Lipitoids--novel cationic lipids for cellular delivery of plasmid DNA in vitro.

BACKGROUND: Although synthetic nonviral vectors hold promise for the delivery of plasmid DNA, their gene-transfer efficiencies are far from matching those of viruses. To systematically investigate the structure-activity relationship of cationic lipids, a small library of cationic lipid-peptoid conjugates (lipitoids) was synthesized. The compounds were evaluated for their ability to form complexes with plasmid DNA and to mediate DNA transfer in vitro. RESULTS: Lipid-peptoid conjugates were conveniently prepared in high yield using solid-phase synthesis. Several lipitoids condensed plasmid DNA into 100 nm spherical particles and protected the DNA and DNase digestion. A subset of lipitoids with a repeated (aminoethyl, neutral, neutral) sidechain trimer motif conjugated with dimyristoyl phosphatidyl-ethanolamine (DMPE) mediated DNA transfer with high efficiency. CONCLUSIONS: Automated solid-phase synthesis of cationic lipids allowed the rapid synthesis of a diverse set of transfection reagents. The most active compound DMPE-(Nae-Nmpe-Nmpe)3 (Nae, N-aminoethyl glycine; Nmpe, N-p-methoxyphenethyl-glycine) is more efficient than lipofectin or DMRIE-C (two commercial cationic lipid transfection reagents) and is active in the presence and absence of serum. The activity in the presence of serum suggests potential for applications in vivo.

A combinatorial approach to the discovery of efficient cationic peptoid reagents for gene delivery.

A family of N-substituted glycine oligomers (peptoids) of defined length and sequence are shown to condense plasmid DNA into small particles, protect it from nuclease degradation, and efficiently mediate the transfection of several cell lines. The oligomers were discovered by screening a combinatorial library of cationic peptoids that varied in length, density of charge, side-chain shape, and hydrophobicity. Transfection activity and peptoid-DNA complex formation are shown to be highly dependent on the peptoid structure. The most active peptoid is a 36-mer that contains 12 cationic aminoethyl side chains. This molecule can be synthesized efficiently from readily available building blocks. The peptoid condenses plasmid DNA into uniform particles 50-100 nm in diameter and mediates the transfection of a number of cell lines with efficiencies greater than or comparable to DMRIE-C, Lipofectin, and Lipofectamine. Unlike many cationic lipids, peptoids are capable of working in the presence of serum.

A statistical analysis of the growth of small abdominal aortic aneurysms.

Screening for abdominal aortic aneurysm has been offered to 13,000 patients. An overall compliance of 76% (range 51-99%) has been achieved. For those with an initial aortic diameter between 29 mm and 45 mm serial ultrasound scans are offered to monitor aortic change. Of the 302 cases followed up by repeated scans, 93 have had more than 5 scans (mean of 7 scans) over a mean time period of 32.1 months (range 15-63 months). The rate of change of all these aneurysms has been calculated using all data points and robust linear regression. It has been possible to postulate an algorithm for aortic growth. The influence of measurement precision and ultrasound quality assurance on the rescan interval is demonstrated and it is suggested that 2 years is a suitable rescan interval for aortas with diameter less than 40 mm.

Prevalence of abdominal aortic aneurysm associated with hypertension in an urban population.

SETTING AND METHOD: Since 1989 13,000 invitations to screening for abdominal aortic aneurysm have been offered to men aged 60 to 75 living within the Birmingham conurbation. RESULTS: The overall attendance was 76.1% (52-99%). The prevalence of aneurysms greater than 29 mm in the whole male population aged 60 to 75 was 7.2%. For those aged 60 to 64 the prevalence was 3.8%. For those aged between 60 and 64 with hypertension, however, the prevalence of abdominal aortic aneurysm was 7.7%. The relative risk of hypertension associated with aneurysm disease is 2.7. CONCLUSIONS: This paper suggests that all men over 65 should be screened for aortic aneurysm, but that those men over 60 with hypertension should be added to the screened cohort.

Radiation-induced point mutations, deletions and micronuclei in lacI transgenic mice.

Ionizing radiation induces gene mutations (point mutations, deletions and insertions) as well as chromosome damage in mammalian cells. Although these effects have been studied extensively in cells in culture, until recently it has not been possible to analyze the mutagenic potential of ionizing radiation in vivo, especially at the molecular level. The development of transgenic mutagenesis systems has now made it possible to study the effects of ionizing radiation at both the molecular and chromosomal levels in the same animal. In this report we present preliminary data on the response of Big Blue lacI transgenic mice to ionizing radiation as measured by lacI mutations and micronuclei. C57Bl/6 transgenic mice were irradiated with 137Cs gamma-rays at doses ranging from 0.1 to 14 Gy, and expression times ranging from 2 to 14 days. Dose-related increases in the mutant frequency were observed after irradiations with longer expression times. Mutant plaques were analyzed by restriction enzyme digestion to detect large structural changes in the target sequence. Of 34 gamma-ray-induced mutations analyzed, 4 were large-scale rearrangements. 3 of these rearrangements were deletions within the lacI gene characterized by the presence of short regions of homology at the breakpoint junctions. The fourth rearrangement was a deletion that extended from within the alpha lacZ gene into downstream sequences and that had 43 bp of homology at the junction. These data indicate that the Big Blue lacI transgenic mouse system in sensitive to the types of mutations induced by ionizing radiation. To determine whether the presence of the transgene affects micronucleus induction we compared the response of nontransgenic to hemizygous transgenic B6C3F1 mice and the response of nontransgenic to hemizygous and homozygous transgenic C57Bl/6 mice. The presence or absence of the lacI transgene had no effect on spontaneous micronucleus frequencies for either strain. However, radiation-induced micronucleus frequencies were significantly higher in hemizygous lacI B6C3F1 mice than in nontransgenic litter mates; the converse was true in C57Bl/6 mice. These data suggest that the lacI transgene does not cause chromosome instability as measured by spontaneous micronucleus levels. However, the response of these transgenic mice to a variety of clastogenic agents needs to be investigated before they are integrated into standard in vivo assays for chromosome damage.

Ultrasonographic screening for abdominal aortic aneurysm in an urban community.

As part of the Birmingham Community Aneurysm Screening Project, 3500 men aged 65-75 years from 20 urban general practices were invited for aortic ultrasonographic screening at their own general practitioner's surgery; 2669 (76.3 per cent) attended. Compliance rates varied between catchment areas, from 52.1 per cent for inner-city areas to 89.6 per cent for suburbs. Successful aortic imaging was achieved in 97.3 per cent of scans. Aortic diameter > 29 mm occurred in 219 patients (8.4 per cent) and 79 (3.0 per cent) with a diameter > 40 mm were referred for vascular surgical assessment; 140 patients with an aortic diameter of 29-40 mm are currently undergoing follow-up by serial ultrasonographic examinations at intervals of 3 months at their doctor's surgery. Risk factor analysis revealed ischaemic heart disease in 21.9 per cent of men with aneurysm, compared with 11.6 per cent in those without (P < 0.001); 18.3 per cent of men with aneurysm had had a previous myocardial infarction and 13.2 per cent had peripheral vascular disease, compared with 7.4 per cent (P < 0.001) and 8.0 per cent (P < 0.01) respectively of those without. No association was found between aneurysm and hypertension or diabetes. Community-based aortic screening is an inexpensive, effective method of diagnosis of aneurysm, with high compliance from the at-risk cohort of an urban population. Such screening programmes may help to reduce the mortality rate from aortic aneurysm rupture.

Two vascular complications of the Ehlers-Danlos syndrome.

Patients with Ehlers-Danlos syndrome type IV have thin walled, friable arteries and veins and are prone to spontaneous arterial rupture. We present a patient who suffered severe damage to the common femoral vein during surgery for varicose veins and who subsequently suffered a spontaneous rupture of the right external carotid artery, treated by ligation and oversewing. Intraarterial radiology and surgery in such patients are hazardous and should be avoided if possible. Where operation is essential, the simplest available procedure should be performed. Patients should be counselled to inform every doctor they consult that they have the Ehlers-Danlos syndrome.

Reperfusion plasma contains a neutrophil activator.

Aortic aneurysm repair produces inflammatory mediators, neutrophil activation, and remote organ injury. Reperfusion plasma from these patients produces microvascular injury in an ex vivo chemotactic model. This study investigates the mechanism of this injury. Vena caval blood was obtained before and 15 minutes after aortic clamp removal (n = 16) or at laparotomy (n = 10). Plasma or saline solution was introduced into unit dose chambers fixed atop dermabrasions on the back of depilated anesthetized rabbits. Animals were treated with intravenous saline solution (n = 4); made neutropenic with nitrogen mustard (n = 4); pretreated with the xanthine oxidase inhibitor allopurinol (n = 4); or cotreated intravenously with the free radical scavengers superoxide dismutase (SOD) and catalase (n = 4). Three hours later neutrophil counts (polymorphonuclear cells [PMN]/mm3) and activity (free radical production by flow cytometry), protein leakage, and inflammatory mediators (thromboxane [TX] and leukotriene B4 [LTB4]) were measured. In contrast to control plasma in untreated rabbits, reperfusion plasma produced TX and LTB4 generation (1090 +/- 105 and 794 +/- 91 pg/ml, respectively, p < 0.01), PMN accumulation (1636 +/- 210/mm3, p < 0.01) and activation (276 +/- 31 mean fluorescent units), and microvascular permeability (554 +/- 90 micrograms/ml, p < 0.01). Neutropenia (3 +/- 1 PMN/mm3) and cotreatment with SOD and catalase abolished these responses, whereas pretreatment with allopurinol did not. Human reperfusion plasma contains a soluble factor that stimulates free radical generation by rabbit neutrophils to produce a microvascular injury characterized by de novo TX production, neutrophil accumulation and activation, and increased microvascular permeability to protein.

Abdominal aortic aneurysms complicated by spontaneous iliocaval or duodenal fistulae.

Seven hundred and twenty one abdominal aortic aneurysms were treated between 1960 and 1985. Twenty one of these (2.9%) were complicated by the development of a spontaneous primary fistula, 16 (2.2%) into the vena cava or iliac veins and 5 (0.7%) into the duodenum. A correct preoperative diagnosis was made in only four instances, two aorto-caval and two aorto-duodenal fistulae. Hospital mortality was 44% for aorto-caval and 60% for aorto-duodenal fistulae. Despite the lack of a precise preoperative diagnosis in the majority of cases, the prognosis for aorto-caval fistula remained comparable to that for patients undergoing emergency surgery for uncomplicated ruptured aortic aneurysms. The mortality of spontaneous aorto-duodenal fistulae was appreciably higher and the aneurysmal contents of 4 out of these 5 cases had positive bacterial cultures.

Anomalous origin of the occipital artery from the cervical internal carotid artery.

A case is reported of the abnormal origin of the occipital artery from the right internal carotid artery in a patient admitted for carotid endarterectomy. The abnormality was demonstrated angiographically and at operation.

Development of a sarcoma in a surgical scar.

The development of a malignant soft tissue tumour (malignant fibrous histiocytoma) in a surgical wound is reported. Although rare, this occurrence has important diagnostic implications and the management is discussed.

Crohn's disease in two married couples.

Only two examples of married couples where both partners have developed Crohn's disease have been reported previously from the United Kingdom. We describe two further examples; one where the index patient had developed Crohn's disease before marriage, and the spouse subsequently developed Crohn's disease and a second example in which both spouses developed Crohn's disease after marriage.

Relationship between adrenocorticotrophin bioactivity in blood and cerebrospinal fluid of rhesus monkeys.

Adrenocorticotrophin levels, measured by a cytochemical bioassay, were determined in the plasma and cerebrospinal fluid (CSF) of adult female rhesus monkeys which were ovariectomized and receiving oestrogen replacement therapy. In control monkeys, ACTH bioactivity was found in both CSF (10.2 +/- 1.8 ng/l) and plasma (186 +/- 51 ng/l) in samples taken at 14.00 h (lights on: 07.00-19.00 h). Dexamethasone treatment (0.2 mg/kg) twice daily for 4 days suppressed plasma ACTH levels (52.8 +/- 25.2 ng/l) but had no effect on CSF levels (7.6 +/- 2.7 ng/l). Raising plasma ACTH, either by daily injections of a long-acting preparation of ACTH (1-24) for 6 days or by bilateral adrenalectomy (and subsequently with-drawing cortisol replacement therapy) also resulted in no detectable changes in ACTH levels in the CSF. A regression analysis between ACTH in the plasma and CSF from samples taken throughout the experiments showed no correlation. In contrast, measurement of ACTH by radioimmunoassay, whilst satisfactory for determination of this peptide in plasma, could not identify authentic ACTH in the CSF. It is concluded that bioactive ACTH does not enter the CSF in detectable quantities from either the peripheral vascular compartment or from the animal's own pituitary gland, and that reducing ACTH secretion from the pituitary also has no effect on levels of ACTH in the CSF. This is in marked contrast to other pituitary peptide hormones, including prolactin, which is secreted together with ACTH during 'stress' but which, unlike ACTH, enters the CSF relatively easily.

Experimental deep venous thrombogenesis by a non-invasive method.

Experimental deep venous thrombi were produced in the untraumatised leg veins of anaesthetised dogs. In place of trauma, the endothelium lining the valve pockets in femoral veins was damaged by hypoxaemia, and the pockets were then intermittently re-perfused with oxygenated blood. Histologically, the experimental thrombi resembled human valve pocket thrombi found at postmortem.

Carotid artery occlusion following endarterectomy: evaluation by spectral analysis of Doppler ultrasound signals.

The clinical results of carotid endarterectomy are well documented, but there is little objective information on the incidence of postoperative occlusion. We have studied the postoperative patency of the internal and external carotid arteries using continuous wave Doppler ultrasound with spectral analysis (Spectrascribe BOC--Medishield). Seventy-nine patients who had had 89 endarterectomies during the past 5 years entered the study. The mean follow-up was 21 months. Fourteen patients had an occluded internal carotid artery of whom 4 had suffered an immediate postoperative stroke and 1 a delayed stroke: transient ischaemic attacks occurred in another 4 patients. Of the 60 patients with a patent internal carotid artery one suffered an immediate postoperative stroke and 2 a delayed stroke; 6 patients had recurrent transient ischaemic attacks. None of the 5 patients who had an external carotid endarterectomy developed postoperative symptoms, although there was occlusion in 2. The carotid artery was occluded in 4 of 43 completely asymptomatic patients. Thus, 18 per cent of all carotid endarterectomies and 9 per cent of asymptomatic patients in this series had postoperative occlusion of the carotid artery.

The PO2 in venous valve pockets: its possible bearing on thrombogenesis.

The PO2 in the lumen and valve pockets of veins in 2 patients and 8 dogs was measured during streamline blood flow and in conditions of intermittent pulsatile flow. The blood within the valve pockets became rapidly hypoxic when undisturbed during streamline flow (i.e. when "static"), but the PO2 in the pockets rose to that of the lumenal blood when the column of venous blood was made to pulsate and so empty the valve pockets at short intervals. The observations suggest that the endothelium covering the valve cusps is entirely dependent on pocket or lumenal blood for its oxygen supply. The endothelium facing the pocket can therefore become hypoxic during non-pulsatile blood flow when that facing the lumen is adequately to oxygenated. Early thrombus formation was seen to develop on a valve cusp after only 2 h non-pulsatile flow. The demonstration that localized hypoxaemia occurs readily and can produce endothelial damage, in circumstances and situations where thrombi are commonly found to originate, is additional circumstantial evidence that hypoxia may trigger thrombogenesis.