Camptothecin resistance from a single mutation changing glycine 363 of human DNA topoisomerase I to cysteine.

A full-length human DNA topoisomerase I complementary DNA clone was mutagenized in vitro and the mutagenized DNA was used to replace wild-type human TOP1 complementary DNA in YCpGAL1-hTOP1, a plasmid constructed for the expression of the human enzyme in yeast. A yeast strain devoid of yeast DNA topoisomerase I and permeable to the anticancer drug camptothecin was transformed with the plasmid pool. Assays of DNA topoisomerase I in lysates of camptothecin-resistant transformants identified one with nearly the same level of the enzyme as transformants of unmutagenized YCpGAL1-hTOP1, and a single mutation changing Gly363 to a cysteine was found in this mutant. The G363C mutant enzyme was overexpressed in yeast and partially purified. It differed significantly from wild-type human DNA topoisomerase I similarly expressed and purified: camptothecin-stimulated cleavage of DNA was observed with the wild-type but not the G363C enzyme, and the DNA relaxation activity of the mutant enzyme, unlike that of the wild-type enzyme, was not significantly stimulated by Mg(II). The positions of the G363C and other previously reported camptothecin resistance mutations in eukaryotic DNA topoisomerase I were discussed in terms of a model in which the active site is an interdomainal cleft.

What is the best management for abdominal aortic aneurysm in patients at high surgical risk? A single-center review.

AIM: To determine the best treatment for high-risk patients with abdominal aortic aneurysms (AAA). METHODS: We reviewed a prospective database of all patients who underwent conventional (OPEN) or endovascular aneurysm repair (EVAR) between January 1998 and December 2002. Patients were preoperatively classified according to the American Society of Anesthesiology (ASA). Comorbidities and medical risk factors were categorized according to the Ad Hoc Committee on Reporting Standards. Perioperative mortality and morbidity rates were analyzed according to the type of surgical procedure (OPEN vs EVAR) and ASA class. Patients in ASA classes I and II were excluded. Continuous data were expressed as mean +/- standard deviation. All data were calculated using the cumulated actuarial method of event outcome probability. Kaplan-Meier curves were constructed and the log-rank statistic and chi squared test were used for comparative data. P values less than 0.05 were considered to indicate statistical significance. RESULTS: Of the total 375 patients who underwent AAA repair, 168 (45%) belonged in ASA classes III and IV (85 submitted OPEN and 83 EVAR to repair). Among general risk factors only coronary artery disease differed significantly between the 4 groups (P = 0.04). The Bonferroni correction identified a statistically significant difference between ASA classes III and IV for the OPEN technique and for EVAR (P = 0.007 and P = 0.012). Neither 30-day morbidity or mortality differed significantly according to ASA class and surgical technique. The median follow-up was 19 months (range 5-60 months). The overall survival was 78% at 60 months. Survival rates during follow-up differed significantly in the 2 risk classes (ASA III 5/123, 4% vs ASA IV 9/38, 24%), (P = 0.0001). The deaths in the ASA class 4 patients (12/14; 86%) were caused by preexisting medical comorbidities (in 9 patients cardiovascular, in 1 cancer and in 2 cirrhosis). CONCLUSIONS: Except patients with small aneurysms (< 6 cm), in whom the risk of death at 1-year due to comorbidities exceeds the risk of a ruptured aneurysm, all patients at high surgical risk (ASA class IV) benefit from AAA repair. Patients with small aneurysms must undergo strict surveillance to assess growth and aneurysmal wall changes to prevent unexpected rupture.

[Atheroembolic syndrome due to isolated infrarenal abdominal aorta stenosis and endovascular treatment: case report and review of literature]. / Ateroembolia da lesione stenosante isolata dell'aorta addominale sottorenale e trattamento endovascolare: caso clinico e revisione della letteratura.

One of the most common source of lower extremity atheroembolization is the aorta and particularly the infrarenal segment. Complex atherosclerotic plaque can lead the patient to gangrene and major amputation. When the origin of embolization is a focal lesion, endoluminal methods could be an alternative to surgical treatment. Although the experience with aortic stent is limited, the results obtained so far seem to be encouraging. The case of a mid-age heavy smoker woman with a history of the abrupt onset of painfull cyanotic toes in the left foot and subsequent complete gangrene of the first digit in the same foot is herein reported. Angiography and CT scan revealed an high-grade calcified aortic infrarenal plaque. Because of the discrete characteristic of the lesion, an endovascular approach with a Palmaz stent was elected. The stenosis was successfully treated: the patient experienced the complete resolution of the toe painfull cyanosis within 3 months, the stent remained patent through a 24 months follow-up and no subsequent embolic episodes were observed.

Preparation and biodistribution of 99m technetium labelled oxidized LDL in man.

Radiolabelled autologous low density lipoprotein (LDL) has previously been used to study in vivo distribution and metabolism of native-LDL. Non-invasive imaging of atherosclerotic lesions using 99mTc-LDL was shown to be feasible in animal models and patients but the clinical utility remains to be assessed. Since recent reports suggest that oxidized LDL may play a major role in the pathogenesis of atherosclerosis, we developed a technique to oxidize autologous LDL and compared the biodistribution of oxidized-LDL with that of native-LDL in man. In addition, we evaluated the uptake in vivo of oxidized- and native-LDL by atherosclerotic plaques. LDL, obtained from human plasma was treated with various combinations of copper ions and H2O2 to induce oxidative modification by increasing the content of lipid peroxidation products and electrophoretic mobility. When LDL (0.3 mg/ml) was incubated with 100 microM Cu2+ and 500 microM H2O2 oxidation occurred rapidly within 1 h, and was labelled with 99mTc efficiently as native LDL. In vivo distribution studies revealed a faster plasma clearance of oxidized-LDL compared to native-LDL, and a higher uptake by the reticuloendothelial system. Tomographic scintigraphy of the neck in patients suffering from transient ischemic attacks, revealed accumulation of radiolabelled LDL preparations in the carotid artery affected by atherosclerotic lesions. We developed a technique to rapidly oxidize LDL using copper and H2O2. Biodistribution data demonstrate that oxidized-LDL is rapidly cleared from circulation, is taken up mostly by organs rich in macrophages, and can be detected at the level of carotid plaques.

Clinical usefulness of technetium-99m-HMPAO-labeled leukocyte scan in prosthetic vascular graft infection.

UNLABELLED: The infection of a prosthetic vascular graft (PVGI), although rare, is the most severe complication in reconstructive vascular surgery. The early diagnosis of this complication reduces the death rate from surgery. Aortofemoral graft infections differ clinically from peripheral graft infections in significant ways. The aim of this article is to evaluate separately the reliability of the 99mTc-HMPAO-labeled leukocyte scan or white blood cell count (WBC) in the early detection of both aortofemoral and peripheral graft infections. METHODS: One hundred sixty-two WBCs were performed on 129 consecutive patients with suspected aortofemoral (122 scans) and peripheral (40 scans) graft infection and in a 12-patient control group. Patients with suspected PVGI were categorized into three groups on the basis of their signs and symptoms on readmission: (a) patients with specific signs of graft infection (Group A); (b) patients with nonspecific signs of graft infection (Group B); and (c) patients with anastomotic aneurysms (Group C). Gram's stains of the perigraft exudate and graft cultures were performed and used as the gold standard in patients who underwent surgery. An 18-mo clinical follow-up was done to assess the presence or absence of graft infection in patients who did not have surgery. RESULTS: In patients with suspected aortofemoral graft infections, the overall sensitivity, specificity and accuracy of WBCs (Groups A, B, C) were 100%, 92.5% and 97.5%, respectively, whereas sensitivity, specificity and accuracy calculated in the patients with nonspecific signs of graft infection (Groups B, C) were 100%, 92.3% and 96.9%, respectively. In patients with suspected peripheral graft infections, sensitivity, specificity and accuracy were 100%. CONCLUSION: The white blood cell scan seems a reliable diagnostic method for early diagnosis of PVGI, and it is more useful in aortofemoral graft infections.

Mechanisms of DNA topoisomerase I-induced cell killing in the yeast Saccharomyces cerevisiae.

DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a mechanism of transient DNA strand cleavage characterized by the formation of a phosphotyrosyl bond between the DNA end and active site tyrosine. Camptothecin reversibly stabilizes the covalent enzyme-DNA intermediate by inhibiting DNA religation. During S-phase, collisions with advancing replication forks convert these complexes into potentially lethal lesions. To define the DNA damage induced by alterations in Top1p catalysis and the cellular processes that mediate the repair of such lesions, the yeast Saccharomyces cerevisiae was used. Substitution of conserved residues N-terminal to the active site tyrosine (Tyr-727) produced alterations in the camptothecin sensitivity or catalytic cycle of DNA Top1. For example, substituting Ala for Thr-722 in Top1T722A increased the stability of the covalent enzyme DNA intermediate. As with camptothecin, Top1T722A-induced cytotoxicity was ascribed to a reduction in DNA religation. By contrast, enhanced covalent complex formation by Top1N726H resulted from a relative increase in the rate of DNA cleavage. Conditional yeast mutants were also selected that exhibit temperature-sensitive growth only in the presence of the self-poisoning Top1T722A enzyme. Subsequent analyses of these tah mutants identified 9 genes whose function suppresses the cytotoxic action of camptothecin and Top1T722A. These include genes encoding essential DNA replication proteins (CDC45 and DPB11) and proteins involved in SUMO- or ubiquitination (UBC9 and DOA4).

Aorto-femoral graft infections: a clinical and microbiological analysis.

A 5-year experience of 19 aortic graft infections is reviewed. Of these, 13 (68%) had a late onset (more than 4 months after graft implantation) and usually presented with anastomotic pseudo-aneurysm or thrombosis. The remaining six infections (32%) had an early onset and presented more often with surgical wound infection. Aorto-enteric fistulae and inguinal sinus tracts were observed in both early and late onset infections. Coagulase-negative staphylococci (all slime negative, oxacillin susceptible strains) were the prevalent pathogens in both groups of infections and were isolated in six (32%) patients. Next most commonly seen were Pseudomonas aeruginosa in four (21%) patients, Enterococcus spp. in three (16%) patients, Staphylococcus aureus in three (16%) patients, other bacteria in six (32%) patients. No organisms were isolated in three (16%) patients. Mortality and major amputation rates were 47.3% and 31.6%, respectively. The therapeutic procedures included total graft removal (15 patients), partial graft excision (two patients), partial graft excision followed by total graft removal (one patient) and local treatment without graft removal (one patient). Six patients recovered, including two who underwent total graft removal associated with a non-conventional 'in situ' graft replacement and one patient treated conservatively with local treatment and antibiotics. The three patients undergoing partial graft excision showed signs of active infection of the residual graft.

A case of aneurysm of a primitive persistent sciatic artery: anatomical-clinical aspects and surgical management.

Aneurysms of the sciatic artery are quite rare. In the literature fewer than 100 cases of gluteal aneurysms have been reported; most have arisen from the superior or inferior gluteal artery; only few cases were aneurysms of an anomalous persistent sciatic artery (6 cases diagnosed angiographically and treated). In our own experience, the overall incidence of this anomaly, in patients undergoing angiography is very low, in the order of 0.025%. In our case the iliac femoral axis showed hypoplasia and required an internal iliac-popliteal bypass. For the location and the propensity atherosclerotic involvement of the persistent sciatic artery, an aneurysm should be considered in all patients with a pulsatile posterior thigh mass, with or not symptoms of peripheral vascular disease.

Echodensitometry: a methodologic approach to the non-invasive diagnosis of carotid atherosclerotic plaques.

The ultrasound image of the fibroatheromatous plaque has to date been unable to reveal the presence or absence of complications as shown by histology, such as thrombosis and intraplaque hemorrhage. We propose a novel densitometric method for determining the composition of the plaque at the echotomography, along with a new classification based on mathematical models drawn from the optical density distribution curve. This approach avails of an extremely high sensitivity. Percent areas occupied by thrombosis, intraplaque hemorrhage and atheroma in histologic sections have been shown to correspond to hyporeflecting areas in echotomographic images, whereas those occupied by calcium and fibrous tissue correspond to reflecting areas with or without acoustic shadow, respectively. This method may prove to have an important role in the non-invasive monitoring of even slight changes during progression or regression of the fibroatheromatous plaque.

Histopathological characterization of carotid plaques echotomography.

The positive correlation between cerebral ischemia and carotid atherosclerosis of extracranial tract has been well established. The reliability of echotomography as diagnostic and prognostic tool in the evaluation of the carotid atherosclerotic lesions is now intensively investigated. Most of the attention has been payed to the accuracy in the determination of the carotid stenosis. A percentage of cerebrovascular accidents do not correlate to the vascular stenosis but to the other modifications induced by the atherosclerotic plaque, such as the release of emboli, acute stenosis for intraplaque haemorrhage or thrombosis. Thus the evaluation of the composition of the plaque may represent a good prognostic tool. In the present study, fifty-two carotid obtained at surgery from patients, who preoperatively underwent ultrasonographic scanning of both carotid arteries, were examined by histological methods. In calcified tissues a significant correlation between findings obtained with both techniques was observed. In the complicated plaques atheromasic gruel, intraplaque haemorrhages and thrombosis could not be discriminated by echotomography.

Clinical and therapeutical evaluation of inflammatory aneurysms of the abdominal aorta.

Unexpected anatomical and clinical features of abdominal aortic aneurysm (AAA) may be encountered by the vascular surgeon creating technical problems that increase the normally low mortality rates of this affection. One such variant is the so called inflammatory aneurysm (IA) as a characteristic fibrosis involving the arterial wall and thus surrounding structure scan be observed. In our series of 525 patients affected by AAA the incidence of IA was about 4% (19 cases). Two groups of patients were considered: group A, including all the atherosclerotic patients, and group B 19 patients affected by IA. The latter group referred to a typical painful symptomatology in 84% of the cases: this element is of interest as only 20% of the cases of group A complained of pain. No other significant clinical or laboratory data were recorded which could allow the surgeon to perform a pre-operative differential diagnosis. In all 19 cases that underwent surgical treatment there was a 2-3 cm thick aneurysmal wall with a shiny white surface adhering to the IV portion of the duodenum, vena cava and iliac vessels and in some cases to the ureters. Histological examination of specimens of the aortic wall showed evident signs of atherosclerosis of the media and marked fibrotic thickening of the adventitia with the presence of lymphocyte aggregates: a sign of chronic inflammation. As what concerns indications and surgical treatment, there are no substantial differences. Pre-operative differential diagnosis can be made with CAT scan and ultrasound and the usual operative manoeuvres of aneurysmorrhaphy should be modified.

Outcome after early treatment of popliteal artery aneurysms.

BACKGROUND: Aneurysms of the popliteal artery are rare events with an estimated incidence of 0.1-2.8%. Their clinical importance depends on their propensity to cause thromboembolic complications or to rupture (18 to 31% of cases). This study was designed to assess the advantages of elective treatment of asymptomatic popliteal artery aneurysms so as to avoid the severe clinical manifestations that eventually arise if they remain untreated. METHODS: From 1980 to 1995, in our department we treated 28 popliteal aneurysms in 23 patients (19 elective operations and 6 emergencies). Three of the aneurysms proved amenable to fibrinolytic therapy alone. Follow-up lasted a mean 48 months (range 3 months to 15 years) in 20 patients (23 revascularizations). RESULTS: Global patency was 91.3%. Two thrombosed bypasses were treated by thrombolysis; in one case it did not resolve the ischaemia and the leg was amputated. In one case only, a graft became infected but this responded to conservative therapy with drainage and antibiotics. CONCLUSIONS: These findings suggest that popliteal aneurysms with good distal run-off should be repaired electively. Completely thrombosed aneurysms with coexistent limb-threatening ischaemia should be treated initially by thrombolysis. This will usually restore run-off and in some cases offers a valid alternative to emergency surgery for limb salvage.

For how long should carotid endarterectomy surveillance be continued?

Early restenosis represent the most important and more common failure after carotid endarterectomy. For this reason, after its first description made in 1976 by Stoney and String, it raised general interest among vascular surgeons. In spite of the efforts to clear the causes of this phenomenon, none of the numerous papers published in the literature has defined a specific cause determining restenosis. Nevertheless, at present, this hyperplastic response of the arterial wall to trauma after operation is generally considered benign because it is rarely responsible for new neurological symptoms or early internal carotid artery occlusion. This unanimous conviction has been achieved after years of instrumental and clinical postoperative follow-up performed all over the world. At the same time and probably for these reasons, recently, a new discussion has begun about the usefulness and cost-effectiveness of prolonged Duplex scanning postoperative surveillance of the endarterectomized carotid arteries. This new question raised our curiosity in verifying the validity of this new approach, so we reviewed accurately our laboratory follow-up registry and the data regarding onset, evolution and clinical outcome of early restenosis. These data associated with a meticulous review of the experience of other authors convinced us that the patients operated on, need, in most cases, a short even aggressive period of careful follow-up (generally the first six months).(ABSTRACT TRUNCATED AT 250 WORDS)

Late results of reconstructive surgery for renovascular hypertension.

In order to evaluate the late results of reconstructive surgery for renovascular hypertension, a review was made on a series of 120 consecutive patients who underwent operations over a 11 year period. There were 82 males (68.3%) and 38 females (31.7%) with a mean age of 48.4 years. Renal artery by-pass grafts were used in 90% (120/133), a thromboendarterectomy in 5.2% (7/133), and other surgical procedures were performed in 4.8% (6/133). Associated vascular procedures were performed in 38.3% (46/120) of patients. Operative mortality was 2.5% (3/120) overall; there was no mortality in the isolated renal artery reconstructions. There was a clinical success (after a mean follow-up of 48 months) in 80.4% of patients. The most important factors influencing clinical result after renal revascularization were: a generalized atherosclerosis (p less than 0.05), duration of hypertension (p less than 0.01) and the early post-operative response of the blood pressure (p less than 0.01). The overall five- and ten-year actuarial survival probabilities were 85 and 68%, respectively. The most common causes of death were myocardial infarction, stroke and cancer. Cox regression analysis for variables influencing survival indicated that persistence of severe hypertension was the major determinant of late survival (p less than 0.05). Hypertension in females is better tolerated, while younger patients appear to have better results and late survival after surgical treatment.

Emergency and early carotid endarterectomy in patients with acute ischemic stroke selected with a predefined protocol. A prospective pilot study.

AIM: The appropriateness of early carotid endarterectomy (CEA) in patients with acute ischemic stroke is still unsettled. The aim of this study was to verify the safety and feasibility of early CEA in a consecutive series of patients with acute ischemic stroke observed in an emergency Department Stroke Unit. METHODS: During a 24-month study, out of 756 patients with acute ischemic stroke 33 (4.4%) were scheduled for early CEA. Endarterectomy procedures were distinguished according to the time between the onset of stroke and operation as emergency (within 8 hours), early CEA (1-18 days). Patients with impaired consciousness or an infarct larger than 2.5 cm on computed tomographic (CT) or magnetic resonance (MR) scans or both were excluded from surgery. All patients underwent spiral CT, echo-color-Doppler (ECD) sonography, transcranial Doppler (TCD) sonography and, when necessary, MR angiography within 6 hours of admission. No patient underwent conventional angiography. Most patients were operated on under cervical block (CB) anesthesia; general anesthesia (GA) was used only for those with an unstable neurological deficit. Selective shunting was used on the basis of intra-operative transcranial Doppler in patients under GA and the onset or worsening of neurological deficit under CB anesthesia. RESULTS: Of the 6 patients operated on within a median 6 hours after the onset of stroke, 1 (16.5%) had a fatal hemorrhagic transformation of the infarct, while the remaining 5 (83.5%) stopped fluctuating or progressing and had a favourable neurological outcome. Of the 16 patients operated on within a median 36 hours and of the 11 patients operated on within 7 days, none deteriorated after operation. CONCLUSION: Emergency CEA is feasible for acute ischaemic stroke provided that strict selection criteria are applied and the door-to-surgery interval is kept short (within 8 hours). Early CEA for secondary prevention is feasible and safe, confirming that a delayed operation is in most cases unwarranted. Large randomized trials are warranted before implementing emergent and early CEA in routine clinical practice.

Preliminary results of a complete study protocol on synthetic vascular graft healing and its complications.

The microscopic and anatomic features and bacteriologic culture results of different portions of single, explanted dacron synthetic vascular grafts (SVG) were studied together with patient clinical data. With this complete study protocol a better understanding of the healing process and its associated pathology can be achieved. We studied three, amply distanced graft portions from each of five patients (15 total graft portions) undergoing revision for infectious and non-infectious reasons. We divided the SVG portions studied into a Group 1, with high degrees of graft healing and into a Group 2, with both infection-dependent, early healing complications and perigraft chronic inflammatory reaction-dependent, late healing complications. These late healing complications were found dependent upon a host vs graft reaction. This study confirmed in humans the important role of an internal and external fibrotic graft incorporation in the definitive healing of a SVG. A host vs graft reaction was suggested to be an alternative to the frequently cited low virulent infection pathogenesis of late SVG healing complications. A sure definition and treatment of late SVG healing complications will only be established by means of a complete study protocol performed on a large number of explanted SVGs.

Indobufen vs acetylsalicylic acid plus dipyridamole in long-term patency after femoropopliteal bypass.

To compare the effects of indobufen (INB) with those of ASA+dipyridamole (DP) on graft patency, 113 patients undergoing femoropopliteal bypass surgery were randomly and blindly assigned to treatment with INB 400 mg daily or with ASA 900 mg daily plus DP 225 mg daily. Treatment started 2 days before surgery and lasted for 12 months. All patients underwent two angiographic examinations: the first early after surgery (mean 6 days) and the second at the end of the study (mean 368 days). The 1 year cumulative patency rate for INB was 60% higher but not statistically different from the ASA-DP group (53.2%). The relative risk (INB/ASA+DP) calculated by the Mantel-Haenszel test was 0.86 (confidence limits 0.54-1.35). Only the site of operation (above-knee or below-knee) has a significant prognostic value on the fate of the graft.

The role of preoperative Tc-99m HMPAO-labeled leukocyte total-body scans in aortic prosthetic reconstruction.

PURPOSE: This study was performed to evaluate the utility of Tc-99m HMPAO-labeled leukocyte total-body scans (TBLS) for detecting remote septic foci before operation in patients undergoing aortofemoral vascular surgery. MATERIALS AND METHODS: Fifty-eight patients were screened before operation for inflammatory or infective disease, and a clinical score was assigned to each patient. Each patient had TBLSs. The relation between the clinical score and the TBLS result was determined. Patients with positive results of the TBLSs were examined, and the causes of these findings were treated before surgery. RESULTS: Ten of the 58 (17%) patients had a positive result of TBLSs. Of 31 patients with a clinical score of zero, 3 had positive findings of TBLS. No TBLS was positive in the 13 patients with a clinical score of 1. TBLS findings were positive in 7 of 14 of the combined patients with clinical scores of 2, 3, or 4. None of the patients showed signs of prosthetic vascular graft infection during the postoperative follow-up period. CONCLUSIONS: In conclusion, TBLSs can be used before aortofemoral vascular surgery, but only in patients with high clinical scores for inflammatory or infective disease to identify relevant infective foci that could cause vascular graft infection.

Selection for surgery and long-term results in renovascular hypertension.

From 1960 to December 1977, 251 patients with renovascular hypertension (RVH) were observed; 219 were operated upon. Long-term results were assessed in 130 patients. Selection for surgery follows three steps: 1) screening of patients with renal artery disease, which is done by angiography; 2) diagnosis of RVH, which is reached mainly by differential renal function studies and renal vein renin measurements (ancillary methods are intravenous pyelography and sequential scintigraphy; the significance of renin measurements is discussed); and 3) prediction of the results of surgery and choice of technique. An original dynamic test of the authors is employed and reconstruction of the renal artery is the procedure of choice. This is done in the majority of cases by aortorenal bypass grafts using dacron prostheses. Techniques and complications are discussed and comparison with venous autograft is made. Hospital mortality was 3.2%. Overall long-term results were favorable in 78%. Long-term mortality was 6% and occurred mainly in patients who remained hypertensive. Results in atherosclerotic patients are compared with those obtained in fibrous stenoses. Results of renal artery reconstructions were far better than those of nephrectomies and lead us to restrict indications for such a procedure.

Emergency carotid surgery.

The authors discuss the indications for emergency carotid endarterectomy, based on their experience between 1956 and 1975 when 15 patients with completed stroke and internal carotid occlusion (Group I) underwent this operation, and after 1975 when emergency revascularization was performed in 22 patients with unstable neurological deficit (Group II) and 21 patients with TIA's associated with preocclusive internal carotid stenosis (Group III). The good early and late results show that surgery was indicated in these cases. An attempt to identify the patients at high risk of acute ischemia on the basis of clinical or anatomical findings is made to ascertain the physiopathologic patterns of cerebral ischemia.