Real-time observations of single bacteriophage λ DNA ejections in vitro

The physical, chemical, and structural features of bacteriophage genome release have been the subject of much recent attention. Many theoretical and experimental studies have centered on the internal forces driving the ejection process. Recently, Mangenot et al. [Mangenot S, Hochrein M, Rädler J, Letellier L (2005) Curr Biol 15:430–435.] reported fluorescence microscopy of phage T5 ejections, which proceeded stepwise between DNA nicks, reaching a translocation speed of 75 kbp/s or higher. It is still unknown how high the speed actually is. This paper reports real-time measurements of ejection from phage {lambda}, revealing how the speed depends on key physical parameters such as genome length and ionic state of the buffer. Except for a pause before DNA is finally released, the entire 48.5-kbp genome is translocated in {approx}1.5 s without interruption, reaching a speed of 60 kbp/s. The process gives insights particularly into the effects of two parameters: a shorter genome length results in lower speed but a shorter total time, and the presence of divalent magnesium ions (replacing sodium) reduces the pressure, increasing ejection time to 8–11 s. Pressure caused by DNA–DNA interactions within the head affects the initiation of ejection, but the close packing is also the dominant source of friction: more tightly packed phages initiate ejection earlier, but with a lower initial speed. The details of ejection revealed in this study are probably generic features of DNA translocation in bacteriophages and have implications for the dynamics of DNA in other biological systems

A point mutation in the Nul gene of bacteriophage λ facilitates phage growth in Escherichia coli with himA and gyrB mutations

A mutant of λ was isolated that grows in the Escherichia coli himAΔ/gyrB-him320 (Ts) double mutant at 42°C; conditions which are non-permissive for wild-type λ growth. The responsible mutation, ohm1 , alters the 40th codon of the Nul reading frame. The Nul and A gene products comprise the terminase protein which cleaves concatameric DNA into unit-length phage genomes during DNA packaging. The Nul-ohm1 gene product acts in trans to support λ growth in the double himA/gyrB mutant, and λ cos154 growth in the single himA mutant. The observation that an alteration in Nul suppresses the inhibition of growth in the double himA/gyrB mutant implicates DNA gyrase, as well as integration host factor, in the DNA: protein interactions that occur at the initiation of packaging.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47567/1/438_2004_Article_BF00322458.pd

Antimicrobial peptide omiganan enhances interferon responses to endosomal toll-like receptor ligands in human peripheral blood mononuclear cells

LL-37 is a cationic antimicrobial peptide and the sole human member of cathelicidins. Besides its bactericidal properties, LL-37 is known to have direct immunomodulatory effects, among which enhancement of antiviral responses via endosomal toll-like receptors (TLRs). Omiganan pentahydrochloride is a synthetic cationic peptide in clinical development. Previously, omiganan was primarily known for its direct bactericidal and antifungal properties. We investigated whether omiganan enhances endosomal TLR responses, similar to LL-37. Human peripheral blood mononuclear cells were treated with endosomal TLR3, -7, -8, and -9 ligands in the presence of omiganan. Omiganan enhanced TLR-mediated interferon-alpha release. Subsequent experiments with TLR9 ligands showed that plasmacytoid dendritic cells were main contributors to omiganan-enhanced IFN production. Based on this type I interferon-enhancing effect, omiganan may qualify as potential treatment modality for virus-driven diseases. The molecular mechanism by which omiganan enhances endosomal TLR responses remains to be elucidated.Drug Delivery Technolog

Clinical, Cellular, and Molecular Effects of Corticosteroids on the Response to Intradermal Lipopolysaccharide Administration in Healthy Volunteers

The intradermal lipopolysaccharide (LPS) challenge in healthy volunteers has proven to be a valuable tool to study local inflammation in vivo. In the current study the inhibitory effects of oral and topical corticosteroid treatment on intradermal LPS responses were evaluated to benchmark the challenge for future investigational drugs. Twenty-four healthy male volunteers received a two-and-a-half-day twice daily (b.i.d.) pretreatment with topical clobetasol propionate 0.05% and six healthy volunteers received a two-and-a-half-day b.i.d. pretreatment with oral prednisolone at 0.25 mg/kg body weight per administration. Participants received one injection regimen of either 0, 2, or 4 intradermal LPS injections (5 ng LPS in 50 µL 0.9% sodium chloride solution). The LPS response was evaluated by noninvasive (perfusion, skin temperature, and erythema) and invasive assessments (cellular and cytokine responses) in suction blister exudate. Both corticosteroids significantly suppressed the clinical inflammatory response (erythema P = 0.0001 for clobetasol and P = 0.0016 for prednisolone; heat P = 0.0245 for clobetasol, perfusion P < 0.0001 for clobetasol and P = 0.0036 for prednisolone). Clobetasol also significantly reduced the number of monocytes subsets, dendritic cells, natural killer cells, and T cells in blister exudate. A similar effect was observed for prednisolone. No relevant corticosteroid effects were observed on the cytokine response to LPS. We successfully demonstrated that the anti-inflammatory effects of corticosteroids can be detected using our intradermal LPS challenge model, validating it for evaluation of future investigational drugs, as an initial assessment of the anti-inflammatory effects of such compounds in a minimally invasive manner

Intradermal lipopolysaccharide challenge as an acute in vivo inflammatory model in healthy volunteers

Aims: Whereas intravenous administration of Toll-like receptor 4 ligand lipopolysaccharide (LPS) to human volunteers is frequently used in clinical pharmacology studies, systemic use of LPS has practical limitations. We aimed to characterize the intradermal LPS response in healthy volunteers, and as such qualify the method as local inflammation model for clinical pharmacology studies. Methods: Eighteen healthy male volunteers received 2 or 4 intradermal 10 ng LPS injections and 1 saline injection on the forearms. The LPS response was evaluated by noninvasive (perfusion, skin temperature and erythema) and invasive assessments (cellular and cytokine responses) in skin biopsy and blister exudate. Results: LPS elicited a visible response and returned to baseline at 48 hours. Erythema, perfusion and temperature were statistically significant (P < .0001) over a 24-hour time course compared to saline. The protein response was dominated by an acute interleukin (IL)-6, IL-8 and tumour necrosis factor response followed by IL-1β, IL-10 and interferon-γ. The cellular response consisted of an acute neutrophil influx followed by different monocyte subsets and dendritic cells. Discussion: Intradermal LPS administration in humans causes an acute, localized and transient inflammatory reaction that is well-tolerated by healthy volunteers. This may be a valuable inflammation model for evaluating the pharmacological activity of anti-inflammatory investigational compounds in proof of pharmacology studies

Pharmacodynamic Effects of Topical Omiganan in Patients With Mild to Moderate Atopic Dermatitis in a Randomized, Placebo-Controlled, Ph

Omiganan is an indolicidin analog with antimicrobial properties that could be beneficial for patients with atopic dermatitis. In this randomized, double-blind, placebo-controlled, phase II trial we explored the efficacy, pharmacodynamics, and safety of topical omiganan once daily in 36 patients with mild to moderate atomic dermatitis. Patients were randomized to apply topical omiganan 1%, omiganan 2.5%, or vehicle gel to one target lesion once daily for 28 consecutive days. Small but significant improvements in local objective SCORing Atopic Dematitis index and morning itch were observed in the omiganan 2.5% group compared with the vehicle gel group (−18.5%; 95% confidence interval, −32.9 to −1.0; P = 0.04; and −8.2; 95% confidence interval, −16.3 to −0.2; P = 0.05, respectively). A shift from lesional to nonlesional skin microbiota was observed in both omiganan treatment groups, in contrast to the vehicle group. Thus, treatment with topical omiganan improved dysbiosis in patients with mild to moderate atopic dermatitis, and small but statistically significant improvements in clinical scores were detected. Our findings warrant further exploration in future clinical trials

Omiganan Enhances Imiquimod-Induced Inflammatory Responses in Skin of Healthy Volunteers

Omiganan (OMN; a synthetic cationic peptide) and imiquimod (IMQ; a TLR7 agonist) have synergistic effects on interferon responses in vitro. The objective of this study was to translate this to a human model for proof-of-concept, and to explore the potential of OMN add-on treatment for viral skin diseases. Sixteen healthy volunteers received topical IMQ, OMN, or a combination of both for up to 4 days on tape-stripped skin. Skin inflammation was quantified by laser speckle contrast imaging and 2D photography, and molecular and cellular responses were analyzed in biopsies. IMQ treatment induced an inflammatory response of the skin. Co-treatment with OMN enhanced this inflammatory response to IMQ, with increases in perfusion (+17.1%; 95% confidence interval (CI) 5.6%–30%; P < 0.01) and erythema (+1.5; 95% CI 0.25%–2.83; P = 0.02). Interferon regulatory factor-driven and NFκB-driven responses following TLR7 stimulation were enhanced by OMN (increases in IL-6, IL-10, MXA, and IFNɣ), and more immune cell infiltration was observed (in particular CD4+, CD8+, and CD14+ cells). These findings are in line with the earlier mechanistic in vitro data, and support evaluation of imiquimod/OMN combination therapy in human papillomavirus-induced skin diseases

No effect of topical digoxin and furosemide gel for patients with external anogenital warts

Drug Delivery Technolog

No effect of topical digoxin and furosemide for patients with actinic keratosis

Drug Delivery Technolog

Results of phase 2 trials exploring the safety and efficacy of omiganan in patients with human papillomavirus-induced genital lesions

AIMS\nMETHODS\nRESULTS\nCONCLUSION\n To assess safety and tolerability and explore pharmacodynamics and efficacy of omiganan in external anogenital warts (AGW) and vulvar high-grade squamous intraepithelial lesions (HSIL).\nTwo randomized controlled trials in patients with external AGW and vulvar HSIL were conducted. Patients received topical omiganan 2.5% or placebo gel once daily for 12 weeks with a follow-up of 12 weeks. Safety and tolerability were monitored and pharmacodynamics and clinical efficacy of omiganan were assessed by analysing lesion count, size and viral load. Self-reported pain, itch and quality of life were assessed by an electronic diary and questionnaire.\nTwenty-four AGW and 12 vulvar HSIL patients were enrolled. All patients had a high treatment adherence (99%). No serious adverse events occurred and all adverse events (n = 27) were mild, transient and self-limiting. The treatment groups were not different in terms of safety and tolerability, lesion count and size, and patient-reported outcomes pain, itch and quality of life. Human papillomavirus load significantly reduced after 12 weeks of treatment with omiganan compared to placebo (-96.6%; 95% confidence interval -99.9 to -7.4%; P = .045) in AGW patients only.\nTopical omiganan appears to be safe in patients with AGW and vulvar HSIL and reduced human papillomavirus load after 12 weeks of treatment in AGW patients.Drug Delivery Technolog