In vitro bactericidal activity of iclaprim in human plasma.

This study evaluated the effect of human plasma on the in vitro bactericidal activity of the novel diaminopyrimidine iclaprim against methicillin (meticillin)-susceptible and -resistant Staphylococcus aureus strains. MICs and minimal bactericidal concentrations (MBCs) of iclaprim, with approximately 93% protein binding, were similar in the absence and in the presence of 50% human plasma; MICs and MBCs ranged from 0.06 to 0.125 microg/ml. Furthermore, the activity of iclaprim was not affected by plasma, with > or = 99.9% reduction in CFU after 5.0 to 7.6 h.

Pathogenomics: an updated European Research Agenda.

The emerging genomic technologies and bioinformatics provide novel opportunities for studying life-threatening human pathogens and to develop new applications for the improvement of human and animal health and the prevention, treatment, and diagnosis of infections. Based on the ecology and population biology of pathogens and related organisms and their connection to epidemiology, more accurate typing technologies and approaches will lead to better means of disease control. The analysis of the genome plasticity and gene pools of pathogenic bacteria including antigenic diversity and antigenic variation results in more effective vaccines and vaccine implementation programs. The study of newly identified and uncultivated microorganisms enables the identification of new threats. The scrutiny of the metabolism of the pathogen in the host allows the identification of new targets for anti-infectives and therapeutic approaches. The development of modulators of host responses and mediators of host damage will be facilitated by the research on interactions of microbes and hosts, including mechanisms of host damage, acute and chronic relationships as well as commensalisms. The study of multiple pathogenic and non-pathogenic microbes interacting in the host will improve the management of multiple infections and will allow probiotic and prebiotic interventions. Needless to iterate, the application of the results of improved prevention and treatment of infections into clinical tests will have a positive impact on the management of human and animal disease. The Pathogenomics Research Agenda draws on discussions with experts of the Network of Excellence "EuroPathoGenomics" at the management board meeting of the project held during 18-21 April 2007, in the Villa Vigoni, Menaggio, Italy. Based on a proposed European Research Agenda in the field of pathogenomics by the ERA-NET PathoGenoMics the meeting's participants updated the established list of topics as the research agenda for the future.

Dihydrofolate reductase inhibitors as antibacterial agents.

Although only a few DHFR inhibitors have progressed as antibiotics to the market there is much renewed interest in the discovery and development of new generation DHFR inhibitors as antibacterial agents. This article describes the success in exploiting DHFR as a drugable target as exemplified by trimethoprim (TMP) and the development of several new diaminopyrimidines. Iclaprim, a recent example of a novel diaminopyrimidine currently in Phase III clinical trials, is also described together with several examples of anti-DHFR antibacterial compounds in pre-clinical development.

Engineered beta-cells secreting dipeptidyl peptidase IV-resistant glucagon-like peptide-1 show enhanced glucose-responsiveness.

Type 2 diabetes is a polygenic disorder characterized by increased insulin resistance, and impaired insulin secretion leading to abnormalities of glucose and lipid metabolism. Reduced responsiveness of the beta-cells to glucose is a critical feature of this syndrome. Glucagon-like peptide 1, a product of the pro-glucagon gene makes beta-cells competent and has many other anti-diabetic properties. We speculated whether GLP-1-based gene therapy could be an approach for treatment of type 2 diabetes. We started with a clone of rat insulinoma cells (S4 cells), which showed reduced responsiveness to glucose in terms of insulin secretion. We transfected these cells with a plasmid encoding a mutated form of GLP-1 (GLP-1-Gly8), which is resistant to the degrading enzyme dipeptidyl-peptidase IV. Activity of secreted GLP-1-Gly8 was assayed using Chinese hamster lung fibroblasts (CHL) cells that expressed cloned GLP-1 receptor and that were transfected with CRE-Luc. Stable cell lines (Glipsulin cells) obtained by this means produced and stored immunoreactive GLP-1-Gly8. In addition to insulin, the Glipsulin cells secreted the GLP-1-Gly8. The secreted GLP-1-Gly8 was active as evidenced by the ability of the conditioned media to elevate cAMP levels in CHL cells expressing GLP-1 receptors. Glipsulin cells responded to glucose with a 6.8 fold increase in insulin secretion compared to a 2.2 fold increase in the control cells. Our results demonstrate that prolonged exposure to GLP-1-Gly8 secreted by increases glucose-responsiveness of these cells. We speculate that engineering GLP-1-Gly8 secretion by beta-cells is a potential gene therapeutic strategy to treat diabetes.

Subtypes A, C, G, and recombinant HIV type 1 are circulating in Bangladesh.

We analyzed the genetic diversity of HIV-1 circulating in Bangladesh by direct sequencing and subsequent phylogenetic analysis of the V3 region of the env gene and p17 fragment of the gag gene from nine unrelated patients. The sequences from one sample grouped into subtype A, five samples grouped into subtype C, and one grouped into subtype G. In addition, two patients appeared to be infected with different recombinant viruses consisting of subtype A and unclassifiable viral sequences. Epidemiological analysis revealed heterosexual transmission in the majority of cases. Furthermore, most subjects had a history of traveling, either to India or to the Arabian Peninsula. This study shows that several HIV-1 subtypes are circulating in Bangladesh, and we conclude that there must have been several introductions of HIV-1 into the Bangladeshi population.

Clinical grade vector production: analysis of yield, stability, and storage of gmp-produced retroviral vectors for gene therapy.

Retroviral vector gene transfer of a therapeutic gene to correct or modify a disease process is a promising strategy for many inherited and acquired diseases. A major obstacle in this process is the large-scale production of the gene transfer vector under good manufacturing practice (GMP) conditions. We have used the CellCube bioreactor system to produce five batches of GMP-grade vector. The production batches were of 10-20 L each, and the titers were around 2 x 10(6) IU/mL. We find that this particular vector is relatively stable with a half-life of about 8 h at 37 degrees C, 40 h at 20 degrees C, and 14 days at 4 degrees C. The half-life during storage at -80 degrees C is around 18 months. The supernatant may be frozen and thawed up to five times without any significant loss of titer. We have also made a comparison between the CellCube bioreactor and the automated roller bottle system RollerCell 40 (RC 40). The yields from the two systems were comparable.

Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis.

Nitric oxide (NO) and other reactive nitrogen species target multiple sites in the mitochondria to influence cellular bioenergetics and survival. Kinetic imaging studies revealed that NO from either activated macrophages or donor compounds rapidly diffuses to the mitochondria, causing a dose-dependent progressive increase in NO-dependent DAF fluorescence, which corresponded to mitochondrial membrane potential loss and initiated alterations in cellular bioenergetics that ultimately led to necrotic cell death. Cellular dysfunction is mediated by an elevated 3-nitrotyrosine signature of the mitochondrial complex I subunit NDUFB8, which is vital for normal mitochondrial function as evidenced by selective knockdown via siRNA. Overexpression of mitochondrial superoxide dismutase substantially decreased NDUFB8 nitration and restored mitochondrial homeostasis. Further, treatment of cells with either necrostatin-1 or siRNA knockdown of RIP1 and RIP3 prevented NO-mediated necrosis. This work demonstrates that the interaction between NO and mitochondrially derived superoxide alters mitochondrial bioenergetics and cell function, thus providing a molecular mechanism for reactive oxygen and nitrogen species-mediated alterations in mitochondrial homeostasis.

Iclaprim, a novel diaminopyrimidine with potent activity on trimethoprim sensitive and resistant bacteria.

Iclaprim, a new selective dihydrofolate inhibitor was synthesized based on rational drug design. Iclaprim's interaction with a resistant Staphylococcus aureus dihydrofolate reductase (DHFR) is outlined in comparison to trimethoprim (TMP). This compound is active against methicillin, TMP and vancomycin resistant strains. Arpida Ltd. is developing Iclaprim for serious hospital infections from Gram-positive pathogens and respiratory tract infections.

Catheter-based transendocardial myocardial gene transfer.

BACKGROUND AND AIM: Local modulation of myocardial function by gene transfer or cell depositions constitutes a potential method of cardiac treatment. This study tested the morphology of myocardial plasmid gene transfer by catheter-based transendocardial injection (NOGA). METHODS: Left ventricular morphology and electrical and mechanical characteristics were mapped in three dimensions. In two pigs, 0.10 mL of toluidine blue was injected at ten sites. In seven pigs, seven to ten injections of 0.10 mL saline containing 0.10 mg pCMV-LacZ expressing the enzyme beta-galactosidase and 0.10 mg phVEGF-A165 were given. The pigs were sacrificed after 3 days and gene expression was determined. RESULTS: Macroscopically on the endocardial surface, all identified spots were located in the target area. However, along the transmyocardial axis, injections with color and plasmid were located randomly throughout the left ventricular wall from the endocardium to the epicardium. In each detected spot, gene expression of beta-galactosidase was observed in an approximate myocardial volume of 5 x 5 x 5 mm. Microscopically, the transfected cells were located typically at the tip of the injection scar. As a rule, 10 to 20 transfected cells were located at the end of the injection scar. In sections where expression of both transcripts was observed, 42% of the cells expressed both beta-galactosidase and vascular endothelial growth factors (VEGF), 32% only beta-galactosidase, and 26% only VEGF. CONCLUSIONS: Myocardial gene transfer following magnetic guidance can be located precisely on the left ventricular inner surface. Within the myocardium, gene expression is local around the distal tip of the injection scar and is located randomly at every level of depth of the left ventricular wall.

Aberrant expression of alpha-Gal on primary human endothelium does not confer susceptibility to NK cell cytotoxicity or increased NK cell adhesion.

The contribution of Gal alpha 1,3Gal (alpha-Gal) to cell-mediated organ xenograft rejection is controversial. We have used recombinant lentiviruses encoding a porcine alpha 1,3 galactosyltransferase (alpha 1,3GalT) to obtain alpha-Gal-expressing primary human aortic endothelial cells (HAEC) at a frequency of 70-90%. These cells were compared to non-transduced and mock-transduced HAEC with regard to their susceptibility to human NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, and support of NK cell adhesion under static and dynamic conditions. Using green fluorescent protein (GFP) as a reporter gene, it was shown that the frequency of green fluorescent HAEC increased until day 5 post-transduction, and at a multiplicity of infection of 2.5, it reached 98%. Lentiviral transduction did not result in activation of HAEC, and transduced HAEC responded as expected to TNF-alpha and IFN-gamma stimulation. No differences were detected between non-alpha-Gal- and alpha-Gal-expressing HAEC in terms of their susceptibility to NK cell-mediated lysis, ability to stimulate IFN-gamma production by NK cells, or ability to support NK cell adhesion under static and dynamic conditions. In conclusion, these data argue against an important role for the alpha-Gal epitope in the direct interaction between endothelium and NK cells and prove that recombinant lentiviruses are efficient gene carriers for primary human endothelial cells.

Electroporation in combination with a plasmid vector containing SV40 enhancer elements results in increased and persistent gene expression in mouse muscle.

Gene transfer into muscle upon injection of plasmid DNA is feasible but occurs with low frequency. However, by using electroporation after injection of plasmid DNA into mouse muscle it has been demonstrated that gene expression can be increased more than 150-fold. In this communication, we have used this technique in combination with plasmids containing a tandem repeat of three 72-bp DNA elements from the SV40 enhancer to study gene expression. Our results show that the combination of electroporation and a plasmid vector carrying these DNA elements results in increased and more persistent gene expression of the luciferase reporter gene in BALB/c mouse muscle. At 14 days after gene delivery, the gene expression was 16-fold higher in muscles injected and electroporated with the plasmid carrying the SV40 enhancers than with control plasmid. We have also studied the effects of the vehicle in which the plasmid was delivered, and the DNase inhibitor aurintricarboxylic acid (ATA), on gene expression. By combining ATA with 150 mM sodium phosphate buffer we were able to obtain a 2-fold increase in gene expression compared to delivery of the plasmid in physiological saline. These results are of importance for the development of efficient delivery techniques for naked DNA.

Vascular endothelial growth factor induced functional and morphologic signs of endothelial dysfunction in isolated arteries from normal pregnant women.

OBJECTIVE: The purpose of this study was to determine the effects of vascular endothelial growth factor on basal tone, endothelium-dependent dilatation, permeability, and morphologic features of endothelium in isolated arteries from normal pregnant women. We hypothesized that vascular endothelial growth factor might induce signs of endothelial dysfunction. STUDY DESIGN: Arteries (approximately 200 microm) were dissected from subcutaneous fat biopsy specimens that were obtained at cesarean delivery and mounted on a pressure arteriograph. Changes in basal tone, dilatation to bradykinin (1 nmol/L to 3 micromol/L) before, during, and after 3 hours of incubation with vascular endothelial growth factor (0.5 or 1 nmol/L), vascular endothelial growth factor (0.5 nmol/L) plus bosentan (a nonselective endothelin receptor A and B antagonist, 1 micromol/L), or vehicle were compared. Scanning electron microscopy was applied for endothelial morphologic features. Permeability to Evans blue dye was evaluated in arteries after incubation with vascular endothelial growth factor, vascular endothelial growth factor plus angiopoietin-1, or vehicle, and in arteries that were obtained from women with preeclampsia. RESULTS: Basal tone was higher after 60 minutes of incubation with vascular endothelial growth factor (0.5 nmol/L) compared with vehicle (29% +/- 5% [n = 10] vs 10% +/- 4% [n = 7], P =.006). Combination of vascular endothelial growth factor with bosentan failed to increase the tone (n = 4). Bradykinin-mediated dilatation was impaired in arteries that were incubated with vascular endothelial growth factor 0.5 nmol/L (max dilatation: 287% +/- 16% vs 160% +/- 23% [n = 10], P =.0001) or vascular endothelial growth factor 1 nmol/L (max dilatation: 207% +/- 21% vs 88% +/- 4% [n = 3], P =.003). Bradykinin-mediated dilatation was similar after incubation with vehicle (n = 7) or the combination of vascular endothelial growth factor plus bosentan (n = 4). Evans blue dye staining was higher after incubation with vascular endothelial growth factor but was reversed by the addition of angiopoietin-1. Scanning electron microscopy demonstrated the development of intercellular gaps. CONCLUSION: Vascular endothelial growth factor impaired bradykinin-mediated dilatation and enhanced basal tone and permeability. This might indicate a potential role for vascular endothelial growth factor in the development of endothelial dysfunction in pregnancy. Angiopoietin-1 inhibited the vascular endothelial growth factor-induced vascular leakage, which may have therapeutic implications in preeclampsia.

Nonsurgical direct delivery of plasmid DNA into rat heart: time course, dose response, and the influence of different promoters on gene expression.

Transfer of genes encoding therapeutic proteins into the myocardium shows great potential for treatment of coronary artery disease. To quantitatively elucidate the behavior of plasmid DNA following cardiac gene transfer, time kinetics, dose-response relationship, systemic spread to the liver, and the influence of different promoters on plasmid DNA gene expression in rat hearts were examined using a novel nonsurgical direct delivery method that enables testing of large numbers of animals. Plasmids encoding either vascular endothelial growth factor A 165 or a fusion protein between enhanced green fluorescent protein (EGFP) luciferase were injected directly in rat hearts under echocardiographic guidance. The results show that gene expression is dose related and that the duration of gene expression is transient. These findings underscore the necessity to explore other efficient vectors or alternative methods of gene delivery to achieve increased and prolonged gene expression.