Using an in vivo phagemid system to identify non‐compatible <i>loxP</i> sequences

The site‐specific recombination system of bacteriophage P1 is composed of the Cre recombinase that recognizes a 34‐bp loxP site. The Cre/loxP system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations. The creation of additional heterologous loxP sequences potentially expands the utility of this system, but only if these loxP sequences do not recombine with one another. We have developed a stringent in vivo assay to examine the degree of recombination between all combinations of each previously published heterologous loxP sequence. As expected, homologous loxP sequences efficiently underwent Cre‐mediated recombination. However, many of the heterologous loxP pairs were able to support recombination with rates varying from 5 to 100%. Some of these loxP sequences have previously been reported to be non‐compatible with one another. Our study also confirmed other heterologous loxP pairs that had previously been shown to be non‐compatible, as well as defined additional combinations that could be used in designing new recombination vectors

Most incompetent calf perforating veins are found in association with superficial venous reflux

AbstractPurpose: The indications for surgical perforator interruption remain undefined. Previous work has demonstrated an association between clinical status and the number of incompetent perforating veins (IPVs). Other studies have demonstrated that correction of IPV physiology results from abolition of saphenous system reflux. The purpose of this study was to identify which, if any, patterns of venous reflux and obstruction are particularly associated with IPV. Patients and Methods: Two hundred thirty patients and subjects (103 men, 127 women, 308 limbs) with varying grades of venous disease were examined both clinically and with duplex ultrasound scan. The odds ratios (ORs) for the presence of IPVs were calculated for different anatomical distributions of main-stem venous reflux and obstruction. The base group are those with no main-stem venous disease. Results: There were no significant associations between the proportions of limbs demonstrating IPVs and patient age or sex. The ORs for the presence of IPVs in association with other venous disease are as follows (age/sex adjusted): long saphenous vein reflux, OR = 1.86, range = 1.32-2.63; short saphenous vein reflux, OR = 1.36, range = 1.02-1.82; deep system venous reflux, OR = 1.61, range = 1.2-2.15; superficial system reflux, OR = 3.17, range = 1.87-5.4; and deep system obstruction, OR = 1.09, range = 0.51-2.33. The ORs for combinations of venous disorders were calculated. Combinations of disease produced higher odds for the presence of IPVs than those above, the highest being long saphenous vein, short saphenous vein, and deep reflux combined, OR = 6.85 (95% CI, 2.97-15.83; P =.0001). Conclusions: Although the presence of IPVs is associated with venous ulceration, the highest ORs for the presence of IPVs were found in patients with superficial disease alone or in combination with deep reflux. Many of these may be corrected by saphenous surgery alone. (J Vasc Surg 2001;34:774-8.

Effect of ramipril on renal function in patients with intermittent claudication

Simon D Hobbs1, Martin W Claridge1, Antonius BM Wilmink1, Donald J Adam1, Mark E Thomas2, Andrew W Bradbury11University Department of Vascular Surgery and 2Department of Nephrology, Heart of England NHS Trust (Teaching), Birmingham, UKBackground: The Heart Outcomes Prevention Study (HOPE) demonstrated that ramipril resulted in a blood-pressure-independent 25% reduction in cardiovascular events in patients with peripheral arterial disease (PAD). Despite this, general practitioners and vascular surgeons remain reluctant to prescribe ACE inhibitors in this group of patients because of concerns about renal artery stenosis (RAS). We aimed to define the effect of ramipril on renal function in patients with intermittent claudication (IC).Methods and Results: Of 132 unselected patients with IC entering the study 78 (59%) were excluded due to: current ACE inhibitor use (38%), renal impairment (serum creatinine above normal range) (15%), known severe RAS (1%) or unwillingness to participate (5%). The remaining 54 patients were titrated to 10 mg ramipril and renal function was monitored at 1, 5, and 12 weeks. Treatment was discontinued during titration in 5 patients due to symptoms (3) or lack of compliance (2). In the remainder, median [IQR] serum creatinine increased (94 [85.8&amp;ndash;103.3] to 98 [88.0&amp;ndash;106.5] &amp;micro;mol/L, p &amp;le; 0.001) and median [IQR] GFR decreased (71.5 [64.6&amp;ndash;82.3] to 68.7 [59.8&amp;ndash;74.7] mL/min per 1.73 m2, p &amp;le; 0.001) between baseline and 5 weeks. These changes were not considered clinically significant. By 12 weeks these values had returned almost to baseline (Cr 95.5 [88.0&amp;ndash;103.25] &amp;micro;mol/L, GFR 71.8 [65.3&amp;ndash;77.4] mL/min). No patient had a serum creatinine rise &amp;gt;30%.Conclusion: Most of patients with IC and a normal serum creatinine can be safely commenced on ramipril provided they are screened, titrated and monitored as described above. Studies in patients with borderline renal impairment (serum creatinine up to 30% above baseline) are on-going.Keywords: peripheral arterial disease, ramipril, renal function, intermittent claudicatio

Development of Phage-Based Single Chain Fv Antibody Reagents for Detection of Yersinia pestis

detection. by flow cytometry and whole-cell ELISA. strains, whereas phage displayed scFvs were found to be easy to purify/label and remarkably stable. Furthermore direct fluorescent labeling of phage displaying scFv allowed for an easy one-step flow cytometry assay. Slight cross-reactivity was observed when fixed cells were used in ELISA. F1 antigen. We describe implementation of different methods for phage-based immunoassay. Based on the success of these methods and the proven stability of phage, we indicate that the use of phage-displayed, rather than phage-free proteins, might generally overcome the shortcomings of scFv antibodies

The relationship between aortic wall distensibility and rupture of infrarenal abdominal aortic aneurysm

AbstractObjective: A more accurate means of prediction of abdominal aortic aneurysm (AAA) rupture would improve the clinical and cost effectiveness of prophylactic repair. The purpose of this study was to determine whether AAA wall distensibility can be used to predict time to rupture independently of other recognized risk factors. Methods: A prospective, six-center study of 210 patients with AAA in whom blood pressure (BP), maximum AAA diameter (Dmax), and AAA distensibility (pressure strain elastic modulus [Ep] and stiffness [β]) were measured at 6 months with an ultrasound scan-based echo-tracking technique. A stepwise, time-dependent, Cox proportional hazards model was used to determine the effect on time to rupture of age, gender, BP, Dmax, BP, Ep, β, and change in Dmax, Ep, and β adjusted for time between follow-up visits. Results: Median (interquartile range) AAA diameter was 48 mm (41 to 54 mm), median age was 72 years (68 to 77 years), and median follow-up period was 19 months (9 to 30 months). In the Cox model, female gender (hazards ratio [HR], 2.78; 95% CI, 1.23 to 6.28; P =.014), larger Dmax (HR, 1.36 for 10% increase in Dmax; 95% CI, 1.12 to 1.66; P =.002), higher diastolic BP (HR, 1.13 for 10% increase in BP; 95% CI, 1.13 to 1.92; P =.004), and a decrease in Ep (increase in distensibility) over time (HR, 1.38 for 10% decrease in Ep over 6 months; 95% CI, 1.08 to 1.78; P =.010) significantly reduced the time to rupture (had a shorter time to rupture). Conclusion: Women have a shorter time to AAA rupture. The measurement of AAA distensibility, diastolic BP, and diameter may provide a more accurate assessment of rupture risk than diameter alone. (J Vasc Surg 2003;37:112-7.

The relationship between aortic wall distensibility and rupture of infrarenal abdominal aortic aneurysm

AbstractObjective: A more accurate means of prediction of abdominal aortic aneurysm (AAA) rupture would improve the clinical and cost effectiveness of prophylactic repair. The purpose of this study was to determine whether AAA wall distensibility can be used to predict time to rupture independently of other recognized risk factors. Methods: A prospective, six-center study of 210 patients with AAA in whom blood pressure (BP), maximum AAA diameter (Dmax), and AAA distensibility (pressure strain elastic modulus [Ep] and stiffness [β]) were measured at 6 months with an ultrasound scan-based echo-tracking technique. A stepwise, time-dependent, Cox proportional hazards model was used to determine the effect on time to rupture of age, gender, BP, Dmax, BP, Ep, β, and change in Dmax, Ep, and β adjusted for time between follow-up visits. Results: Median (interquartile range) AAA diameter was 48 mm (41 to 54 mm), median age was 72 years (68 to 77 years), and median follow-up period was 19 months (9 to 30 months). In the Cox model, female gender (hazards ratio [HR], 2.78; 95% CI, 1.23 to 6.28; P =.014), larger Dmax (HR, 1.36 for 10% increase in Dmax; 95% CI, 1.12 to 1.66; P =.002), higher diastolic BP (HR, 1.13 for 10% increase in BP; 95% CI, 1.13 to 1.92; P =.004), and a decrease in Ep (increase in distensibility) over time (HR, 1.38 for 10% decrease in Ep over 6 months; 95% CI, 1.08 to 1.78; P =.010) significantly reduced the time to rupture (had a shorter time to rupture). Conclusion: Women have a shorter time to AAA rupture. The measurement of AAA distensibility, diastolic BP, and diameter may provide a more accurate assessment of rupture risk than diameter alone. (J Vasc Surg 2003;37:112-7.