A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis.

Vascular injury is a serious complication of sepsis due to the gram-negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non-LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E-selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS-deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor-κB (NF-κB)]. In this study, we show that the particular propensity for N. meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen-activated protein (MAP) kinases, on the activity of the E-selectin promoter. Furthermore, inhibition of E-selectin expression in response to the lpxA- strain by a p38 inhibitor indicates a significant role of a p38-dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally.

Inhibition of neointimal hyperplasia in a rabbit vein graft model following non-viral transfection with human iNOS cDNA.

Vein graft failure caused by neointimal hyperplasia (IH) after coronary artery bypass grafting with saphenous veins is a major clinical problem. The lack of safe and efficient vectors for vascular gene transfer has significantly hindered progress in this field. We have developed a Receptor-Targeted Nanocomplex (RTN) vector system for this purpose and assessed its therapeutic efficacy in a rabbit vein graft model of bypass grafting. Adventitial delivery of ß-Galactosidase showed widespread transfection throughout the vein wall on day 7, estimated at about 10% of cells in the adventitia and media. Vein grafts were then transfected with a plasmid encoding inducible nitric oxide synthase (iNOS) and engrafted into the carotid artery. Fluorescent immunohistochemistry analysis of samples from rabbits killed at 7 days after surgery showed that mostly endothelial cells and macrophages were transfected. Morphometric analysis of vein graft samples from the 28-day groups showed approximately a 50% reduction of neointimal thickness and 64% reduction of neointimal area in the iNOS-treated group compared with the surgery control groups. This study demonstrates efficacy of iNOS gene delivery by the RTN formulation in reducing IH in the rabbit model of vein graft disease.

Non-invasive liposome-mediated gene delivery can correct the ion transport defect in cystic fibrosis mutant mice.

We report gene transfer to the Edinburgh insertional mutant mouse (cf/cf), delivering CFTR cDNA-liposome complexes into the airways by nebulization. We show full restoration of cAMP related chloride responses in some animals and demonstrate, in the same tissues, human CFTR cDNA expression. Overall, a range of correction was seen with restoration of about 50% of the deficit between wild type mice and untreated cf/cf controls. We report modest correction in the intestinal tract following direct instillation and provide initial encouraging safety data for both the respiratory and intestinal tract following the liposome mediated gene delivery. The non-viral nature and potentially lower immunogenicity of DNA-liposomes suggest that this may offer a therapeutic alternative to adenoviral therapies.

Elemental imaging of MRI contrast agents: benchmarking of LA-ICP-MS to MRI.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been used to map the spatial distribution of magnetic resonance imaging (MRI) contrast agents (Gd-based) in histological sections in order to explore synergies with in vivo MRI. Images from respective techniques are presented for two separate studies namely (1) convection enhanced delivery of a Gd nanocomplex (developmental therapeutic) into rat brain and (2) convection enhanced delivery, with co-infusion of Magnevist (commercial Gd contrast agent) and Carboplatin (chemotherapy drug), into pig brain. The LA technique was shown to be a powerful compliment to MRI not only in offering improved sensitivity, spatial resolution and signal quantitation but also in giving added value regarding the fate of administered agents (Gd and Pt agents). Furthermore simultaneous measurement of Fe enabled assignment of an anomalous contrast enhancement region in rat brain to haemorrhage at the infusion site.

Peptide and nucleic acid-directed self-assembly of cationic nanovehicles through giant unilamellar vesicle modification: Targetable nanocomplexes for in vivo nucleic acid delivery.

One of the greatest challenges for the development of genetic therapies is the efficient targeted delivery of therapeutic nucleic acids. Towards this goal, we have introduced a new engineering initiative in self-assembly of biologically safe and stable nanovesicle complexes (∼90 to 140nm) derived from giant unilamellar vesicle (GUV) precursors and comprising plasmid DNA or siRNA and targeting peptide ligands. The biological performance of the engineered nanovesicle complexes were studied both in vitro and in vivo and compared with cationic liposome-based lipopolyplexes. Compared with cationic lipopolyplexes, nanovesicle complexes did not show advantages in transfection and cell uptake. However, nanovesicle complexes neither displayed significant cytotoxicity nor activated the complement system, which are advantageous for intravenous injection and tumour therapy. On intravenous administration into a neuroblastoma xenograft mouse model, nanovesicle complexes were found to distribute throughout the tumour interstitium, thus providing an alternative safer approach for future development of tumour-specific therapeutic nucleic acid interventions. On oropharyngeal instillation, nanovesicle complexes displayed better transfection efficiency than cationic lipopolyplexes. The technological advantages of nanovesicle complexes, originating from GUVs, over traditional cationic liposome-based lipopolyplexes are discussed. STATEMENT OF SIGNIFICANCE: The efficient targeted delivery of nucleic acids in vivo provides some of the greatest challenges to the development of genetic therapies. Giant unilamellar lipid vesicles (GUVs) have been used mainly as cell and tissue mimics and are instrumental in studying lipid bilayers and interactions. Here, the GUVs have been modified into smaller nanovesicles. We have then developed novel nanovesicle complexes comprising self-assembling mixtures of the nanovesicles, plasmid DNA or siRNA, and targeting peptide ligands. Their biophysical properties were studied and their transfection efficiency was investigated. They transfected cells efficiently without any associated cytotoxicity and with targeting specificity, and in vivo they resulted in very high and tumour-specific uptake and in addition, efficiently transfected the lung. The peptide-targeted nanovesicle complexes allow for the specific targeted enhancement of nucleic acid delivery with improved biosafety over liposomal formulations and represent a promising tool to improve our arsenal of safe, non-viral vectors to deliver therapeutic cargos in a variety of disorders.

An RGD-oligolysine peptide: a prototype construct for integrin-mediated gene delivery.

We have synthesized a linear, bifunctional peptide that comprises an integrin-targeting domain containing an arginine-glycine-aspartic acid tripeptide motif and a DNA-binding moiety consisting of a short stretch of 16 lysine residues. This peptide can form distinctive, condensed complexes with DNA and is capable of mediating its delivery and expression in a variety of mammalian cells in culture. Internalization is mediated by cell surface integrin receptors via a mechanism that is known to be phagocytic. We have analyzed the relationship between DNA and peptide and have investigated the conditions suitable for optimal gene delivery. The formation of condensed peptide DNA complexes leads to resistance to nuclease degradation. The level of reporter gene expression obtained is dependent on the peptide-to-DNA ratio and is enhanced in the presence of the endosomal buffer chloroquine, polyethyleneimine, and deactivated adenovirus during gene delivery. Under optimal conditions the levels of reporter gene expression obtained approach or even exceed those obtained with DNA delivered with the commercial liposome Lipofectamine. The ability to produce an efficient gene delivery system using small, easily modified, and well-defined constructs that have no constraint of particle size demonstrates the advantages of integrin-targeting peptides for gene transfer.

High-titer recombinant adeno-associated virus production from replicating amplicons and herpes vectors deleted for glycoprotein H.

Production of high-titer rAAV is essential for in vivo clinical application. One limiting factor may be the failure of existing systems to replicate the packaging genome in such a way that expression of Rep and Cap proteins is coordinately amplified. DISC-HSV (disabled single-cycle virus) is a genetically modified herpes simplex virus (HSV) that by deletion of glycoprotein H (gH) is infectious only if propagated in a complementing cell line. In this study, we have used DISC-HSV as a helper for rAAV replication, and have simulated to some extent the amplication of the rep and cap genomes seen in wtAAV infection by incorporating both these and vector sequences in HSV amplicons. Facilitated production of AAV Rep and Cap proteins translates into a considerably improved recovery of rAAV, which transduces cells of the neuroretina in vivo with high efficiency. The potential for contamination with infectious herpes particles is eliminated by the use of noncomplementing (gH-) cell lines to propagate the virus, and by standard purification methods. The use of DISC-HSV and herpes-derived amplicons for production of rAAV may be a useful strategy for future in vivo studies and for clinical application.

Lipid-mediated enhancement of transfection by a nonviral integrin-targeting vector.

Nonviral vectors consisting of integrin-targeting peptide/DNA (ID) complexes have the potential for widespread application in gene therapy. The transfection efficiency of this vector, however, has been limited by endosomal degradation. We now report that lipofectin (L) incorporated into the ID complexes enhances integrin-mediated transfection, increasing luciferase expression by more than 100-fold. The transfection efficiency of Lipofectin/Integrin-binding peptide/DNA (LID) complexes, assessed by beta-galactosidase reporter gene expression and X-gal staining, was improved from 1% to 10% to over 50% for three different cell lines, and from 0% to approximately 25% in corneal endothelium in vitro. Transfection complexes have been optimized with respect to their transfection efficiency and we have investigated their structure, function, and mode of transfection. Both ID and LID complexes formed particles, unlike the fibrous network formed by lipofectin/DNA complexes (LD). Integrin-mediated transfection by LID complexes was demonstrated by the substantially lower transfection efficiency of LKD complexes in which the integrin-biding peptide was substituted for K16 (K). Furthermore, the transfection efficiency of complexes was shown to be dependent on the amount of integrin-targeting ligand in the complex. Finally, a 34% reduction in integrin-mediated transfection efficiency by LID complexes was achieved with a competing monoclonal antibody. The role of lipofectin in LID complexes appears, therefore, to be that of a co-factor, enhancing the efficiency of integrin-mediated transfection. The mechanism of enhancement is likely to involve a reduction in the extent of endosomal degradation of DNA.

Adhesion molecules and gene transfer.

Cell adhesion molecules are a large group of molecules involved in a variety of cell-to-cell and cell-to-extra-cellular matrix (ECM) interactions. Apart from their cellular function these molecules are exploited by a number of pathogenic micro-organisms as receptors for cell entry. Discovery of the use of adhesion molecules for binding and internalisation by naturally occurring pathogens has fuelled much research, in recent years, into the utilisation of these molecules for the targeting and uptake of both gene and drug delivery systems. This review describes the development of such systems and their potential advantages over other receptor-targeted delivery systems.

Differential antinociceptive effects of morphine and methylmorphine in the formalin test.

The antinociceptive activities of morphine, and its quaternary analogue methylmorphine, have been compared after intraperitoneal and intracerebroventricular administrations in the mouse paw formalin test. Systemic morphine inhibited both the early and late phases of the formalin-induced licking response and this activity was naloxone sensitive. In contrast, systemic methylmorphine inhibited only the late phase, and this activity was blocked by pre-treatment with methylnaloxone. Central administration of either morphine or methylmorphine inhibited the early phase of the licking response partially and the late phase completely. Systemic naloxone inhibited the central action of both opioids, whilst systemic methylnaloxone did not affect the central action of methylmorphine. The results indicate that the early phase of the response to formalin in the mouse may be inhibited by stimulation of central opioid receptors whilst inhibition of the late phase may involve both peripheral and central opioid receptors.

Involvement of histamine in naloxone-resistant and naloxone-sensitive models of swim stress-induced antinociception in the mouse.

The antinociceptive activity of histamine in male mice has been demonstrated using chemical and thermal noxious stimuli and its involvement in naloxone-sensitive and naloxone-insensitive models of stress-induced antinociception investigated. In the abdominal constriction test, histamine and dimaprit but not histidine, induced antinociception. Compound 48/80 and H1 antagonists (diphenhydramine, mepyramine and promethazine) and large doses of H2 antagonists (cimetidine and zolantidine) produced antinociception in this test. Antinociception induced by histamine was refractory to mepyramine, metiamide and naloxone. Histamine and non-antinociceptive doses of its antagonists had no influence on the naloxone-resistant warm water swim stress-induced antinociception. In the hot-plate test, histamine agonists, except the H3 agonist (R) alpha-methyl histamine (alpha-MeHA), were antinociceptive but all these agents augmented the naloxone-sensitive room temperature swim stress-induced antinociception, after either intraperitoneal or intraventricular injection. The antinociceptive action of dimaprit was not antagonized by zolantidine which, like other histamine antagonists excluding metiamide, also produced antinociception and enhanced room temperature swim stress-induced antinociception. These findings suggest that histamine is involved in pathways mediating antinociception in the mouse and that such pathways are activated in a naloxone-sensitive model of stress-induced antinociception but not in a naloxone-insensitive model.

Effects of 6-hydroxydopamine-induced lesions of the paraventricular nucleus, and of prazosin, on the corticosterone response to restraint in rats.

In rats, with bilateral lesions of the paraventricular nuclei induced by 6-hydroxydopamine, restraint stress failed to elevate levels of corticosterone in plasma, although the resting levels in lesioned animals were similar to those of control rats. Prazosin inhibited the plasma-corticosterone response to restraint in intact rats, at doses which did not by themselves influence resting hormone levels. These results suggest a positive role for noradrenaline, acting through alpha-adrenoceptors, on the stress-induced output of corticotrophin.

Recombinant HMG1 protein produced in Pichia pastoris: a nonviral gene delivery agent.

This paper describes the production of a recombinant protein from the expression system based on the methylotrophic yeast Pichia pastoris. Efficient production of rat high-mobility-group 1 (HMG1) protein was obtained using the system. Two forms of HMG1 were secreted into the culture medium: a 24.5-kDa species corresponding to the native HMG1 and a 32-kDa glycosylated derivative. Non-glycosylated recombinant HMG1 was purified easily and shown to possess the same DNA-binding properties as HMG1 purified from calf thymus. Plasmid DNA complexed to the recombinant HMG1 is taken up by a variety of mammalian cells in culture. Transient expression of a luciferase reporter gene was observed. Under selective conditions, stable expression of a neomycin gene was established as a result of integration into the genome. HMG1-mediated gene delivery was as efficient as calcium phosphate-mediated transfection but without associated cell damage. In addition, stable transfectants obtained after selection for G418 resistance usually integrated only one copy of the transfected DNA in contrast to the high unpredictable number obtained by the calcium phosphate method. HMG1 transfection complexes were not toxic to cultured cells, even at high concentrations.

The actions of prostaglandins E2 and F2alpha on human foetal intestine.

Human foetal small and large intestine responded to prostaglandins E(2) and F(2alpha), the gestational age of the tissue being between 14 and 26 weeks. The predominant response to both prostaglandins was a contraction of each region of the intestine studied. The response to both prostaglandins was antagonized selectively by poly.phloretin phosphate.

Vasopressin and stress-induced antinociception in the mouse.

1. Arginine vasopressin produced antinociception in the hot-plate test after intracerebroventricular injection (0.5 micrograms) and in the acetic acid abdominal constriction test after intraperitoneal injection (0.1 mg kg-1). 2. The antinociception produced by arginine vasopressin was sensitive to deamino(CH2)5Tyr(Me) arginine vasopressin (0.5 micrograms i.c.v.; 0.1 mg kg-1 i.p.) but not to naloxone (5 micrograms i.c.v.; 2 mg kg-1 i.p.) 3. Arginine vasopressin when administered by the intracerebroventricular route, but not by the intraperitoneal route, produced characteristic behaviour which was sensitive to deamino(CH2)5Tyr(Me) arginine vasopressin (0.5 micrograms, i.c.v.). 4. A 3 min swim at 20 degrees C produced antinociception on the hot-plate which was sensitive to naloxone (0.4 mg kg-1, i.p.) but not to deamino(CH2)5Tyr(Me) arginine vasopressin (0.5 micrograms, i.c.v.). 5. The reduction in the number of acetic acid-induced abdominal constrictions produced by a 30 s swim at 30 degrees C was not sensitive to either naloxone (2 mg kg-1, i.p.) or deamino(CH2)5Tyr(Me) arginine vasopressin (0.1 mg kg-1, i.p.). 6. Arginine vasopressin, at high doses, is antinociceptive in the mouse but does not appear to mediate stress-induced antinociception in this species.

Adrenoceptors in the human foetal small intestine.

Human foetal small intestine was shown to contain both alpha- and beta-adrenoceptors with a predominance of beta-adrenoceptors. The tissue examined was obtained from foetuses of gestational age between 8 and 26 weeks.

Leucine-enkephalin and methionine-enkephalin produce opposing effects on plasma corticosterone levels in ether-stressed mice.

The inhibitory effects of intracerebroventricular administration of saline on the plasma corticosterone response to ether stress in mice was reduced by Met-enkephalin and enhanced by Leu-enkephalin. When administered simultaneously the effects of the two peptides opposed each other. Met and Leu-enkephalin may subserve different physiological functions in the response of the hypothalamus-pituitary-adrenal system to stress.

Different effects of current strength on inhibitory responses of the mouse vas deferens to methionine- and leucine-enkephalin.

The inhibitory potency of methionine (Met)-enkephalin on the field-stimulated mouse vas deferens was greatly increased by a reduction in current strength whilst that of leucine (Leu)-enkephalin increased only slightly. All currents were submaximal and all muscle twitches were neuronally evoked. These results suggest that inhibitory effects of Met- and Leu-enkephalin in the mouse vas deferens are not commonly mediated and provide a rapid method for ascertaining heterogeneity of enkephalin extracts.

The effects of opiate receptor agonists and antagonists on the stress-induced secretion of corticosterone in mice.

1 Intraperitoneal administration of normorphine, morphine or naloxone or exposure to ether vapour for 1 min, elevated plasma corticosteroid concentrations in mice. 2 Injection of saline or exposure to ether vapour rendered mice less sensitive to a subsequent exposure to ether vapour 15 min later. 3 Treatment with normorphine (50 mg/kg) potentiated the corticosteroid response to ether stress whilst pentazocine (20 mg/kg), naltrexone (10 mg/kg), morphine (24 mg/kg), levorphanol (20 mg/kg) and naloxone (50 mg/kg) prevented the stress-induced elevation of plasma corticosteroids. 4 Both naloxone and morphine inhibited the potentiation by normorphine of the response to ether, the dose of naloxone required being higher than that for inhibition of normorphine analgesia. 5 It is concluded that endogenous opioid peptides may be involved in the control of the response to ether stress in mice.