Search

Serological assays have been widely employed during the coronavirus disease 2019 (COVID-19) pandemic to measure antibody responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and to track seroconversion in populations. However, currently available assays do not allow determination of neutralization capacity within the assay protocol. Furthermore, commercial serology assays have a high buy-in cost that is inaccessible for many research groups. We have replicated the serological enzyme-linked immunosorbent assay for the detection of SARS-CoV-2 antibody isotypes, developed at the Icahn School of Medicine at Mount Sinai, New York. Additionally, we have modified the protocol to include a neutralization assay with only a minor modification to this protocol. We used this assay to screen local COVID-19 patient sera ( n = 91) and pre-COVID-19 control sera ( n = 103), and obtained approximate parity with approved commercial anti-nucleoprotein-based assays with these sera. Furthermore, data from our neutralization assay closely aligns with that generated using a spike-based pseudovirus infection model when a subset of patient sera was analyzed.

If thermoplasmonic applications such as heat-assisted magnetic recording are to be commercially viable, it is necessary to optimize both thermal stability and plasmonic performance of the devices involved. In this work, a variety of different adhesion layers were investigated for their ability to reduce dewetting of sputtered 50 nm Au films on SiO 2 substrates. Traditional adhesion layer metals Ti and Cr were compared with alternative materials of Al, Ta, and W. Film dewetting was shown to increase when the adhesion material diffuses through the Au layer. An adhesion layer thickness of 0.5 nm resulted in superior thermomechanical stability for all adhesion metals, with an enhancement factor of up to 200× over 5 nm thick analogues. The metals were ranked by their effectiveness in inhibiting dewetting, starting with the most effective, in the order Ta > Ti > W > Cr > Al. Finally, the Au surface-plasmon polariton response was compared for each adhesion layer, and it was found that 0.5 nm adhesion layers produced the best response, with W being the optimal adhesion layer material for plasmonic performance.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans., Competing Interests: TV, SS, JM, ZN, DS, CS, MG, CS, AB, AE, HS, GW, AM, KF, SR, MD, YS No competing interests declared, JL was an employee at Sygnature Discovery Limited at the time of the study, performing surface plasmon resonance experiments as part of a paid service, SP is an employee at Sygnature Discovery Limited, performing surface plasmon resonance experiments as part of a paid service, RL is CSO and shareholder of Aplagon Oy, Helsinki, Finland, AJ receives research funding from Aplagon Oy, Helsinki, Finland, DO, TH, HM, JW, CG is an employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service, RR is employee at Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service, WA is CEO of Peak proteins Limited, performing crystallography and protein expression studies as part of a paid service, (© 2019, Vögtle et al.)

The use of a metallic adhesion layer is known to increase the thermo-mechanical stability of Au thin films against solid-state dewetting, but in turn results in damping of the plasmonic response, reducing their utility in applications such as heat-assisted magnetic recording (HAMR). In this work, 50 nm Au films with Ti adhesion layers ranging in thickness from 0 to 5 nm were fabricated, and their thermal stability, electrical resistivity, and plasmonic response were measured. Subnanometer adhesion layers are demonstrated to significantly increase the stability of the thin films against dewetting at elevated temperatures (>200 °C), compared to more commonly used adhesion layer thicknesses that are in the range of 2-5 nm. For adhesion layers thicker than 1 nm, the diffusion of excess Ti through Au grain boundaries and subsequent oxidation was determined to result in degradation of the film. This mechanism was confirmed using transmission electron microscopy and X-ray photoelectron spectroscopy on annealed 0.5 and 5 nm adhesion layer samples. The superiority of subnanometer adhesion layers was further demonstrated through measurements of the surface-plasmon polariton resonance; those with thinner adhesion layers possessed both a stronger and spectrally sharper resonance. These results have relevance beyond HAMR to all Ti/Au systems operating at elevated temperatures.

Purified recombinant proteins are key reagents in academic and industrial research. The ability to make these proteins quickly often relies on the availability of higher eukaryotic cell hosts such as insect and mammalian cells where there is a very wide range of post-translational modifications, protein folding and trafficking pathways. This enables the generation of many proteins that cannot be made in microbial hosts. In this article we outline some of the most commonly used methods to express recombinant proteins in insect and mammalian cells., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Certain recombinant proteins are deemed "difficult to express" in mammalian expression systems requiring significant cell and/or process engineering to abrogate expression bottlenecks. With increasing demand for the production of recombinant proteins in mammalian cells, low protein yields can have significant consequences for industrial processes. To investigate the molecular mechanisms that restrict expression of recombinant proteins, naturally secreted model proteins were analyzed from the tissue inhibitors of metalloproteinase (TIMP) protein family. In particular, TIMP-2 and TIMP-3 were subjected to detailed study. TIMP proteins share significant sequence homology (∼50% identity and ∼70% similarity in amino acid sequence). However, they show marked differences in secretion in mammalian expression systems despite this extensive sequence homology. Using these two proteins as models, this study characterized the molecular mechanisms responsible for poor recombinant protein production. Our results reveal that both TIMP-2 and TIMP-3 are detectable at mRNA and protein level within the cell but only TIMP-2 is secreted effectively into the extracellular medium. Analysis of protein localization and the nature of intracellular protein suggest TIMP-3 is severely limited in its post-translational processing. To overcome this challenge, modification of the TIMP-3 sequence to include a furin protease-cleavable pro-sequence resulted in secretion of the modified TIMP-3 protein, however, incomplete processing was observed. Based on the TIMP-3 data, the protein engineering approach was optimized and successfully applied in combination with cell engineering, the overexpression of furin, to another member of the TIMP protein family (the poorly expressed TIMP-4). Use of the described protein engineering strategy resulted in successful secretion of poorly (TIMP-4) and non-secreted (TIMP-3) targets, and presents a novel strategy to enhance the production of "difficult" recombinant targets. Biotechnol. Bioeng. 2017;114: 2348-2359. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the crystal structure of inhibitor-bound hFEN1, which shows a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein-substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the necessary unpairing of substrate DNA. Other compounds were more competitive with substrate. Cellular thermal shift data showed that both inhibitor types engaged with hFEN1 in cells, and activation of the DNA damage response was evident upon treatment with inhibitors. However, cellular EC50 values were significantly higher than in vitro inhibition constants, and the implications of this for exploitation of hFEN1 as a drug target are discussed.

Transient expression of heterologous proteins in mammalian systems is a powerful way to generate protein reagents quickly. However, it has historically suffered from poor yields in comparison to methods where the recombinant gene is stably integrated into the genome and high expressing clones isolated. Transient methods have been well described for HEK-based systems. In this paper we show the use of a design of experiments (DoE) approach to quickly analyse the effect of a range of different parameters on protein expression from a CHO-based transient system. We show that this system is amenable to a very simple transfection procedure by independent direct addition of DNA and transfection reagent to the culture vessel. In addition we show that expression can be improved by reducing the temperature of the culture conditions post-transfection. The process is demonstrated to be transferrable from 3 ml cultures in deep 24-well plates through cultures in CultiFlask Bioreactors, shake flasks and up to 25 L culture in Wave Bioreactors. Data are shown to illustrate the utility of the system with a number of different classes of protein., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A number of different methods are commonly used to map the fine details of the interaction between an antigen and an antibody. Undoubtedly the method that is now most commonly used to give details at the level of individual amino acids and atoms is X-ray crystallography. The feasibility of undertaking crystallographic studies has increased over recent years through the introduction of automation, miniaturization and high throughput processes. However, this still requires a high level of sophistication and expense and cannot be used when the antigen is not amenable to crystallization. Nuclear magnetic resonance spectroscopy offers a similar level of detail to crystallography but the technical hurdles are even higher such that it is rarely used in this context. Mutagenesis of either antigen or antibody offers the potential to give information at the amino acid level but suffers from the uncertainty of not knowing whether an effect is direct or indirect due to an effect on the folding of a protein. Other methods such as hydrogen deuterium exchange coupled to mass spectrometry and the use of short peptides coupled with ELISA-based approaches tend to give mapping information over a peptide region rather than at the level of individual amino acids. It is quite common to use more than one method because of the limitations and even with a crystal structure it can be useful to use mutagenesis to tease apart the contribution of individual amino acids to binding affinity., (© 2014 John Wiley & Sons Ltd.)

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays.

The activity of kinases is regulated by phosphorylation on Ser, Thr or Tyr residues within the activation loop. The ability to produce these enzymes recombinantly with a specific phosphorylation status is essential in order to understand structure and function. In this paper we describe a screening approach to co-express different phosphatases together with a kinase in the baculovirus expression system. This enabled the testing of different phosphatases as well as different levels of both phosphatase and kinase by varying the multiplicity of infection (MOI) of the different baculoviruses. This approach translated well to a larger scale. An unexpected observation was that co-expression of the phosphatase could have profound effects on expression levels even of heterologous target proteins that would not be a substrate for the phosphatase. This was most apparent with lambda phosphatase, an enzyme that removes phosphorylation from Ser and Thr residues, where expression was almost completely abolished for all proteins, even at modest MOIs. The effect of lambda phosphatase was observed irrespective of whether co-expression was from two separate baculoviruses or from two genes on the same vector. The effect was shown to be due, in part at least, to a decrease in transcription., (Copyright © 2012 Elsevier Inc. All rights reserved.)

IL-17A is a pro-inflammatory cytokine produced by the newly identified Th17 subset of T-cells. We have isolated a human monoclonal antibody to IL-17A (CAT-2200) that can potently neutralize the effects of recombinant and native human IL-17A. We determined the crystal structure of IL-17A in complex with the CAT-2200 Fab at 2.6 A resolution in order to provide a definitive characterization of the epitope and paratope regions. Approximately a third of the IL-17A dimer is disordered in this crystal structure. The disorder occurs in both independent copies of the complex in the asymmetric unit and does not appear to be influenced by crystal packing. The complex contains one IL-17A dimer sandwiched between two CAT-2200 Fab fragments. The IL-17A is a disulfide-linked homodimer that is similar in structure to IL-17F, adopting a cystine-knot fold. The structure is not inconsistent with the previous prediction of a receptor binding cavity on IL-17 family members. The epitope recognized by CAT-2200 is shown to involve 12 amino acid residues from the quaternary structure of IL-17A, with each Fab contacting both monomers in the dimer. All complementarity-determining regions (CDRs) in the Fab contribute to a total of 16 amino acid residues in the antibody paratope. In vitro affinity optimization was used to generate CAT-2200 from a parental lead antibody using random mutagenesis of CDR3 loops. This resulted in seven amino acid changes (three in VL-CDR3 and four in VH-CDR3) and gave an approximate 30-fold increase in potency in a cell-based neutralization assay. Two of the seven amino acids form part of the CAT-2200 paratope. The observed interaction site between CAT-2200 and IL-17A is consistent with data from hydrogen/deuterium exchange mass spectrometry and mutagenesis approaches.

The ADAMTS (a disintegrin-like and metalloproteinase domain with thrombospondin type I motifs) family of proteases plays a role in pathological conditions including arthritis, cancer, thrombotic thrombocytopenic purpura and the Ehlers-Danlos type VIIC and Weill-Marchesani genetic syndromes. Here, we report the first crystal structures for a member of the ADAMTS family, ADAMTS-1. Originally cloned as an inflammation-associated gene, ADAMTS-1 has been shown to be involved in tissue remodelling, wound healing and angiogenesis. The crystal structures contain catalytic and disintegrin-like domains, both in the inhibitor-free form and in complex with the inhibitor marimastat. The overall fold of the catalytic domain is similar to related zinc metalloproteinases such as matrix metalloproteinases and ADAMs (a disintegrin and metalloproteinases). The active site contains the expected organisation of residues to coordinate zinc but has a much larger S1' selectivity pocket than ADAM33. The structure also unexpectedly reveals a double calcium-binding site. Also surprisingly, the previously named disintegrin-like domain showed no structural homology to the disintegrin domains of other metalloproteinases such as ADAM10 but is instead very similar in structure to the cysteine-rich domains of other metalloproteinases. Thus, this study suggests that the D (for disintegrin-like) in the nomenclature of ADAMTS enzymes is likely to be a misnomer. The ADAMTS-1 cysteine-rich domain stacks against the active site, suggesting a possible regulatory role.

Background: In response to a Congressional mandate to compare risk-adjusted surgical outcomes from Department of Veterans Affairs (VA) hospitals with those from private-sector hospitals, the National Surgical Quality Improvement Program was initiated in the VA system and then was developed in a select group of university medical centers in the private sector. This article analyzes risk-adjusted outcomes after vascular surgical operations in men performed at VA hospitals as compared with private-sector hospitals., Study Design: This is a prospective cohort study of a sample of vascular surgical operations in men performed at 128 VA medical centers as compared with 14 university medical centers from October 1, 2001 to September 30, 2004. Patient and operative characteristics, and both unadjusted and risk-adjusted 30-day postoperative morbidity and mortality outcomes were compared., Results: Data from 30,058 vascular operations in men at VA hospitals were compared with 5,174 cases performed at private-sector hospitals. The unadjusted 30-day mortality rate was notably lower in the VA system as compared with the private-sector group (3.4% versus 4.2%, p = 0.004). After risk-adjustment, there was no marked difference in mortality between the two hospital types. The unadjusted 30-day morbidity rate was also considerably lower in the VA hospitals as compared with the private sector (17.3% versus 22.3%, p < 0.0001). After risk-adjustment, morbidity in the VA system remained considerably lower than in the private sector, with an odds ratio of 0.84 (95% CI, 0.78 to 0.92)., Conclusions: In vascular surgical operations in men, the VA hospitals demonstrated a lower risk-adjusted 30-day morbidity rate than the private-sector group. There is no marked difference in adjusted mortality rates between the two types of institutions.

Background: Despite advances by surgeons in assessing quality and safety, the traditional surgical morbidity and mortality (M&M) conference has mostly remained unchallenged and unchanged. The goal of this study was to compare data as reported in a traditional M&M conference to data collected using the American College of Surgeons-National Surgical Quality Improvement Program (ACS-NSQIP) techniques., Study Design: A retrospective study was performed comparing data from the M&M conference in a general surgery division, in which complications and deaths were identified by residents or attendings, to data compiled by a nationally audited nurse reviewer from the ACS-NSQIP from July 1, 2002, to June 30, 2003., Results: Mortality rates calculated by traditional M&M conference (53 deaths in 5,905 patients), compared with the ACS-NSQIP nurse reviewer (28 deaths in 1,439 patients; 24% sample), were 0.9% versus 1.9%, respectively (p=0.001). Complication rates reported in M&M were 6.4% versus 28.9% ACS-NSQIP (p<0.0001). Subgroup analyses showed that mortality rates, as reported in conference, were substantially lower for both in-hospital and postdischarge patients, when compared with ACS-NSQIP. All subclassifications of complications, as presented in conference, were also lower, compared with ACS-NSQIP., Conclusions: Traditional surgical M&M reporting considerably underreports both in-hospital and postdischarge complications and deaths as compared with ACS-NSQIP. Approximately one of two deaths and three of four complications were not reported in the M&M conference at our institution. A Web-based reporting system based on an ACS-NSQIP platform was created to automate, facilitate, and standardize data on surgical morbidity and mortality.

Objective: To assess the impact of the 80-hour resident workweek restrictions on surgical residents and attending surgeons., Summary Background Data: The ACGME mandated resident duty hour restrictions have required a major workforce restructuring. The impact of these changes needs to be critically evaluated for both the resident and attending surgeons, specifically with regards to the impact on motivation, job satisfaction, the quality of surgeon training, the quality of the surgeon's life, and the quality of patient care., Methods: Four prospective studies were performed at a single academic surgical program with data collected both before the necessary workforce restructuring and 1 year after, including: 1) time cards to assess changes in components of daily activity; 2) Web-based surveys using validated instruments to assess burnout and motivation to work; 3) structured, taped, one-on-one interviews with an external PhD investigator; and 4) statistical analyses of objective, quantitative data., Results: After the work-hour changes, surgical residents have decreased "burnout" scores, with significantly less "emotional exhaustion" (Maslach Burnout Inventory: 29.1 "high" vs. 23.1 "medium," P = 0.02). Residents have better quality of life both in and out of the hospital. They felt they got more sleep, have a lighter workload, and have increased motivation to work (Herzberg Motivation Dimensions). We found no measurable, statistically significant difference in the quality of patient care (NSQIP data). Resident training and education objectively were not statistically diminished (ACGME case logs, ABSITE scores). Attending surgeons perceived that their quality of their life inside and outside of the hospital was "somewhat worse" because of the work-hour changes, as they had anticipated. Many concerns were identified with regards to the professional development of future surgeons, including a change toward a shift-worker mentality that is not patient-focused, less continuity of care with a loss of critical information with each handoff, and a decrease in the patient/doctor relationship., Conclusion: Although the mandated restriction of resident duty hours has had no measurable impact on the quality of patient care and has led to improvements for the current quality of life of residents, there are many concerns with regards to the training of professional, responsible surgeons for the future.

Objective: To compare laparoscopic versus open gastric bypass procedures with respect to 30-day morbidity and mortality rates, using multi-institutional, prospective, risk-adjusted data., Summary Background Data: Laparoscopic Roux-en-Y gastric bypass for weight loss is being performed with increasing frequency, partly driven by consumer demand. However, there are no multi-institutional, risk-adjusted, prospective studies comparing laparoscopic and open gastric bypass outcomes., Methods: A multi-institutional, prospective, risk-adjusted cohort study of patients undergoing laparoscopic and open gastric bypass procedures was performed from hospitals (n = 15) involved in the Private Sector Study of the National Surgical Quality Improvement Program (NSQIP). Data points have been extensively validated, are based on standardized definitions, and were collected by nurse reviewers who are audited for accuracy., Results: From 2000 to 2003, data from 1356 gastric bypass procedures was collected. The 30-day mortality rate was zero in the laparoscopic group (n = 401), and 0.6% in the open group (n = 955) (P = not significant). The 30-day complication rate was significantly lower in the laparoscopic group as compared with the open group: 7% versus 14.5% (P < 0.0001). Multivariate logistic regression analysis was performed to control for potential confounding variables and showed that patients undergoing an open procedure were more likely to develop a complication, as compared with patients undergoing an laparoscopic procedure (odds ratio = 2.08; 95% confidence interval, 1.33-3.25). Propensity score modeling revealed similar results. A prediction model was derived, and variables that significantly predict higher complication rates after gastric bypass included an open procedure, a high ASA class (III, IV, V), functionally dependent patient, and hypertension as a comorbid illness., Conclusions: Multicenter, prospective, risk-adjusted data show that laparoscopic gastric bypass is safer than open gastric bypass, with respect to 30-day complication rate.

Background: Carotid endarterectomy (CEA) is the gold standard for the treatment of carotid stenosis, but carotid angioplasty and stenting has been advocated in high-risk patients. The definition of such a population has been elusive, particularly because the data are largely retrospective. Our study examined results for CEA in the National Surgical Quality Improvement Program database (both Veterans Affairs and private sector)., Methods: National Surgical Quality Improvement Program data were gathered prospectively for all patients undergoing primary isolated CEA during the interval 2000 to 2003 at 123 Veterans Affairs and 14 private sector academic medical centers. Study end points included the 30-day occurrence of any stroke, death, or cardiac event. A variety of clinical, demographic, and operative variables were assessed with multivariate models to identify risk factors associated with the composite (stroke, death, or cardiac event) end point. Adjudication of end points was by trained nurse reviewers (previously validated)., Results: A total of 13,622 CEAs were performed during the study period; 95% were on male patients, and 91% of cases were conducted within the Veterans Affairs sector. The average age was 68.6 +/- 0.1 years, and 42.1% of the population had no prior neurologic event. The composite stroke, death, or cardiac event rate was 4.0%; the stroke/death rate was 3.4%. Multivariate correlates of the composite outcome were (odds ratio, P value) as follows: deciles of age (1.13, .018), insulin-requiring diabetes (1.73, <.001), oral agent-controlled diabetes (1.39, .003), decade of pack-years smoking (1.04, >.001), history of transient ischemic attack (1.41, >.001), history of stroke (1.51, >.001), creatinine >1.5 mg/dL (1.48, >.001), hypoalbuminemia (1.49, >.001), and fourth quartile of operative time (1.44, >.001). Cardiopulmonary comorbid features did not affect the composite outcome in this model. Regional anesthesia was used in 2437 (18%) cases, with a resultant relative risk reduction for stroke (17%), death (24%), cardiac event (33%), and the composite outcome (31%; odds ratio, 0.69; P = .008)., Conclusions: Carotid endarterectomy results across a spectrum of Veterans Affairs and private sector hospitals compare favorably to contemporary studies. These data will assist in selecting patients who are at an increased risk for adverse outcomes. Use of regional anesthetic significantly reduced perioperative complications in a risk-adjusted model, thus suggesting that it is the anesthetic of choice when CEA is performed in high-risk patients.

Transient transfection of mammalian cells with episomal vectors is a very useful method for producing high levels of recombinant proteins. Transient systems remove the need for the laborious and time-consuming process of creating stable cell lines. Here, we describe the optimisation and evaluation of a high-throughput transient expression system in HEK293-EBNA cells. The process was developed for the expression of 10 constructs simultaneously in deep-well plates and subsequent purification using 96-well plate affinity chromatography. This enabled multiple combinations of different constructs, vectors, and expression conditions to be studied in parallel.

Recombinant baculoviruses have proved to be a very useful means to express many proteins over the last 20 years. Since their introduction, there have been a number of significant improvements that have simplified and speeded up the construction of baculoviruses. One of the most commonly used methods relies upon recombination with the baculovirus genome maintained in Escherichia coli. In this paper, we report the conversion of nearly all the steps in this process including the expression testing and purification to a multi-well plate format. This enables a significant increase in the number of constructs that can be processed in a shorter period of time and an order of magnitude increase in the number of expression conditions that can be analysed. A key step in our process is that the transfection is done in suspension rather than adherent cells, which gives a much higher virus titre than in the standard methods.

Background: There remains no consensus on the appropriate application of endovascular abdominal aortic aneurysm repair (EVAR). Information from administrative databases, industry-sponsored trials, and single institutions has inherent deficiencies. This study was designed to compare early outcomes of open (OPEN) versus EVAR in a contemporary (2000 to 2003) large, multicenter prospective cohort., Methods: Fourteen academic medical centers contributed data to the National Surgical Quality Improvement Program-Private Sector (NSQIP-PS), which ensures uniform, comprehensive, prospective, and previously validated data entry by trained, independent nurse reviewers. A battery of clinical and demographic features was assessed with multivariate analysis for association with the principal study end points of 30-day operative mortality and morbidity., Results: One thousand forty-two patients underwent elective infrarenal abdominal aortic aneurysm (AAA) repairs: 460 EVAR and 582 OPEN. EVAR patients were older (74 vs 71 years, P < .0001), included more men (84.6% vs 79.6%, P < .05), and had a higher incidence of chronic obstructive pulmonary disease (25.4% vs 17.9%, P < .01). EVAR resulted in significantly reduced overall morbidity (24% vs 35%, P < .0001) and hospital stay (4 vs 9 days, P < .0001). Cardiopulmonary and renal function-related comorbidities had the expected significant impact on mortality for both procedures at univariate analysis ( P < .05). While crude mortality rates between EVAR and OPEN did not differ significantly (2.8% vs 4.0%) ( P = 0.32). After multivariate analysis, correlates of operative mortality included OPEN (odds ratio [OR], 2.44; 95% confidence interval [CI], 1.03 to 5.78; P < .05), advanced age (OR, 1.11; P < .001), history of angina (OR, 5.54; P < .01), poor functional status (OR, 5.78; P < .001), history of weight loss (OR, 7.42; P < .01), and preoperative dialysis (OR, 51.4; P < .0001). EVAR also compared favorably to OPEN (OR, 2.14; 95% CI, 1.58 to 2.89; P < .0001) for overall morbidity., Conclusion: Significant morbidity accompanies AAA repair, even at major academic medical centers. These data strongly endorse EVAR as the preferred approach in the presence of significant cardiopulmonary or renal comorbidities, or poor preoperative functional status.

Objectives: Carotid endarterectomy (CEA) is supported by level 1 evidence as the standard treatment of severe carotid stenosis in both symptomatic and asymptomatic patients. As interventional techniques are emerging for treatment of this disease, this study was undertaken to provide a contemporary surgical standard for comparison to carotid stenting., Patients and Methods: During the interval 1989 to 1999, 2236 isolated CEAs were performed on 1897 patients (62% male, 36% symptomatic, 4.6% reoperative procedures). Study endpoints included perioperative events, patient survival, late incidence of stroke, anatomic durability of CEA, and resource utilization changes during the study. Variables associated with complications, long-term and stroke free survival, restenosis, and resource utilization were analyzed by univariate and multivariate analysis., Results: Perioperative complications occurred in 5.5% of CEA procedures, including any stroke/death (1.4%), neck hematoma (1.7%), cardiac complications (0.5%), and cranial nerve injury (0.4%). Actuarial survival at 5 and 10 years was 72.4% (95% confidence interval [CI] 69.3-73.5) and 44.7% (95% CI 41.7-47.9) respectively, with coronary artery disease (P < 0.0018), chronic obstructive pulmonary disease (P < 0.00018) and diabetes mellitus (P < 0.0011) correlating with decreased longevity. The age- and sex-adjusted incidence of any stroke during follow-up was reduced by 22% (upper 0.35, lower 0.08) of predicted with the patient classification of hyperlipidemia (P < 0.0045) as the only protective factor. Analysis of CEA anatomic durability during a median follow-up period of 5.9 years identified a 7.7% failure rate (severe restenosis/occlusion, 4.5%; or reoperative CEA, 3.2%) with elevated serum cholesterol (P < 0.017) correlating with early restenosis. Resource utilization diminished (first versus last 2-year interval periods) for average hospital length of stay from 10.3 +/- 1.5 days to 4.3 +/- 0.7 days (P < 0.01) and preoperative contrast angiography from 87% +/- 1.4% to 10.3% +/- 4%., Conclusions: These data delineate the safety, durability, and effectiveness in long-term stroke prevention of CEA. They provide a standard to which emerging catheter-based therapies for carotid stenosis should be compared.

Popliteal artery embolism has been a focus of study at the Massachusetts General Hospital for over 60 years. It is a formidable vascular problem with significant limb loss and mortality. To assess the impact of advances in cardiac and vascular therapies, we reviewed our outcomes over 12 years. A retrospective review from our databases identified 66 patients with 72 popliteal artery emboli between January 1989 and October 2000. Patients undergoing nonsurgical therapy or with in situ atherosclerotic thrombosis were excluded. Demographics, comorbidities, presentation, duration, etiology, treatment, and outcomes were analyzed. Patients were classified into those with acute (AP; symptoms <7 days; 59 of 72, 82%) or delayed (DP; symptoms >7 days; 13 of 72, 18%) presentation. The presentation was typically acute ischemia (85%) in the AP group and claudication (69%), rest pain (15.5%), or gangrene (15.5%) in the DP group. The most common etiology was embolism secondary to atrial fibrillation (17 of 72, 24%). Femoral artery access (15 of 72, 21%) was more prevalent than in our prior experience. In the AP group, 9 of 59 (15%) were treated with a femoral artery approach, 44 of 59 (75%) with a popliteal artery approach, and 6 of 59 (10%) with bypass. In the DP group, 11 of 13 (85%) were treated with a popliteal approach and 2 of 13 (15%) with bypass. Completion angiography was done in 17 cases (24%). Limb salvage rate was 88% (88% AP, 85% DP); the rate was 94% with angiography and 85% without it (p > 0.1). There were seven deaths (10%). The mortality rate was 33% after amputation and 7% after limb salvage (p < 0.05). Except for a greater prevalence of femoral artery access as an etiology, the demographics of patients with popliteal embolism were similar to those of prior reports. Although a femoral approach may be appropriate in select AP cases, a popliteal approach is preferred in most patients and is necessary in DP cases. Completion angiography should be performed to ensure adequacy of the embolectomy. Outcomes are unchanged. Future therapies should aim to improve limb salvage and mortality rates.

Objectives: We evaluated mid-term results of the multicenter EVT/Guidant aortouniiliac endograft (AI) trial and ascertained the durability of this endovascular technique in patients unable to undergo standard bifurcated endografting., Methods: From November 1996 to December 1998, 121 patients were enrolled to receive the AI device on the basis of complex iliac artery anatomy contraindicating bifurcated endografting. Clinical data were centrally collected, and radiographic data were evaluated by core facility., Results: AI placement was technically successful in 113 of 121 patients. At operation, patients who underwent AI had significantly more arrhythmias, congestive heart failure, and peripheral occlusive disease (P <.05) compared with patients who underwent open aneurysmorrhaphy in the EVT/Guidant trials, indicating comorbid features in this anatomic cohort. Distal AI attachment was performed to the external iliac artery in 40 (36%) patients. Median follow-up was 38 months. In the AI group, overall aneurysm diameter decreased over the duration of study from 54.4 +/- 9.6 mm to 44.4 +/- 16.4 mm (P =.004). At 24 and 36 months after repair, reduction in aneurysm size was associated with absence of endoleak (P =.003 and P =.008, respectively). Aneurysms shrunk or remained stable in 109 (96.5%) patients. Endoleak was identified in 52.3% of patients at discharge, and at follow-up in 30.9% at 1 year, 34.8% at 2 years, 28.6% at 3 years, and 30.4% at 4 years. Type II endoleak predominated. Leak from failure to completely occlude contralateral iliac flow accounted for 8 of 58 endoleaks (13.8%) at discharge. Sixteen patients (14.2%) underwent postoperative endoleak treatment; in one of these patients open conversion was necessary at 20 months. Post-procedure thigh or buttock claudication developed in 16 patients (14.2%). Thirteen patients (81.3%) had either distal attachment in the external iliac artery or contralateral type IIA occlusion. Fifteen patients (13.3%) required intervention because of reduced limb flow; one of these patients underwent open conversion at 27 months, and another underwent axillofemoral grafting at 28 months. Device migration was confirmed in 2 (1.8%) patients, without current clinical sequelae. Whereas no femorofemoral graft thromboses occurred, graft infection developed in 3 patients (2.6%). During follow-up, aneurysm in 2 patients ruptured. Late death occurred in 41 patients (36.3%). Twenty-four patients (58.5%) died of cardiopulmonary disease; one death was endograft-related after aneurysm rupture; and one death was related to femorofemoral bypass infection. Actuarial survival was 78.4% (95% confidence interval [CI], 71%-86%) at 2 years and 63.4% (95% CI, 54%-73%) at 4 years., Conclusions: In patients with significant comorbid conditions and complex iliac anatomy unfavorable for bifurcated endografting, AI with femorofemoral bypass grafting is safe and effective. In most patients this endovascular option provides satisfactory mid-term results.

Historically, immune-mediated degradation and subsequent aneurysm formation have limited the usefulness of cryopreserved arterial allografts. This study tested the hypothesis that modern cryopreserved arterial allografts are protected from immune-mediated dilation. Abdominal aortas were harvested from anesthetized rats (Lewis and Brown-Norway) for immediate implantation or cryopreservation. Subsequently, Lewis rats underwent infrarenal aortic replacement with either an acutely harvested or a cryopreserved graft. There were four experimental groups: (1) acutely harvested isografts (Iso; n = 6), (2) cryopreserved isografts (C-Iso; n = 6), (3) cryopreserved allografts (C-Allo; n = 6), and (4) acutely harvested allografts (Allo; n = 6). All grafts were explanted at 8 weeks. A video camera and edge detection software were used to measure systolic and diastolic in vivo graft diameter (d). Measurement of arterial blood pressure (p) allowed calculation of compliance (Dd/Dp). Tail-cuff plethysmography was used to assess graft patency at 1 week. Graft diameter and blood pressure measurements were repeated at harvest. All harvested grafts were examined histologically. Our results showed that cryopreservation prevented immune-mediated dilation in arterial allografts in our 8-week rat implant model. Furthermore, the compliance of the cryopreserved grafts and was similar to that of controls. Further investigation is needed to delineate the exact mechanism of these potential clinically significant findings.

Objective: Despite well-documented good early results and benefits of endoluminal stent graft repair of abdominal aortic aneurysm (J Vasc Surg 2002;35:1137-44.)(AAA), the long-term outcome of this method of treatment remains uncertain. In particular, concern exists that late effectiveness and durability are inferior to that of open repair. To determine the incidence and causes of clinical failures of endovascular AAA repair, a 7-year experience with 362 primary AAA endografts was reviewed., Methods: Clinical failures were defined as deaths within 30 days of the procedure, conversions (early and late) to open AAA repair, AAA rupture after endoluminal treatment, or AAA sac growth of more than 5 mm in maximal diameter despite endograft repair. Endoleak status per se was not considered unless it resulted in an adverse event. If clinical problems arose but could be corrected with catheter-based therapies or limited surgical procedures, thereby maintaining the integrity of successful stent graft treatment of the AAA, such cases were considered as primary assisted success and not classified as clinical failures., Results: The average follow-up period was 1.5 years. Six deaths (1.6%) occurred after the procedure, all in elderly patients or patients at high risk. Five patients (1.4%) needed early conversion (immediate, 2 days) to open repair for access problems or technical difficulties with deployment, resulting in an implantation success rate of 98.6%. Eight patients (2.2%) underwent late conversion for a variety of problems, including AAA expansion (n = 4), endograft thrombosis (n = 1), secondary graft infection (n = 2), and rupture at 3 years (n = 1). Rupture occurred in an additional two patients for a total incidence rate of 0.8%. AAA sac growth of greater than 5 mm was observed in 20 patients (5.6%), four of whom have undergone successful catheter-based treatments to date. Overall, 39 patients (10.7%) needed catheter-based (n = 45) or limited surgical (n = 4) reinterventions for a variety of late problems that were successful in 92%., Conclusion: In our 7-year experience, one or more clinical failures of endovascular AAA repair were observed in 31 patients (8.3%). Reinterventions were necessitated in a total of 10.7% of patients but were usually successful in maintaining AAA exclusion and limiting AAA growth. These results emphasize that endovascular repair provides good results and many benefits for most properly selected patients but is not as durable as standard open repair.

Human procathepsin S and cathepsin S were expressed as inclusion bodies in Escherichia coli. Following solubilization of the inclusion body proteins, fractional factorial protein folding screens were used to identify folding conditions for procathepsin S and cathepsin S. A primary folding screen, including eight factors each at two levels, identified pH and arginine as the main factors affecting procathepsin S folding. In a second simple screen, the yields were further improved. The in vitro folding of mature cathepsin S has never been reported previously. In this study we used a series of fractional factorial screens to identify conditions that enabled the active enzyme to be generated without the prodomain although the yields were much lower than achieved with procathepsin S. Our data show the power of fractional factorial screens to rapidly identify folding conditions even for a protein that does not easily fold into its active conformation., (Copyright 2002 Elsevier Science (USA).)

The medium chain mu 2 subunit (AP50) of the clathrin-associated adapter protein complex 2 (AP-2) interacts specifically with the tyrosine-based signals of several integral membrane proteins through the consensus sequence YXXPhi, where X can be any residue and Phi is a large hydrophobic residue. Using surface plasmon resonance combined with structural information, we have analysed the interaction of AP50 with peptides derived from the cytoplasmic tail of cytotoxic T-lymphocyte antigen 4 (CTLA-4). The crystal structure of AP50 in complex with a CTLA-4-derived peptide was determined to 3.6 A (1 A=0.1 nm) resolution. The binding domain of AP50 (residues 164-435) was expressed in Escherichia coli and purified. In agreement with previous reports, the AP50 domain bound to residues 152-174 of CTLA-4, but not to the same peptide that was phosphorylated at the single tyrosine residue (position 165). The interaction exhibited fast kinetics with rapid on and off rates and a K(d) of 0.7 microM. In order to further understand why AP50 binds to CTLA-4, but not to the homologous receptor CD28, a comparison of binding of AP50 with five peptides with single changes in and around the YXXPhi motif to the equivalent residues of CD28 was made. T162H greatly reduced binding, whereas T161L had little effect. Mutations G163S, V164D and K167N all exhibited reduced binding. Modelling of the single amino acid changes using structural information, was in broad agreement with the binding data, demonstrating that residues outside of the YXXPhi motif are also important in the interaction of membrane proteins with AP50.

Introduction: Evidence exists that an ideal bypass conduit should have a functional endothelial cell surface combined with mechanical properties similar to those of native arteries. We hypothesized that the effect of combined arterial levels of pulsatile shear stress, flow, and cyclic strain would enhance saphenous venous endothelial cell nitric oxide (NO) production, and that variations in these "ideal" conditions could impair this function. We studied NO production as a measure of endothelial function in response to different hemodynamic conditions., Methods: Human adult saphenous venous endothelial cells were cultured in 10-cm silicone tubes, similar in diameter (5 mm) and compliance (6%) to a medium-caliber peripheral artery (eg, popliteal). Tube cultures were exposed to arterial conditions: a combined pressure (120/80 mm/Hg; mean, 100 mm/Hg), flow (mean, 115 mL/min) and cyclic strain (2%), with a resultant pulsatile shear stress of 4.8 to 9.4 dyne/cm2 (mean, 7.1). Identical tube cultures were used to study variations in these conditions. Modifications of the system included a noncompliant system, a model with nonpulsatile flow, and a final group exposed to pulsatile pressure with no flow. NO levels were measured with a fluorometric nitrite assay of conditioned media collected at 0, 0.25, 0.5, 1, 2, and 4 hours. Experimental groups were compared with cells exposed to nonpulsatile, nonpressurized low flow (shear stress 0.1 dyne/cm2) and static cultures., Results: All experimental groups had greater rates of NO production than cells under static conditions (P <.05). Cells exposed to ideal conditions produced the greatest levels of NO. Independent decreases in compliance, flow, and pulsatility resulted in significantly lower rates of NO production than those in the group with these conditions intact (vs noncompliant P <.05, vs nonflow P <.05, and vs nonpulsatile P <.05)., Conclusions: Our results show that in the absence of physiologically normal pulsatility, cyclic strain, and volume flow, endothelial NO production does not reach the levels seen under ideal conditions. Pulsatile flow and compliance (producing flow with cyclic stretch) play a key role in NO production by vascular endothelium in a three-dimensional hemodynamically active model. This correlates biologically with clinical experience linking graft inflow and runoff and the mechanical properties of the conduit to long-term patency.

Purpose: External pneumatic compression (EPC) is an effective means of prophylaxis against deep venous thrombosis. However, its mechanism remains poorly understood. Understanding of the biological consequences of EPC is an important goal for optimizing performance of the EPC-generating device and providing guidance for clinical use. We present a new in vitro cell culture system (Venous Flow Simulator) that simulates blood flow and vessel collapse conditions during EPC, and we examine the influence of these factors on endothelial cell (EC) fibrinolytic activity and vasomotor function., Methods: An in vitro cell culture system was designed to replicate the hemodynamic shear stress and vessel wall strain associated with induced blood flow during different modes of EPC. Human umbilical vein endothelial cells were cultured in the system and subjected to intermittent flow, vessel collapse, or a combination of the two. The biologic response was assessed through changes in EC morphology and the expression of fibrinolytic factors tissue plasminogen activator, plasminogen activator inhibitor type 1, profibrinolytic receptor (annexin II), and vasomotor factors endothelial nitric oxide synthase and endothelin-1., Results: The cells remained attached and viable after being subjected to intermittent pulsatile flow (F) and tube compression (C). In F and F + C, cells aligned in the direction of flow after 6 hours. Northern blot analysis of messenger RNA shows that there is an upregulation of tissue plasminogen activator expression (1.95 +/- 0.19 in F and 2.45 +/- 0.46 in FC) and endothelial nitric oxide synthase expression (2.08 +/- 0.25 in F and 2.11 +/- 0.21 in FC). Plasminogen activator inhibitor type 1, annexin II, and endothelin 1 show no significant change under any experimental conditions. The results also show that pulsatile flow, more than vessel compression, influences EC morphology and function., Conclusion: Effects on ECs of intermittent flow and vessel collapse, either individually or simultaneously, were simulated with an in vitro system of new design. Initial results show that intermittent flow associated with EPC upregulates EC fibrinolytic potential and influences factors altering vasomotor tone. The system will facilitate future studies of EC function during EPC.

Purpose: We describe a technique for end-to-end sleeve anastomosis that may enable minimally invasive bypass grafting and characterize the tensile strength, stenosis rate, durability, and healing of the anastomosis., Method: An anastomotic device assembly consisting of a cable tie-type band with mobile teeth elements is mounted on the outer surface of a polytetrafluoroethylene graft. The graft is drawn over the artery resulting in a sleeve of vessel within the graft. As the band is tightened over an intraluminal obturator, the independently mobile anchoring teeth are driven through the graft into the artery. The tensile strength of the anastomosis was compared with sutured anastomosis during in vitro studies using cadaveric human femoral arteries. For in vivo studies on pigs and goats, we used a proximal exovascular sleeve anastomosis along with a distal sutured aortic anastomosis. Survival animals were studied by angiogram postoperatively and at the time of explantation., Results: In vitro studies showed no difference in maximal tensile strength between sutured and exovascular anastomoses (10.5 +/- 2.7 lb vs 10.2 +/- 3.0 lb, P =.83). However, loss of continuous graft to artery interface occurred at lower loads in the sutured anastomoses (6.5 +/- 0.6 lb, P <.05). In total, all 24 pigs tested in nonsurvival or survival settings sustained a successful and leakproof anastomosis. Of the 13 nonsurvival cases, maximal epinephrine-induced hypertension sustained over 15 minutes (peak systolic blood pressure > 210-250 mm Hg) was tolerated without leakage in a subgroup of five animals (100%). All 11 survival pigs had no pseudoaneurysms or stenosis relative to sutured control anastomoses at 6 week explantation (8.2 +/- 1.25 mm vs 8.5 +/- 1.6 mm, P =.21). The three long-term survival goats had no pseudoaneurysm or stenosis after 40 weeks. Histologic examination confirmed healing of the aorta to graft with minimal neointimal hyperplasia., Conclusion: Exovascular sutureless anastomosis appears comparable to sutured anastomosis in stenosis rate, healing, and durability, with some advantages in tensile strength and rapidity of application.

We describe here the expression of a C-terminally truncated form of human procollagenase-3 in Escherichia coli. The protein was found almost exclusively in inclusion bodies that were solubilized and refolded by two separate methods and then purified on Ni-NTA agarose. The purified proenzyme could be activated with either trypsin or APMA and active enzyme could be purified on a peptidic hydroxamate affinity column. Competitive elution from the affinity matrix yielded a highly purified preparation., (Copyright 2000 Academic Press.)

Objective: The purpose of this study was to determine the early efficacy of endovascular aortouniiliac stent grafts with femorofemoral bypass graft in the treatment of aortoiliac aneurysmal disease., Methods: We analyzed 51 consecutive patients from January 1997 to March 1999 with a mean follow-up of 15.8 months. Patients ranged in age from 44 to 93 years (mean, 75 years) with a mean aortic aneurysm diameter of 6.2 cm. Technical success was achieved in 50 patients; one patient required conversion to open repair intraoperatively. We placed 28 custom-made and 22 commercial devices. The mean operative time was 223 minutes. The endograft was extended to the external iliac artery in 42% of cases. The contralateral common iliac artery was occluded using either a closed covered stent or intraluminal coils., Results: The median hospital stay was 4 days with an average intensive care unit stay of 0.25 days. There were no operative mortalities. Two patients died during follow-up from unrelated conditions. Endoleaks occurred in 11 patients (22%); seven patients (14%) required intervention (four catheter based, three operative). Other complications occurred in 38% of patients but were largely remote or wound related. One femorofemoral bypass graft occluded immediately postoperatively as a result of an intraprocedural external iliac dissection yielding a 98% primary patency and 100% secondary patency. Clinical success was achieved in 88% of patients., Conclusions: These data suggest that this strategy represents a reliable method of repair of aortoiliac aneurysmal disease and extends the capability of an endoluminal approach to patients with complex iliac anatomy.

Purpose: To determine the clinical outcome of hypogastric artery occlusion in patients who underwent endovascular treatment of aortoiliac aneurysmal disease., Material and Methods: From January 1994 to March 1998, 94 patients underwent endovascular treatment of aneurysmal diseases involving the infra-abdominal aorta or iliac arteries. Preoperative and intraoperative radiologic data were reviewed. Discharge summaries, clinic visits, and phone calls formed the basis for clinical follow-up, with a mean follow-up period of 7.3 months (range, 1-24 months)., Results: Because of the anatomy of the aneurysms, 28 patients required occlusion of one or more hypogastric arteries. One of the 28 patients died of unrelated causes before follow-up. Seven (26%) of the remaining 27 patients developed symptoms attributable to the hypogastric artery occlusions. Five patients developed new buttock or thigh claudication; of these five patients, three with initially mild symptoms noted complete or near complete resolution of symptoms upon follow-up. One patient with originally significant claudication at 2-year follow-up noted near resolution of symptoms. The other patient with severe pain did not improve significantly on final 1-year follow-up before his death (of unrelated causes). Other clinical complications were worsening sexual function in one patient and a nonhealing sacral decubitus ulcer that developed in a debilitated patient in the postoperative setting, which required surgery. No bowel ischemia was observed., Conclusion: When treating aortoiliac aneurysmal disease through an endovascular approach, the occlusion of internal iliac artery is often necessary but carries with it a small but finite chance of morbidity.

Adult human saphenous vein endothelial cells (HVEC) were cultured in a compliant tubular device and evaluated by Northern hybridization for the effects of combined pressurized pulsatile flow and cyclic strain on the expression of mRNAs for endothelin-1 (ET-1), endothelial cell nitric oxide synthase (ecNOS), tissue plasminogen activator (tPA), and plasminogen activator inhibitor type 1 (PAI-1). The hemodynamic environment was designed to mimic shear stress conditions at the distal anastomosis of a saphenous vein graft, a common site of intimal proliferation. Steady-state mRNA levels in experimental tubes were expressed relative to that in controls. No changes were observed in ET-1 mRNA after 1 and 24 hr, but a 50% decrease in experimental cultures was observed after 48 hr in the vascular simulating device. Similar results were obtained for ecNOS mRNA, although a subgroup (4 of 11) showed a significant decrease (>50%) by 24 hr. For tPA mRNA, no change was observed after 1 hr, but a significant decrease (>60%) was measured after 24 hr and no message was detectable after 48 hr. Steady-state levels for PAI-1 mRNA remained unchanged through 48 hr of treatment. These results show that pressure, pulsatile flow, and cyclic strain, when applied in concert, differentially alter vasoactive and fibrinolytic functions in HVEC. Moreover, the dramatic decrease in steady-state levels of tPA mRNA is consistent with a shift toward an increased thrombotic state.

Purpose: This trial was designed to identify factors affecting patency rates of primary prosthetic above-knee femoropopliteal bypass grafts at 5 years., Methods: A multi-institutional, prospective trial randomized 240 patients to compare patency rates of Gore-tex and Hemashield above-knee femoropopliteal bypass grafts at 5 years. Univariate comparisons of patency between levels of each prognostic variable were made with the Kaplan-Meier method. Variables that had a univariate P value less than.25 or those known to be important were submitted to a Cox regression analysis., Results: The patient survival rate at 5 years was 59.4%. There were no differences in primary or secondary patency rates at 5 years between the two graft materials (primary, 45% vs 43% and secondary, 68% vs 68%). The risk for graft occlusion was significantly increased for patients younger than 65 years (2.1; P =.001) and for grafts with a diameter less than 7 mm (1.65; P =.0219). Variables with no apparent independent effect on patency rates were smoking status, runoff, diabetes mellitus, sex, presenting symptoms, and postoperative treatment with aspirin or Coumadin. Noninvasive test results were not predictive of subsequent graft function., Conclusion: Although the type of prosthetic used for above-knee femoropopliteal bypass grafts does not affect 5-year patency rates, age and graft size do influence results. These factors should be considered before a prosthetic bypass grafting procedure. Furthermore, these data should serve as a contemporary standard, with which evolving and conventional procedures can be compared.

In this work we have undertaken a comparative study of human umbilical vein endothelial cells (HUVECs) and human saphenous vein endothelial cells (HSVECs) with respect to functional and antigenic tissue factor (TF), tissue factor pathway inhibitor (TFPI), and TF mRNA. Monolayers of each cell type (passage 2, except where specified) were grown to confluence and then activated for 4 h with either 50 U/mL IL-1-alpha or 10 microg/mL tumor necrosis factor-alpha. Activated factor X appearing in supernatant was measured using a chromogenic assay, and both Northern blots and quantitative RT-PCR were performed to assess concentrations of TF mRNA accompanying activation. The role of TFPI was separately determined by ELISA for supernatant TFPI antigen, and by measurements of production of activated factor X in the presence of 0, 5, 15, or 50 microg/mL of an antibody directed against TFPI. To address a non-TF pathway endothelial cell function, antigenic concentrations of tissue plasminogen activator for both cell types was also determined by ELISA. HUVECs were found to produce 2.4- to 3.5-fold more functional TF. No significant HUVEC-HSVEC differences were detected in TF antigen, supernatant TFPI, anti-TFPI affinity for endothelial cell-associated TFPI, TF mRNA or its amplification products, and tissue plasminogen activator. Immunostaining for TF antigen, however, may have failed to detect a modest HUVEC-HSVEC difference. Our finding with respect to functional TF indicates that HUVECs and HSVECs are not equivalent in terms of models for endothelial cell function in small children versus adults.

Purpose: Contrast arteriography is the accepted gold standard for diagnosis and treatment planning in patients with atherosclerotic renovascular disease (RVD). In this study, the results of a selective policy of surgical renal artery reconstruction (RAR) with magnetic resonance angiography (MRA) as the sole preoperative imaging modality are reviewed., Methods: From May 1993 to May 1998, 25 patients underwent RAR after clinical evaluation, and aortic/renal MRA performed with a gadolinium-enhanced and 3-dimensional phase contrast technique. Clinical presentations suggested severe RVD in all patients and included poorly controlled hypertension (16 patients), hospitalization for hypertensive crises and/or acute pulmonary edema (13), and deterioration of renal function within one year of operation (15). Thirteen patients had associated aortic pathologic conditions (12 aneurysms, 1 aortoiliac occlusive disease), and eight of these patients also underwent noncontrast computed tomography scans. Significant renal dysfunction (serum creatinine level, >/=2.0 mg/dL) was present in all but 4 patients with 14 of 25 patients having extreme (creatinine level, >/=3.0 mg/dL) dysfunction., Results: Hemodynamically significant RVD in the main renal artery was verified at operation in 37 of 38 reconstructed main renal arteries (24/25 patients). A single accessory renal artery was missed by MRA. RAR was comprehensive (bilateral or unilateral to a single-functioning kidney) in 21 of 25 patients and consisted of hepatorenal bypass graft (3 patients), combined aortic and RAR (13 patients), isolated transaortic endarterectomy (8 patients), and aortorenal bypass graft (1 patient). Early improvement in both hypertension control and/or renal function was noted in 21 of 25 patients without operative deaths or postoperative renal failure. Sustained favorable functional results at follow-up, ranging from 5 months to 4 years, were noted in 19 of 25 patients., Conclusion: MRA is an adequate preoperative imaging modality in selected patients before RAR. This strategy is best applied in circumstances where the clinical presentation suggests hemodynamically significant bilateral RVD and/or in patients at substantial risk of complications from contrast angiography.

A tissue engineering approach was developed to produce arbitrary lengths of vascular graft material from smooth muscle and endothelial cells that were derived from a biopsy of vascular tissue. Bovine vessels cultured under pulsatile conditions had rupture strengths greater than 2000 millimeters of mercury, suture retention strengths of up to 90 grams, and collagen contents of up to 50 percent. Cultured vessels also showed contractile responses to pharmacological agents and contained smooth muscle cells that displayed markers of differentiation such as calponin and myosin heavy chains. Tissue-engineered arteries were implanted in miniature swine, with patency documented up to 24 days by digital angiography.