Variability in the presentation of complicated jejunal diverticulosis.

Jejunal diverticulosis is a rare disease which normally presents for the first time with acute complications, often requiring surgical intervention. The diverticulae are acquired, occurring more commonly after middle age, but their aetiology is unclear. We discuss this condition in the context of four cases which presented to our hospital as emergencies over a five year period: small bowel obstruction, gastrointestinal haemorrhage, small bowel volvulus, and visceral perforation. Our aim is to encourage clinicians to include jejunal diverticular disease as a differential diagnosis in patients with abdominal symptoms.

Device-specific outcomes following endovascular aortic aneurysm repair.

OBJECTIVE: To compare aneurysm morphology, initial outcomes and mid-term results in patients receiving Talent or Zenith grafts for elective endovascular aneurysm repair (EVR). METHODS: Over a 6-year time period ending in 2007, 286 patients underwent elective EVR of infra-renal abdominal aortic aneurysms using Talent or Zenith devices. Patient demographics, aneurysm morphology and initial outcomes (primary-assisted technical success rates, 30-day limb occlusion, re-intervention and mortality) were compared using chi-squared tests or Student's t-tests. Kaplan-Meier curves were calculated to compare cumulative rates of freedom from type I or III endoleak, re-intervention, endograft patency and overall survival over mid-term follow-up. RESULTS: Adverse aneurysm morphology was more common in patients receiving Zenith stent grafts, with a greater proportion of shorter neck lengths (<10mm, 12.9% vs 0%; p60 degrees , 25.0% vs 10.3%; p=0.002). Equivalent primary-assisted technical success rates were achieved with both Talent and Zenith grafts (94.0% vs 96.1%; p=0.41). A significant number of adjunctive procedures were required in both groups to obtain a proximal endograft seal, with relatively more procedures performed in the Talent group (28.6% vs 12.4%; p=0.003). Early outcomes were similar for 30-day re-intervention (5.3% vs 3.9%; p=0.91), 30-day limb occlusion (1.5% vs 2.6%; p=0.51), 30-day morbidity (6.8% vs 11.8%; p=0.15) and 30-day mortality (4.5% vs 3.9%; p=0.80). The cumulative incidence of freedom from re-intervention was 88.3+/-2.9%, 86.1+/-3.3% and 84.1+/-3.9% at 1, 2 and 3 years respectively. There were no significant differences between Talent and Zenith groups for re-intervention, type I or III endoleak or limb occlusion rates over the same time period. Overall patient survival was 88.4+/-2.85% at 1 year, 83.7+/-4.0% at 2 years and 78.9+/-5.5% at 3 years. CONCLUSIONS: Equivalent primary-assisted technical success rates can be achieved using either Talent or Zenith endografts for endovascular aneurysm repair, but operating teams should be prepared to perform additional adjunctive procedures to obtain a primary proximal seal with either stent. The Zenith endograft performed well in the context of less favourable pre-operative aneurysm morphology. Both Talent and Zenith endografts appeared equally durable in the medium term.

Objective risk-scoring systems for repair of abdominal aortic aneurysms: applicability in endovascular repair?

OBJECTIVES: Recent studies propose the use of objective risk-scoring systems as a clinical tool for selecting patients for open or endovascular abdominal aortic aneurysm repair (EVR). The aim of this study was to evaluate four established risk-scoring systems for accuracy of prediction of early mortality and morbidity following EVR. PATIENTS AND METHODS: 266 consecutive patients undergoing elective EVR at St. George's Vascular Institute between July 2001 and January 2007 were studied using a prospective database. The Glasgow Aneurysm Score (GAS), the Vascular Physiology and Operative Severity Score for the enUmeration of Mortality and Morbidity (V-POSSUM), the modified Customised Probability Index (m-CPI) and the Customised Probability Index (CPI) were applied for prediction of 30-day mortality and morbidity. Accuracy of prediction was compared using receiver operating characteristics (ROC) curve analyses. RESULTS: 30-day mortality and morbidity rates were 4% (11/266) and 8% (22/266) respectively. For prediction of mortality, GAS, V-POSSUM, m-CPI and CPI ROC curve analyses showed areas under the curves (AUCs) of 0.68 (95% confidence interval (CI), 0.48-0.87; p=0.046), 0.66 (95% CI, 0.51-0.81; p=0.067), 0.63 (95% CI, 0.45-0.81; p=0.148) and 0.65 (95% CI, 0.49-0.80; p=0.101) respectively. Corresponding AUCs for prediction of morbidity were 0.64 (95% CI, 0.51-0.76; p=0.511), 0.62 (95% CI, 0.51-0.74; p=0.505), 0.54 (95% CI, 0.41-0.67; p=0.416) and 0.55 (95% CI, 0.42-0.68; p=0.451). CONCLUSIONS: GAS, V-POSSUM, m-CPI and CPI were poor predictors of early mortality and morbidity following EVR in this series. Caution should be applied to the use of these scoring systems for pre-operative risk stratification and treatment selection for endovascular repair of abdominal aneurysms.

Endovascular management of chronic type B dissecting aortic aneurysm utilizing aortic and renal stents.

Over the last 10 years endovascular stent-graft placement has been increasingly used to treat complicated acute Type B thoracic aortic dissections. While studies have demonstrated the use of additional aortic stent-grafts to treat continued false lumen perfusion and case reports have detailed the use of renal artery stents to treat renal ischemia related to aortic dissection, to our knowledge the adjuvant use of renal artery stents to reduce false lumen perfusion has not been reported. We present the case of a 72-year-old male who had previously undergone endovascular repair of a complicated Type B thoracic aortic dissection and presented with an expanding false lumen in the peridiaphragmatic aorta despite coverage of the entire thoracic aorta. This was treated by closure of a right renal fenestration using a renal stent.

Is it acceptable to approach colorectal cancer patients at diagnosis to discuss genetic testing? A pilot study.

In this pilot study, the acceptability of approaching 111 newly diagnosed colorectal cancer patients with the offer of genetic testing for hereditary nonpolyposis colorectal cancer (HNPCC) was assessed. A total of 78% of participants found it highly acceptable to have the information about HNPCC brought to their attention at that time.

Characterisation of novel point mutations in the survival motor neuron gene SMN, in three patients with SMA.

We report two novel mutations in three cases of spinal muscular atrophy (SMA), including two distant cousins who followed an unexpectedly severe course. Diagnosis was confirmed by reduced SMN protein and full-length SMN mRNA levels. Sequencing of the non-deleted SMN1 gene revealed a single G insertion at the end of exon 1 in the two cousins and a novel G275S exon 6 missense mutation in the milder case.

Overexpressed human survival motor neurone isoforms, SMNDeltaexon7 and SMN+exon7, both form intranuclear gems but differ in cytoplasmic distribution.

Homozygous mutations of the telomeric survival motor neurone gene (SMN1) cause spinal muscular atrophy (SMA). The centromeric copy gene (SMN2) generally skips exon 7 during splicing and fails to compensate for SMN1 deficits, so SMA cells have reduced SMN protein and few nuclear gems. To investigate the role of exon 7 in SMN localisation, cDNAs for full-length SMN and SMNDeltaexon 7 were overexpressed in COS cells, neurones and SMA fibroblasts. Both constructs formed discrete intranuclear bodies colocalising with p80-coilin, but produced more cytoplasmic aggregates in cells overexpressing exon 7. Hence, the exon 7 domain enhances SMN aggregation but is not critical for gem formation.

Nuclear gems and Cajal (coiled) bodies in fetal tissues: nucleolar distribution of the spinal muscular atrophy protein, SMN.

SMN, the affected protein in spinal muscular atrophy (SMA), is a cytoplasmic protein that also occurs in nuclear structures called "gems" and is involved in snRNP maturation. Coilin-p80 is a marker protein for nuclear Cajal bodies (coiled bodies; CBs) which are also involved in snRNP maturation, storage or transport. We now show that gems and CBs are present in all fetal tissues, even those that lack gems/CBs in the adult. Most gems and CBs occur as separate nuclear structures in fetal tissues, but their colocalization increases with fetal age and is almost complete in the adult. In adult tissues, up to half of all gems/CBs are inside the nucleolus, whereas in cultured cells they are almost exclusively nucleoplasmic. The nucleolar SMN is often more diffusely distributed, compared with nucleoplasmic gems. Up to 30% of cells in fetal tissues have SMN distributed throughout the nucleolus, instead of forming gems in the nucleoplasm. The results suggest a function for gems distinct from Cajal bodies in fetal nuclei and a nucleolar function for SMN. Spinal cord, the affected tissue in SMA, behaves differently in several respects. In both fetal and adult motor neurons, many gems/CBs occur as larger bodies closely associated with the nucleolar perimeter. Uniquely in motor neurons, gems/CBs are more numerous in adult than in fetal stages and colocalization of gems and CBs occurs earlier in development. These unusual features of motor neurons may relate to their special sensitivity to reduced SMN levels in SMA patients.

Intramuscular injection of a plasmid vector expressing human apolipoprotein E limits progression of xanthoma and aortic atheroma in apoE-deficient mice.

Apolipoprotein-E (apoE) protects against coronary artery disease via hepatic removal of atherogenic remnant lipoproteins, sequestration of cholesterol from vessel walls and local anti-oxidant, anti-platelet and anti-inflammatory actions. ApoE gene transfer may thus ameliorate a hyperlipidaemic profile and have beneficial effects at lesion sites to prevent or regress atherosclerosis, a concept endorsed by adenoviral-mediated hepatic expression studies. Here, using plasmid vectors expressing allelic human apoE2 or apoE3 isoforms, skeletal muscle was evaluated as an effective secretory platform for apoE gene augmentation. Transfected myoblasts and myotubes were found to efficiently secrete recombinant apoE in vitro as spherical 10-16 nm lipoprotein particles with pre-beta mobility. Intramuscular plasmid injection in apoE(-/-) mice, which develop spontaneous atherosclerotic plaque and xanthoma resulted in expression and secretion of apoE. Human apoE mRNA was detected by RT-PCR in injected muscles and, although concentrations of apoE3, which is rapidly cleared from plasma, were near ELISA detection limits, levels of plasma apoE2 were measurable (17.5 +/- 4.3 ng/ml). To assess whether muscle-based expression of apoE2 could inhibit atherogenesis, long-term follow-up studies were conducted. Although hyperlipidaemia was not reduced in treated animals, end-point pathology showed clear retardation of atherosclerotic and xanthomatous lesions. Up to 9 months following a single apoE2 plasmid administration, atherosclerotic lesion coverage in proximal aorta was significantly reduced by 20-30% (P < 0.01), whereas development of gross dorsal xanthoma (>5 mm diameter) was effectively reduced to zero. We conclude that expression of apoE from ectopic muscle sites has therapeutic potential to limit progression of atherosclerosis.

Lipofection of cultured mouse muscle cells: a direct comparison of Lipofectamine and DOSPER.

Cationic lipid-DNA complexes (lipoplexes) have been widely used as gene transfer vectors which avoid the adverse immunogenicity and potential for viraemia of viral vectors. With the long-term aim of gene transfer into skeletal muscle in vivo, we describe a direct in vitro comparison of two commercially available cationic lipid formulations, Lipofectamine and DOSPER. Optimisation of transfection was performed in the C2C12 mouse muscle cell line, before further studies in primary mouse myoblasts and C2C12 myotubes. Reporter gene constructs expressing either E. coli beta-galactosidase or green fluorescent protein (GFP) were used in order to evaluate transfection efficiency by histochemical staining or FACS analysis, respectively. Both lipid formulations were able to promote efficient, reproducible gene transfer in C2C12 cells, and to transfect primary mouse myoblast cultures successfully. However, DOSPER exhibited the important advantage of being able to transfect cells in the presence of serum of both bovine and murine origin. This feature allowed increased cell survival during in vitro transfections, and may be advantageous for direct in vivo gene transfer efficacy.

Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides.

Deletions and point mutations in the gene encoding the cytoskeletal protein dystrophin and its isoforms cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy (BMD), largely depending on whether the reading frame is lost or maintained respectively. Frameshift mutations tend to result in a lack of dystrophin at the sarcolemma, destabilization of the membrane and degeneration of skeletal muscle. The mdx mouse is a valuable animal model of DMD as it bears a nonsense point mutation in exon 23 of the murine DMD gene leading to an absence of dystrophin expression in the muscle sarcolemma and muscular dystrophy. This report represents a novel approach to correct dystrophin deficiency at the post-transcriptional level by transfection of muscle cells with antisense RNA. Essentially, 2'- O -methyl oligoribonucleotides (2'OMeRNA) were delivered to the nuclei of primary mdx myoblasts in culture. Dystrophin expression was observed in the sarcolemma of transfected mdx myotubes after transfection by an oligonucleotide complementary to the 3' splice site of murine dystrophin intron 22. Direct sequencing of RT-PCR products from these cells revealed precise splicing of exon 22 to exon 30, skipping the mutant exon and creating a novel in-frame dystrophin transcript. As patients with comparable in-frame internal deletions show relatively mild myopathic symptoms, this may in the future offer a therapeutic approach for DMD, as well as for other inherited disorders.

Efficient coexpression and secretion of anti-atherogenic human apolipoprotein AI and lecithin-cholesterol acyltransferase by cultured muscle cells using adeno-associated virus plasmid vectors.

Plasma apolipoprotein AI (apoAI) and lecithin-cholesterol acyltransferase (LCAT) play important roles in reverse cholesterol transport, promoting the removal of excess cholesterol from peripheral cells and reducing formation of atherosclerotic lesions. Gene augmentation of either apoAI or LCAT, or both, are thus attractive targets for prevention or treatment of atherosclerosis. With the eventual aim of safe and efficient gene delivery to skeletal muscle, our chosen secretory platform for systemic delivery of anti-atherogenic proteins, we have constructed conventional and AAV-based plasmid vectors containing human apoAI or LCAT cDNAs; their efficacy was tested by lipoplex transfection of mouse C2C12 muscle cells or human 293 cells. The secretion of apoAI or LCAT by transduced cultures was two- to five-fold higher using AAV-based plasmid vectors than conventional plasmid vectors. Additionally, cells transfected with a bicistronic AAV-based vector containing an internal ribosome entry site (IRES) efficiently expressed both apoAI and LCAT simultaneously. Furthermore, AAV-based vector sequences were retained by host cells, whereas those of conventional plasmid vectors were lost. These studies indicate that ectopic overexpression of apoAI and LCAT in muscle tissue using AAV-based plasmid vectors might provide a feasible anti-atherogenic strategy in vivo.

Becker muscular dystrophy presenting with complete heart block in the sixth decade.

Becker muscular dystrophy may be associated with myocardial abnormalities which are usually diagnosed after the onset of weakness. We present a patient who developed complete heart block 6 years before the onset of muscle weakness which occurred unusually late at the age of 62 years.

Independent localization of dystrophin N- and C-terminal regions to the sarcolemma of mdx mouse myofibres in vivo.

Dystrophin has been proposed to associate with the skeletal muscle membrane by way of a glycoprotein complex that interacts with its C-terminal domains. Transfection of mdx mouse myotubes in culture or myofibres in vivo with recombinant genes encoding human dystrophin deletion mutants shows, however, that not only the C terminus of dystrophin but also its N-terminal actin-binding domain can locate independently to the muscle sarcolemma. This observation suggests that lack of sarcolemma-associated dystrophin in Duchenne muscular dystrophy (DMD) muscle may result from enhanced degradation of truncated mutation products rather than their inability per se to associate with the sarcolemma.

A comparative evaluation of three transfection procedures as assessed by resistance to G418 conferred to HEPG2 cells.

Three commonly used transfection techniques (electroporation, calcium phosphate precipitation and scrape loading) and a novel procedure combining the latter two methods were evaluated and conditions optimised for successful transfection of human HepG2 cells with plasmid DNA incorporating a mouse MHC Class I gene and a selectable marker (neomycin transferase gene) conferring resistance to G418. While transfection with linear DNA by scrape-loading gave satisfactory results, transfer of cloned circular DNA by electroporation, calcium phosphate precipitation or a combined use of scrape-loading and calcium phosphate gave best results for HepG2 cells.

Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo.

At the cellular level, the primary pathology in Duchenne muscular dystrophy (DMD) is caused by deficiency of the sarcolemmal-associated protein, dystrophin, in the striated musculature. Here we describe the somatic transfer and long-term expression of a human dystrophin minigene corresponding to a mild Becker muscular dystrophy (BMD) phenotype in skeletal muscle tissues of the dystrophin-deficient mdx mouse by direct retroviral transduction. Following a single intramuscular injection of recombinant retrovirus, sarcolemmal expression of dystrophin was observed in an average of approximately 6% of myofibres in treated tibialis anterior muscles and was associated with activated reappearance of at least one component (43kD) of the dystrophin-glycoprotein membrane complex (DGC). Furthermore, expression of recombinant dystrophin was observed in muscle tissues up to 9 months after treatment and a significant enhancement of retrovirus-mediated myofibre transduction was obtained in mdx muscle undergoing experimentally-induced regeneration. The results clearly demonstrate that retroviral transduction of activated satellite cells in regenerating skeletal muscle is a feasible route for direct and stable dystrophin gene transfer into muscle tissues in vivo.