Efficacy of piperacillin in combination with novel ß-lactamase inhibitor IID572 against ß-lactamase-producing strains of Enterobacteriaceae and Staphylococcus aureus in murine neutropenic thigh infection models.

OBJECTIVES: The neutropenic murine thigh infection model was used to assess the effectiveness of IID572, a novel ß-lactamase inhibitor, in rescuing piperacillin activity against bacterial strains expressing various ß-lactamase enzymes. METHODS: Mice (n = 4/group) were inoculated with Enterobacteriaceae or Staphylococcus aureus bacterial strains expressing a range of ß-lactamases via intramuscular injection. Two hours after bacterial inoculation, subcutaneous treatment with piperacillin/IID572 or piperacillin/tazobactam every 3 h was initiated. Animals were euthanized via CO2 24 h after the start of therapy and bacterial cfu (log10 cfu) per thigh was determined, and the static dose was calculated. RESULTS: In a dose-dependent manner, piperacillin/IID572 reduced the thigh bacterial burden in models established with Enterobacteriaceae producing class A, C and D ß-lactamases (e.g. ESBLs, KPC, CMY-2 and OXA-48). Piperacillin/IID572 was also efficacious against MSSA strains, including one producing ß-lactamase. Static doses of piperacillin/IID572 were calculable from animals infected with all strains tested and the calculated static doses ranged from 195 to 4612 mg/kg/day piperacillin, the active component in the combination. Of the 13 strains investigated, a 1 log10 bacterial reduction was achieved for 9 isolates and a 2 log10 reduction was achieved for 3 isolates; piperacillin/tazobactam was not efficacious against 6 of the 13 isolates tested. CONCLUSIONS: In contrast to tazobactam, IID572 was able to rescue piperacillin efficacy in murine thigh infection models established with ß-lactamase-producing strains of Enterobacteriaceae and S. aureus, including those expressing ESBLs or serine carbapenemases.

Pharmacokinetics and pharmacodynamics of the novel monobactam LYS228 in a neutropenic murine thigh model of infection.

Objectives: The neutropenic murine thigh infection model and a dose-fractionation approach were used to determine the pharmacokinetic/pharmacodynamic (PK/PD) relationship of LYS228, a novel monobactam antibiotic with activity against Enterobacteriaceae including carbapenem-resistant strains. Methods: Mice (n = 4 per group) were inoculated with Enterobacteriaceae strains via intramuscular injection. Two hours post-bacterial inoculation, treatment with LYS228 was initiated. Animals were euthanized with CO2 24 h after the start of therapy and bacterial counts (log10 cfu) per thigh were determined. PK parameters were calculated using free (f) plasma drug levels. Results: Following a dose-fractionation study, non-linear regression analysis determined that the predominant PK/PD parameter associated with antibacterial efficacy of LYS228 was the percentage of the dosing interval that free drug concentrations remained above the MIC (%fT>MIC). In a dose-dependent manner, LYS228 reduced the thigh bacterial burden in models established with Enterobacteriaceae producing ß-lactamase enzymes of all classes (e.g. ESBLs, NDM-1, KPC, CMY-2 and OXA-48). The range of the calculated static dose was 86-649 mg/kg/day for the isolates tested, and the magnitude of the driver of efficacy was 37-83 %fT>MIC. %fT>MIC was confirmed as the parameter predominantly driving efficacy as evidenced by a strong coefficient of determination (r2 = 0.68). Neutrophils had minimal impact on the effect of LYS228 in the murine thigh infection model. Conclusions: LYS228 is efficacious in murine thigh infection models using ß-lactamase-producing strains of Enterobacteriaceae, including those expressing metallo-ß-lactamases, ESBLs and serine carbapenemases, with the PK/PD driver of efficacy identified as %T>MIC.

The effect of P38 MAP kinase inhibition in a mouse model of influenza.

PURPOSE: Influenza viruses are a common cause of human respiratory infections, resulting in epidemics of high morbidity and mortality. We investigated the effect of a novel mitogen-activated protein kinase (MAPK) inhibitor in vitro and in a murine influenza model to further explore whether p38 MAPK inhibition could reduce viral replication. METHODS: In vitro, the antiviral effect of p38 MAPK inhibitor BCT194 was evaluated in differentiated human bronchial epithelial cells (HBECs); in vivo, female BALB/c mice were infected intranasally with 150 pfu of influenza H1N1 A/Puerto Rico/8/34 and treated with BCT197 (a closely related p38 MAPK inhibitor with an IC50 value of<1 µM, currently in clinical testing), dexamethasone or oseltamivir (Tamiflu) starting 24 h post infection. Body weight, bronchoalveolar lavage cells, cytokines, total protein and lactate dehydrogenase as well as serum cytokines were measured; a subset of animals was evaluated histopathologically.Results/Key findings. p38MAP kinase inhibition with BCT194 had no impact on influenza replication in HBECs. When examining BCT197 in vivo, and comparing to vehicle-treated animals, reduced weight loss, improvement in survival and lack of impaired viral control was observed at BCT197 concentrations relevant to those being used in clinical trials of acute exacerbations of chronic obstructive pulmonary disease; at higher concentrations of BCT197 these effects were reduced. CONCLUSIONS: Compared to vehicle treatment, BCT197 (administered at a clinically relevant concentration) improved outcomes in a mouse model of influenza. This is encouraging given that the use of innate inflammatory pathway inhibitors may raise concerns of negative effects on infection regulation.

Investigation into the biological stability of collagen/chondroitin-6-sulphate gels and their contraction by fibroblasts and keratinocytes: the effect of crosslinking agents and diamines.

Artificial skin substitutes based on autologous keratinocytes cultured on collagen-based substrata are being developed for grafting onto patients with severe burns. The properties of the substratum can be manipulated by crosslinking the collagen with the glysocaminoglycan, chondroitin-6-sulphate (Ch6SO4), carbodiimides and polyamines. Biological stability, assessed by resistance to collagenase, was increased by incorporation of Ch6SO4, but crosslinking with the carbodiimides, 1-ethyl-3-(dimethylaminopropyl)carbodiimide and 1,1-carbonyldiimidazole or the polyamines, putrescine or diaminohexane, had little further benefit. Contraction of the collagen gels occurred to a greater extent when seeded with fibroblasts than with keratinocytes. The extent of contraction by either cell type was not influenced by the presence of Ch6SO4 in the gel, but the carbodiimides, and to a lesser extent the polyamines, limited cell-mediated contraction, particularly that mediated by fibroblasts. Optimum substratum composition for artificial skin substitutes will involve a compromise between the desired attributes of biological stability, rate of contraction, mechanical strength, biocompatibility and promotion of cell growth.

Investigation into the tensile properties of collagen/chondroitin-6-sulphate gels: the effect of crosslinking agents and diamines.

Artificial skin substitutes based on autologous keratinocytes are being developed for grafting onto burns patients. In order to be used successfully in the clinic, these skin substitutes need to have sufficient strength to allow ease of handling. This may be achieved by crosslinking the collagen substratum on which the cells are cultured. The influence of potential crosslinking agents on the tensile properties of acellular collagen gels has been investigated, including the glycosaminoglycan, chondroitin-6-sulphate (Ch6SO4), the water-soluble carbodiimide crosslinking agents 1-ethyl-3-(3-diaminopropyl) carbodiimide (EDAC), and 1,1-carbonyldiimidazole (CDI), and the polyamines, putrescine and diaminohexane. Values for Young's modulus, maximum load, stress, displacement and percentage strain at maximum load were generated by subjecting the samples to a tear propagation test. Incorporation of 20% Ch6SO4 into collagen gels caused a significant increase in the Young's modulus, maximum load and stress at maximum load. Crosslinking treatment with EDAC, CDI or polyamines had little further benefit, and in many cases resulted in a decrease in particular parameters. In terms of mechanical strength, the best crosslinking combination proved to be the combination of CDI and diaminohexane, with results either improved or maintained when compared with the control no treatment variants. However, previous experience suggests that the use of CDI as a crosslinking reagent may inhibit infiltration and proliferation of fibroblasts in the substratum and it may be necessary to reach a compromise to obtain the best combination of biological and mechanical properties for artificial skin substitutes.

Epidermal-dermal interactions regulate gelatinase activity in Apligraf, a tissue-engineered human skin equivalent.

BACKGROUND: Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have important functions during skin development, repair and maintenance. MMP-2 and MMP-9 (gelatinase A and gelatinase B) are involved in regulating keratinocyte migration. OBJECTIVES: To analyse whether Apligraf, a bilayered tissue-engineered human skin equivalent (HSE), produces gelatinases and TIMPs and whether or not epidermal-dermal interactions regulate MMP activity. METHODS: The tissue distribution of MMP-2, MMP-9, TIMP-1, TIMP-2 and fibronectin was analysed by immunohistochemistry. Secreted MMP activity was quantified by a fluorimetric assay and gelatin zymography was used to monitor gelatinases in tissue culture supernatants. RESULTS: Apligraf expressed MMP-2 and MMP-9 and contained immunohistochemically detectable amounts of TIMP-1 and TIMP-2. The gelatinases were predominantly produced in the epidermis, whereas immunostaining of TIMP-1 and TIMP-2 was largely confined to the dermal component of the HSE. Fibronectin was expressed only in the dermis. Gelatin zymography demonstrated that intact Apligraf produced both MMP-2 and MMP-9, the latter predominantly in its latent form. Separation of the dermis from the epidermis resulted in an enhanced production and activation of MMP-9 by the epidermal layer, and secretion of latent and active MMP-2 by the dermal layer. Moreover, the incubation media of the separated epidermis demonstrated significantly stronger MMP activity than did intact Apligraf or its dermal component. CONCLUSIONS: These observations provide evidence that epidermal-dermal interactions suppress epidermal gelatinase activity. In addition, coexpression of TIMPs and fibronectin in the Apligraf dermis suggests that the product has the potential to counteract the imbalance between matrix production and degradation in chronic wounds and thus may support wound re-epithelialization.

Transcriptional regulation of mouse granulocyte-macrophage colony-stimulating factor/IL-3 locus.

Granulocyte-macrophage (GM)-CSF and IL-3 are hemopoietic growth factors whose genes are closely linked in both humans and mice. In humans, the GM-CSF and IL-3 genes are regulated by a cyclosporin A-inhibitable enhancer located 3 kb upstream of the GM-CSF gene that is inducible by signals that mimic TCR activation. To search for a murine homologue of this enhancer we probed mouse genomic DNA and located a 400-bp element 2 kb upstream of the mouse GM-CSF gene that was 76% homologous with the human GM-CSF enhancer. Like the human GM-CSF enhancer, this element formed a cyclosporin A-inhibitable DNase I-hypersensitive site in the murine T cell line EL4 upon activation with phorbol ester and calcium ionophore. Transient transfection assays showed that this homologue of the human enhancer acted as an inducible enhancer of the thymidine kinase promoter, the mouse IL-3 promoter, and the human GM-CSF promoter. We observed, however, that the mouse GM-CSF promoter was significantly more active than the human GM-CSF promoter and found that it supported a level of activity equivalent to the combination of the human GM-CSF promoter and the human GM-CSF enhancer. Consequently, the activity of mouse GM-CSF promoter was not significantly elevated in the presence of the mouse GM-CSF enhancer. Because the mouse GM-CSF enhancer is considerably less active than its human homologue we suggest that the mouse GM-CSF gene has evolved with less dependence upon the upstream enhancer for its activation.

Investigation into cell growth on collagen/chondroitin-6-sulphate gels: the effect of crosslinking agents and diamines.

Artificial skin substitutes based on cultured autologous keratinocytes need to have sufficient strength and ease of handling to be utilized successfully by surgeons in the clinic. This may be achieved by crosslinking the collagen substratum on which the cells are cultured, which in this case is a collagen gel. Increased strength must be attained without detrimental effect on cell growth. The influence of potential crosslinking agents including the glycosaminoglycan, chondroitin-6-sulphate (Ch6SO4), the water soluble carbodiimide crosslinking agents 1-ethyl-3-(3-diaminopropyl) carbodiimide (EDAC), and 1,1-carbonyldiimidazole (CDI), and the polyamines putrescine, spermine and diaminohexane, on cell growth rate has been investigated. Incorporation of 20% Ch6SO4 into collagen gels caused an approximately 16% increase in keratinocyte growth, but had no significant effect on that of dermal fibroblasts. Pre-formed collagen gels (+/- Ch6SO4) were treated with the carbodiimides. This crosslinking treatment markedly inhibited fibroblast growth (EDAC 45% inhibition, CDI 70%), without affecting that of keratinocytes. Pre-formed collagen gels (+/-Ch6SO4 and carbodiimide) were treated with 0.1 M, 0.5 M or 1.0 M polyamine. Spermine inhibited the growth rate of both cell types at all concentrations tested, whereas putrescine and diaminohexane had little effect. The mechanical strength of these crosslinked gels is currently being assessed to determine the optimum composition in terms of cell growth and biocompatibility, and strength.

Antifungal drug response in an in vitro model of dermatophyte nail infection.

Despite terbinafine being fungicidal against Trichophyton rubrum in standard NCCLS assays and rapidly accumulating in nails in vivo, onychomycosis patients require prolonged terbinafine treatment to be cured. To investigate this, we developed a more clinically relevant onychomycosis in vitro test model. Human nail powder inoculated with T. rubrum and incubated in liquid RPMI 1640 salt medium, which did not support growth alone, developed extensive and invasive mycelial growth. Antifungal drugs were added at different concentrations and cultures incubated for 1 to 4 weeks. Fungal survival was determined by spreading cultures on PDA plates without drug and measuring CFU after 1 to 4 weeks incubation. Drug activity was expressed as the nail minimum fungicidal concentration (Nail-MFC) required for 99.9% elimination of viable fungus. Terbinafine Nail-MFC was 4 microg/ml after 1 week exposure, decreasing to 1 microg/ml after 4 weeks exposure, much higher than MFCs < or = 0.03 microg/ml determined in standard NCCLS MIC assays. In contrast, other clinically used drugs were unable to kill T. rubrum after 4 weeks incubation in this model. Invasive mycelial growth on nail appears to protect T. rubrum from the cidal action of systemic drugs, thus providing a rationale for the long treatment periods in onychomycosis.

Regulation of GM-CSF gene transcription by core-binding factor.

GM-CSF gene activation in T cells is known to involve the transcription factors nuclear factor-kappa B, AP-1, NFAT, and Sp1. Here we demonstrate that the human GM-CSF promoter and enhancer also encompass binding sites for core-binding factor (CBF). Significantly, the CBF sites are in each case contained within the minimum essential core regions required for inducible activation of transcription. Furthermore, these core regions of the enhancer and promoter each encompass closely linked binding sites for CBF, AP-1, and NFATp. The GM-CSF promoter CBF site TGTGGTCA is located 51 bp upstream of the transcription start site and also overlaps a YY-1 binding site. A 2-bp mutation within the CBF site resulted in a 2-3-fold decrease in the activities of both a 69-bp proximal promoter fragment and a 627-bp full-length promoter fragment. Stepwise deletions into the proximal promoter also revealed that the CBF site, but not the YY-1 site, was required for efficient induction of transcriptional activation. The AML1 and CBF beta genes that encode CBF each have the ability to influence cell growth and differentiation and have been implicated as proto-oncogenes in acute myeloid leukemia. This study adds GM-CSF to a growing list of cytokines and receptors that are regulated by CBF and which control the growth, differentiation, and activation of hemopoietic cells. The GM-CSF locus may represent one of several target genes that are dysregulated in acute myeloid leukemia.

Amelioration of retroviral vector silencing in locus control region beta-globin-transgenic mice and transduced F9 embryonic cells.

Retroviral vectors are transcriptionally silenced in hematopoietic stem cells, and this phenomenon must be overcome for effective gene therapy of blood diseases. The murine stem cell virus (MSCV) vector completely silences beta-globin reporter genes regulated by locus control region (LCR) elements 5'HS2 to 5'HS4 in seven of eight transgenic mice. Here, we show that no single known MSCV silencer element is sufficient for complete LCR beta-globin transgene silencing. However, partial silencing of high-copy transgenes is conveyed by the MSCV direct repeat and promoter elements. The CpG methylation pattern of silenced and expressed MSCV promoter transgenes is virtually identical, demonstrating that silencing does not absolutely correlate with methylation status. Combined mutations in all four MSCV silencer elements leads to expression of beta-globin in 6 of 10 transgenic mice. The same mutations incorporated into the HSC1 retrovirus vector direct neo gene expression in 71% of transduced F9 embryonic carcinoma cells. These studies demonstrate that combined mutation of four retroviral silencer elements relieves complete silencing in most transgenic mice and transduced F9 cells and suggests that novel silencer elements remain. Enhanced expression of the HSC1 vector in primitive stem cells is well suited for blood gene therapy applications.

Generation of an improved luciferase reporter gene plasmid that employs a novel mechanism for high-copy replication.

We have engineered the reporter gene plasmid pXPG by incorporating a novel high-copy origin of replication and a modified luciferase gene into a pXP1-derived vector that efficiently blocks read-through transcription in eukaryotic cells. pXPG contains the Luc+ luciferase gene derived from pGL3 that lacks a peroxisomal targeting sequence, thereby allowing accumulation of luciferase protein in the cytoplasm rather than subcellular organelles of transfected eukaryotic cells. pXPG has distinct advantages over pGL3, because it contains SV40 polyadenylation signals that appear to be more efficient at blocking read-through transcription than the synthetic polyadenylation signal present in pGL3. pXPG contains a novel mutation near the origin of replication that increases plasmid copy number in Escherichia coli. This mutation alters the -10 sequence in the RNA II promoter of the ColE1 origin of replication from TAATCT to TAATAT. As this sequence is a closer match to the consensus -10 element, we suggest that the mutation increases copy number by increasing the rate of transcription of the RNA II replication primer. This novel mechanism for increasing copy number may have more widespread applications than the commonly used pUC high-copy origin of replication mutation. Unlike pUC, which reverts to low copy number at 30 degrees C, the pXPG mutation supports a higher copy number at both 37 and 30 degrees C.

Retrovirus vector silencing is de novo methylase independent and marked by a repressive histone code.

Retrovirus vectors are de novo methylated and transcriptionally silent in mammalian stem cells. Here, we identify epigenetic modifications that mark retrovirus-silenced transgenes. We show that murine stem cell virus (MSCV) and human immunodeficiency virus type 1 (HIV-1) vectors dominantly silence a linked locus control region (LCR) beta-globin reporter gene in transgenic mice. MSCV silencing blocks LCR hypersensitive site formation, and silent transgene chromatin is marked differentially by a histone code composed of abundant linker histone H1, deacetylated H3 and acetylated H4. Retrovirus-transduced embryonic stem (ES) cells are silenced predominantly 3 days post-infection, with a small subset expressing enhanced green fluorescent protein to low levels, and silencing is not relieved in de novo methylase-null [dnmt3a-/-;dnmt3b-/-] ES cells. MSCV and HIV-1 sequences also repress reporter transgene expression in Drosophila, demonstrating establishment of silencing in the absence of de novo and maintenance methylases. These findings provide mechanistic insight into a conserved gene silencing mechanism that is de novo methylase independent and that epigenetically marks retrovirus chromatin with a repressive histone code.

LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1.

We have previously shown correction of X-linked severe combined immunodeficiency [SCID-X1, also known as gamma chain (gamma(c)) deficiency] in 9 out of 10 patients by retrovirus-mediated gamma(c) gene transfer into autologous CD34 bone marrow cells. However, almost 3 years after gene therapy, uncontrolled exponential clonal proliferation of mature T cells (with gammadelta+ or alphabeta+ T cell receptors) has occurred in the two youngest patients. Both patients' clones showed retrovirus vector integration in proximity to the LMO2 proto-oncogene promoter, leading to aberrant transcription and expression of LMO2. Thus, retrovirus vector insertion can trigger deregulated premalignant cell proliferation with unexpected frequency, most likely driven by retrovirus enhancer activity on the LMO2 gene promoter.