics. The postantibiotic sub-MIC effect (PA-SME) is a combination of the PAE and SME. LTX-109 had an average PAE of 5·51 h vs 1·04 h for mupirocin. The PA-SME of LTX-109 ranged from 2·51 to 9·33 h as the concentration increased from 0·2 to 0·4 times the minimal inhibitory concentration (MIC). The PA-SME range for mupirocin was 0·93-2·58 h. LTX-109, as compared to mupirocin, demonstrated prolonged time of effect for these pharmacodynamic parameters, which supports persistent activity for several hours after the drug is no longer present or is below the MIC. The pharmacodynamic parameters studied here suggest that LTX-109 is less likely than mupirocin to generate resistance to S. aureus. SIGNIFICANCE AND IMPACT OF THE STUDY: Resistant bacterial infections continue to be a challenge for clinicians. Identification of antibiotics with pharmacodynamic advantages may be beneficial in the treatment of these infections. An antibiotic with a longer postantibiotic effect may be able to be administered less frequently resulting in improved adherence. In this study, a new synthetic antimicrobial peptide, LTX-109, demonstrated a more prolonged time for LTX-109 than mupirocin against methicillin-resistant Staphylococcus aureus.

Early cell kinetic effects of a single dose of monochromatic ultraviolet B irradiation on hairless mouse epidermis.

Hairless mice were exposed to a single erythemic (25 mJ/cm2) or suberythemic dose (12.5 mJ/cm2) of ultraviolet B (UVB) irradiation at 297 nm. The cell kinetic changes were observed at several times during the first 7 d after the irradiation. The mitotic count, the mitotic rate (stathmokinetic method), and the number of suprabasal and basal cells were scored in histologic sections. The incorporation of [3H]thymidine was measured after pulse labeling, and the DNA distribution pattern was studied by flow cytometry. Initially, both UVB-doses induced a block or delay in the cell proliferation. The rate of entrance of cells into mitosis and the uptake of [3H]thymidine were reduced, and cells accumulated in the S phase of the cell cycle. Hence, during the first period after irradiation, UVB seemed to interfere with the DNA synthesis by inducing a prolonged S phase duration. The DNA synthesis rate was reduced to the same degree after both UVB-doses. From 24 h after irradiation rapid regenerative proliferation took place, most pronounced after the highest UVB-dose. Waves of proliferation seemed to arise from partially synchronized cohorts of cells proceeding through the cell cycle at a higher speed than normal. Thus, the present study indicates that UVB irradiation is comparable with the cell kinetic effects following both chemical skin carcinogens and non-carcinogenic skin irritants. UVB induces an inhibitory effect on the DNA synthesis activity, in addition to regenerative cell proliferation subsequent to cell toxicity.

UVB-induced epidermal hyperproliferation is modified by a single, topical treatment with a mitosis inhibitory epidermal pentapeptide.

A single application of a water-miscible cream base containing the recently identified mitosis inhibitory epidermal pentapeptide pyroGlu-Glu-Asp-Ser-GlyOH (EPP) to hairless mouse skin is followed by a long-lasting period of reduced epidermal cell proliferation. To examine if a similar growth inhibition could be achieved in stimulated and rapidly proliferating epidermis, EPP was applied at two different concentrations, 0.005 or 0.02%, to hairless mouse skin immediately after exposure of the left flank to an erythemic dose of ultraviolet B light (UVB). This dose of UVB alone induces a sustained period of rapid epidermal cell proliferation, starting at about 18 h after the irradiation. Epidermal cell proliferation was followed from 18 to 54 h (0.005% cream) or from 18 to 30 h (0.02% cream) after the treatment by estimating the rate of G2-M cell flux (the mitotic rate) by means of Colcemid, and epidermal DNA synthesis by counting labeled cells after pulse-labeling with 3H-thymidine. The unirradiated side of the mice was used as reference. The results showed that topical treatment with a 0.02% EPP cream partially inhibited UVB-induced epidermal hyperproliferation, while the 0.005% EPP cream inhibited as well as stimulated the UVB-induced hyperproliferation. Thus, EPP is effective even in rapidly proliferating epidermal cell populations, but the outcome is obviously dose-dependent in this test system.

Accelerated hematopoietic recovery with the hemoregulatory peptide dimer SK&F 107647 in bone marrow transplantation.

The effect of a novel hemoregulatory peptide SK&F107647 on recovery of hematopoiesis was studied in murine bone marrow transplantation (BMT) models. In the first model, lethally irradiated recipient mice were transplanted with 10(6) bone marrow cells from donors given a single injection of either SK&F107647 or PBS 2 days prior to transplantation. The femoral content of GM-CFC was shown to be almost twice as high in mice treated with SK&F107647 compared with controls. Both the marrow GM-CFC progenitor cell population and Mac-1 positive cells in blood were shown to recover faster in recipients of SK&F107647 treated donors. The use of SK&F107647 treated donors did not result in significantly faster blood cell count recovery in the recipient mice. In the second model, lethally irradiated recipients were transplanted with normal bone marrow cells (10(5)-10(6) and then given daily injections of SK&F107647 for 10 days starting 1 day after transplantation. The results show significantly enhanced recovery of GM-CFC in spleen and bone marrow and enhanced numbers of mature Mac-1 positive cells in spleen and peripheral blood. In addition, Thy1.2 positive cells recovered faster in SK&F107647 treated animals compared with controls. The results from these models suggest a role for SK&F107647 in BMT either as a primer of the marrow before transplantation or as an enhancer of hematopoietic recovery when given therapeutically after transplantation.

The epidermal cell kinetic response to ultraviolet B irradiation combines regenerative proliferation and carcinogen associated cell cycle delay.

The cell cycle traverse of epidermal basal cells 24 h after in vivo exposure of ultraviolet B (UVB) irradiation was studied by immunochemical staining of incorporated bromodeoxyuridine (BrdU) and bivariate BrdU/DNA flow cytometric analysis. The results were compared with the cell kinetic patterns following topical application of the skin carcinogen methylnitrosourea (MNU) as well as the skin irritant cantharidin. Hairless mice were injected intraperitoneally with BrdU 24 h after treatment of their back skin with either a minimal erythema dose of UVB, or a single application of MNU or cantharidin dissolved in acetone. The cell cycle traverse of the BrdU-labelled cohorts of epidermal basal cells were then followed for the subsequent 12 h. At 6 h after BrdU-injection, when all labelled cells in the control group as well as in the cantharidin group had left the S phase, the bivariate distributions of the UVB-exposed and the MNU group showed that BrdU-positive cells were still present in S phase. Hence, UVB irradiation, similar to the carcinogen MNU, prolonged the S phase duration in some of the basal cells. At 12 h after pulse labelling, however, BrdU-positive cells from UVB-exposed mice were re-entering S phase from G1 phase, indicating that UVB irradiation induced a shortening of the cell cycle time as well, similar to the response observed after cantharidin. The present data can not tell whether these cells also were delayed in S phase. Thus, the cell cycle traverse in hairless mouse epidermis 24 h after in vivo exposure to UVB seemed to be a combination of the cell kinetic effects following chemical skin carcinogens and skin irritants. UVB irradiation induced both a delay in transit time through S phase, probably due to DNA damage and subsequent repair, as well as a reduction in the total cell cycle time consistent with rapid regenerative proliferation.

Early cell kinetic effects of a single dose of narrow-banded ultraviolet B irradiation on the rat corneal epithelium.

The right eyes of 40 rats were exposed to a single erythemogenic dose of ultraviolet B irradiation (UVB) at 297 nm. The irradiation was directed perpendicular to the center of the cornea. The left eyes served as controls. The animals were randomly assigned into 10 groups. The labeling index (LI) after pulse labeling with tritiated thymidine and the mitotic rate (MR) after Colcemid administration were registered in the corneal epithelium at predetermined intervals up to 96 h after the irradiation. A mathematical method was used to correlate corresponding corneal areas from the different animals. In the central cornea the LI was considerably reduced up to 36 h after the irradiation. The LI increased toward the peripheral cornea and reached normal values at the limbal area. The MR was also reduced up to 36 h. However, this reduction was over the entire epithelium. The block in cell proliferation was followed by increased proliferation.

Ultraviolet B irradiation induces epidermal regeneration with rapidly cycling cells.

The left flank of hairless mouse skin was irradiated with a minimal erythema dose of ultraviolet B (UVB) light at 297 nm (25 mJcm-2), while the right flank served as untreated control. The alterations in epidermal growth kinetics induced by this UVB dose were studied with the percentage of labelled mitoses (PLM) technique during the period of increased proliferation. Thirty hours after irradiation, when a large cohort of cells appears in S phase, each animal was injected intra-peritoneally with 50 microCi tritiated thymidine [( 3H]-TdR). The number of labelled basal and suprabasal cells, as well as their localization in epidermis were registered in histological sections at short intervals up to 48 h after the [3H]-TdR pulse. Labelled mitoses were also counted in the same specimens. The results showed a four-fold increase of the high initial number of labelled cells in UVB-exposed epidermis within 18 h of the pulse injection, and a six-fold increase after 36 h. In control epidermis, where the starting value of the labelling index was much lower, there was only a three to four-fold increase in the number of labelled cells during the period studied. The PLM and the labelling index data were consistent with an average cell cycle time of approximately 10-12 h for UVB-exposed cells, in contrast to about 30 h for the fastest cycling population in control epidermis. The PLM curve also indicated a prolonged S phase duration in UVB-exposed epidermis compared with controls. In addition, labelled cells were seen in the suprabasal layer as early as 6 h after the [3H]-TdR injection and within 36 h labelled cells had reached the outermost layer of nucleated cells, indicating a reduced transit time through epidermis. The present study shows that a minimal erythema dose of UVB light at 297 nm induced a period of increased transit time through the S phase, combined with rapid cell proliferation, leading to an overall shortening of the epidermal cell cycle time. The cohort of cells labelled with [3H]-TdR 30 h after irradiation seemed to proceed as a wave of partially synchronized cells through the cell cycle for more than two rounds, which is comparable with the cell kinetic perturbations observed in regenerating mouse epidermis.

Effects of 3H-thymidine on preimplantation mouse embryos in vitro.

The effect of 3H-thymidine on in vitro development of preimplantation mouse embryos was studied. Two-cell and 4-8-cell embryos from B6CBA/F1 mice were continuously exposed to 3H-thymidine in medium containing 3H-thymidine in concentrations ranging from 10-500 nCi/ml. The effect of the radioactive precursor on embryo development to the blastocyst stage was studied by morphological observation, counting the blastocyst cell number and measuring 3H-thymidine incorporation. The continuous presence of 3H-thymidine significantly inhibited development of 2-cell and 4-8-cell embryos to the blastocyst stage. Embryos cultured from the 2-cell stage were more sensitive to 3H-thymidine than those exposed from the 4-8-cell stage. Even in morphologically normal blastocysts the cell number was significantly reduced. A 2 hr pulse of 100 nCi/ml 3H-thymidine at the blastocyst stage, did not affect the blastocyst formation or the blastocyst cell number and the amount of incorporated 3H-thymidine was sufficient to provide a reliable quantitation of DNA synthesis during the culture of preimplantation embryos in vitro. Continuous incubation with 3H-thymidine in order to measure DNA synthesis of preimplantation mouse embryos should be avoided when DNA synthesis is used as a means of evaluating toxic effect of an agent. Adverse radiation effects by 3H-thymidine on preimplantation mouse embryos during toxicity testing can be avoided by pulse labelling.

Effect of Shigella toxin on preimplantation mouse embryos in vitro.

Preimplantation mouse embryos at the 4-cell to 8-cell stage were exposed to Shigella dysenteriae toxin at concentrations of 0.001-100 pg/ml in vitro. The effect of the toxin was studied by morphological observation of the embryos to the blastocyst stage, by assessing protein synthesis with 14C-leucine incorporation, and by measuring embryonic adenosine triphosphate (ATP) content. Preimplantation mouse embryos were highly sensitive to the toxin. All variables investigated were adversely influenced by the toxin. After a lag period of 24 hr, 0.01 pg/ml toxin inhibited development to the blastocyst stage and protein synthesis. Toxin concentrations of 1.0 pg/ml resulted in a significant decrease in ATP content.

Cell kinetic effects of low doses of the skin carcinogen 7,12-dimethylbenz[a]anthracene on hairless mouse epidermis.

Groups of hairless (hr/hr) mice were given a single, topical skin application of either 50, 5 or 0.5 micrograms 7,12-dimethylbenz[a]anthracene (DMBA). At different times up to 3 days after treatment epidermal DNA distribution patterns were determined by flow cytometry, and sp. act. of DNA and labeling indices were obtained based on incorporation of [3H]thymidine. Mitotic rates were determined by the Colcemid method, and the number of basal and suprabasal cells were scored in histological sections. All three doses of DMBA led to an early depression in the uptake of [3H] thymidine, associated with an accumulation of cells with S phase DNA content peaking at 16 h. By combining the methods for studying DNA synthesis, it can be concluded that the alterations observed probably were due to a slow rate of DNA synthesis in the S phase cells, rather than to a block at the entrance of cells into the S phase. Three days after the application, DMBA still maintained an effect on the cell cycle progression, at least in the S phase. There was an obvious dose-response relationship in the inhibition of epidermal DNA synthesis. Six hours after application of the two highest doses of DMBA an early increase in the mitotic rate was observed. This short-lasting high mitotic rate was followed by a transient, very brief increase in the number of suprabasal cells. Thereafter a decrease in both the mitotic rate and the number of suprabasal cells occurred, probably caused by the alterations in DNA synthesis. After the lowest dose there was no such early increase in the mitotic rate and no initial, short-lasting increase in the number of suprabasal cells. Hence, this study shows that decreasing doses of DMBA provoke decreasing degrees of the same type of cell kinetic perturbations in the epidermal cell cycle.

A comparison between the epidermal regenerative responses provoked by a skin irritant and a tumor promoter using anti-BrdUrd/DNA flow cytometry.

The proliferative responses induced in hairless mouse epidermis after application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and the skin irritant cantharidin were investigated. Doses known to give the same degree of hyperplasia were chosen. Mice were pulse-labeled with bromodeoxyuridine (BrdUrd) 30 min prior to or 24 h after a single application of either cantharidin or TPA, and thereafter killed at various time intervals. The basal cells were isolated from epidermis, fixed in 70% ethanol and prepared for bivariate BrdUrd/DNA flow cytometric analysis. Cells pulse-labeled in S phase 30 min prior to treatment with cantharidin or TPA were slightly delayed in their progression through S phase and temporarily blocked in G2 phase. However, they were still able to re-enter S phase 18 h later, indicating a shortening of the G1 phase. Cells pulse-labeled 24 h after treatment had a considerably reduced cell cycle time, i.e. reduced G1 transit time. Hence, the wave of cells entering S phase from 16 h after injury could be explained by an immediate reduction in G1 transit time, without assuming recruitment of temporarily resting G0/G1 cells. Although cantharidin caused the longest initial delay in cell cycle progression, the subsequent proliferative response was less pronounced than that provoked by TPA. Rapid proliferation may allow for clonal expansion of initiated cells. The higher ability of TPA to induce rapid proliferation, apparently without causing any severe initial cell damage, may thus be related to its higher tumor promoting ability.

Some acute effects of monochromatic ultraviolet B irradiation on mouse epidermis measured by the tetrazolium-reduction test and determination of DT-diaphorase activity, with reference to carcinogenesis.

Some acute epidermal effects of monochromatic ultraviolet B (UVB) irradiation on hairless mouse skin were measured by the tetrazolium test (TZT), and by determining the DT-diaphorase activity in epidermal cells. Dose response and time course studies were carried out after UVB irradiation at 280, 290, 297 and 302 nm. Appropriate UV doses at all the wavelengths increased the cellular deposition of formazan (TZT). At higher doses the epidermal cells became too injured to react. Wavelengths at 280 and 290 nm seemed more injurious than those at 297 and 302 nm. There was, however, no increase in DT-diaphorase activity after UVB irradiation. This indicates that the increased formazan deposition (TZT) after UVB is more likely to be caused mainly by membrane effects. Detoxification mechanisms which activate DT-diaphorase, as often seen after cellular contact with chemical carcinogens, are not involved.

The early effects of a single application of acetone and various doses of 7,12-dimethylbenz(alpha)anthracene on CD-1 and hairless mouse epidermis. A cell kinetic study of so-called initiation and complete carcinogenesis (initiation plus promotion) in chemical skin tumor induction.

To study the early kinetic effects of different single doses of 7,12-dimethylbenz(alpha)anthracene (DMBA) on hairless mouse epidermis, about 1,800 female CD-1 mice and 340 hairless mice of both sexes were treated with a topical skin application of acetone alone or with various doses of from 4.3 to 200 micrograms DMBA in 200 microliter reagent grade acetone for the CD-1 mice, and with 0.5, 5 and 50 micrograms DMBA for the hairless mice. Five cell kinetic variables were measured at different times, from 6 h up to 14 days, after the application: cell counts in 40 vision fields of basal and suprabasal cells, [3H]TdR-labeling indices, DNA-specific radioactivity, and mean grain count after flash labeling with [3H]TdR, fractions of cells in S and G2 by flow cytometry, and the mitotic rate with the Colcemid method. The idea was to see whether there are significant (qualitative, or clearly stepwise) differences between small (allegedly) only initiating single doses of DMBA and larger, completely carcinogenic (and hence allegedly also promoting) doses of the same carcinogen. Higher doses of DMBA (25.6-200 micrograms) led, as expected, to an initial reduction in both DNA synthesis and mitotic activity. The reduction in DNA synthesis seemed more to be due to a low rate of cellular DNA synthesis than to a block at the entrance of cells into the S phase. The decreases lasted for about 3-5 days, and was followed by a period of increased rate of cell proliferation. The number of suprabasal cells (after a very short initial peak after 51.2 micrograms DMBA) was then reduced, probably due to toxic cell death. Subsequently a pronounced hyperplasia developed, which was dose-dependent as regards both size and time pattern. At medium doses, 8.5-12.8 micrograms DMBA, the pattern of reaction changed gradually. The reduction in DNA synthesis and mitotic activity was less pronounced and lasted somewhat shorter, and the ensuing hyperplasia was more moderate. After the lowest doses, 4.3 and 6.4 micrograms DMBA, an initial, short-lasting decrease in the mitotic rate only was observed, but an initial inhibition of DNA synthesis could not be observed in the CD-1 mice. Instead, an early, moderate increase in overall cellular DNA synthesis and in the fraction of G2 cells was observed, concomitant with an almost immediate increase in the number of suprabasal cells. Hence, after these small doses a primary moderate increase in the number of suprabasal cells developed, remaining for at least 10 days. After a temporary reduction the MR increased to normal or slightly above.(ABSTRACT TRUNCATED AT 400 WORDS)

UVB-induced (6-4) photoproducts in hairless mouse epidermis studied by quantitative immunohistochemistry.

DNA photoproducts in epidermal basal cells caused by in vivo exposure to various wavelengths of ultraviolet B (UVB) light were studied by immunohistochemical and microfluorometric methods. Hairless mice were irradiated with UVB doses ranging between 6.3 and 100 mJ/cm2 at 280, 290, 297, 302 and 313 nm. A dose of 25 mJ/cm2 at 297 nm corresponded to a minimal erythema dose in the Hr/Hr mice. Immediately after the exposure, frozen skin sections were stained in situ with affinity-purified antiserum having a major specificity against UV-induced (6-4) photoproducts. Microfluorometric measurements of nuclear immunofluorescence intensities were then performed to quantitate the DNA lesions in epidermal basal cell nuclei. All wavelengths except 313 nm produced DNA photoproducts, and the induction was both dose and wavelength dependent. The most effective wavelength was 290 nm, followed by 297, 280 and 302 nm. The quantitative determinations indicated variations in the DNA photoproduct content between nuclei exposed to the same dose and wavelength. This may be due to shielding by other nuclei or to differences in the sensitivity to UVB irradiation among basal cells, resulting in non-random distribution of DNA-damaged cells. Hence, measurable amounts of UVB-induced (6-4) photoproducts are produced in hairless mouse epidermis following in vivo exposure with biologically relevant doses of narrow-banded UVB light.

UVB-induced formation of (6-4) photoproducts in the rat corneal epithelium.

The induction of DNA photoproducts in rat corneal epithelium was studied after in vivo exposure to different doses of ultraviolet B light at 297 nm. Affinity-purified antibodies with a major specificity against UV-induced (6-4) photoproducts were used. The results indicate a dose dependent formation of (6-4) photoproducts. Even a minimal erythema dose (25 mJ/cm2) produced (6-4) photoproducts, demonstrating that DNA damage occurs in corneal tissue following exposure to biologically relevant doses of UVB light.

Inhibition of enriched stem cells in vivo and in vitro by the hemoregulatory peptide SK&F108636.

Replacement of the labile sulfhydryl group (-SH) of the hemoregulatory peptide monomer pyroGluGluAspCysLys (HP5b) with an isosteric methylene group yields a chemically stable compound, SK&F108636. In this study, we describe the effects of SK&F108636 on highly enriched Lin-Sca1+ hematopoietic stem cells. SK&F108636 significantly reduced the fraction of cycling progenitor cells, granulocyte macrophage colony-forming cells (GM-CFC), in vitro and in vivo. There was no effect on GM-CFC or Mix-CFC colony formation. SK&F108636 significantly inhibited proliferation of high proliferative potential (HPP)-CFC in semisolid agar cultures stimulated by stem cell factor + interleukin 3 (IL-3) + IL-1, but had no effect in cultures stimulated with M-CSF + IL-3 + IL-1. SK&F108636 was shown to act directly on the stem cells since SK&F108636 inhibited proliferation of Lin-Sca1+ cells in single cell assays. Administration of SK&F108636 to lethally irradiated mice transplanted with 2000 Lin-Sca1+ cells significantly inhibited proliferation/differentiation of cells developing into colony forming units-spleen (CFU-S) (preCFU-S) and the reconstitution of HPP-CFC and GM-CFC. There was no effect of SK&F108636 on CFU-S colony formation or mature cell regeneration in bone marrow, spleen and blood. Hence, the hemoregulatory peptide monomer SK&F108636 is a potent primitive stem cell inhibitor in vivo and in vitro. Inhibition of stem cell proliferation by small specific inhibitors may protect hematopoiesis from myelotoxic side effects during chemotherapy treatment.

Mannuronan enhances survival of lethally irradiated mice and stimulates murine haematopoiesis in vitro.

Mannuronan (poly-beta-(1-->4)-D-mannuronate or poly-M), produced by Pseudomonas aeruginosa as a mucoid exopolysaccharide, has previously been shown to exhibit immunostimulating activity. The authors investigated the in vivo and in vitro effects of mannuronan on murine haematopoiesis. In vivo, prophylactic (-24 h, intraperitoneal) administration of mannuronan enhanced survival of lethally irradiated mice from zero day 40 survivors (NaCl) to 20, 80 and 70% survival at 0.5, 1 and 2 mg/kg bw mannuronan, respectively. In vitro, primary stromal cultures stimulated with mannuronan produced high levels of interleukin(IL)-1, IL-6 and colony stimulating activity. Mannuronan alone did not have any colony stimulating activity on GM-CFC, BFU-E, Mix-CFC or HPP-CFC progenitors in clonogenic assays, but acted synergistically with suboptimal amounts of growth factors on GM-CFC, Mix-CFC and HPP-CFC colony formation. Limiting dilution analysis showed that 1 of 423 bone marrow cells formed colonies in response to suboptimal GM-CSF plus mannuronan compared to 1 of 592 for suboptimal GM-CSF alone. The primitive Lin-Sca-1+ haematopoietic progenitors showed increased day 10 colony size in the presence of mannuronan in single cells assays. These stimulating effects of mannuronan on haematopoiesis may prove to have clinical importance.