Uptake kinetics of monoclonal antibodies by human malignant melanoma multicell spheroids.

Detailed uptake kinetics by multicell spheroids of three tumor associated monoclonal antibodies was investigated. The spheroids were established from a human melanoma cell line and the human colon adenocarcinoma cell line HT29 as in vitro models of poorly vascularized micrometastases in vivo. The selected antibodies 96.5, 140.240, and OST15 showed a wide range of reactivity against the melanoma cell but they all had negligible binding with the colon cancer cell. Uptake of the antibodies by small spheroids (about 300 micron diameter) was generally sigmoidal in shape with respect to incubation time, and amount of uptake followed the same trend of immunoreactivity of the antibodies with single cells. The correlation was weaker for spheroids with diameter greater than 500 micron presumably due to the increasing size of the necrotic core. By varying the concentration of the antibodies in the incubation medium from tracer dose (0.2 microgram/ml) to a higher dose (3 micrograms/ml), negligible changes in the amount of antibodies bound with their target spheroids were observed. Nonspecific binding between antibodies and spheroids, however, resulted in proportional increase in uptake.

LaPlace transform analysis of femoral artery Doppler signals: the state of the art.

A followup study was conducted to validate our previous experience with the LaPlace Transform Analysis (LTA) method for processing Doppler ultrasound signals from the common femoral artery to detect significant stenosis of the aorto-iliac segment. The first phase used the same instantaneous mean velocity signal processor as used in the prior study. A comparison of the Doppler examinations with angiograms in 98 legs yielded a sensitivity = 92% and sensitivity = 94% in the identification of 50% or greater stenosis of the aorta-iliac segment, results almost identical to the last study. Because of theoretical disadvantages of using the instantaneous mean velocity signal we carried out a second phase using a peak velocity detector. In 148 limbs sensitivity = 87% and specificity = 98%. The presence or absence of superficial femoral artery occlusion did not affect the accuracy of the waveform analysis in the detection of proximal disease. The LTA parameter related to distal resistance, G, was not found to have clinical value in the assessment of the femoral-popliteal segment.

Differential diagnosis of arterial disease of the lower limb using Doppler ultrasound.

Doppler arterial waveforms from the femoral and ankle arteries of 55 normal control subjects and 76 arteriopaths were analysed for Laplace transform coefficients, delta and omega omicron, and pulsatility index (PI). At the femoral artery delta was superior to PI in accurately detecting mild and moderate aortoiliac stenosis, as assessed from arteriograms. In the femoropopliteal segment, the ratio of omega omicron from femoral to ankle level compared favourably with PI damping factor in the detection of occlusive disease. The Laplace transform system provides a helpful diagnostic method in lower limb arterial disease.

Physiological interpretation of Doppler shift waveforms: the femorodistal segment in combined disease.

A new method is presented for assessing the femorodistal segment in multisegmental arterial disease, using the Laplace transform technique of Doppler waveform analysis. Blood velocity/time waveforms were obtained at femoral and ankle levels in three groups of limbs--50 without arterial disease, 12 with isolated aortoiliac stenoses, and 32 with femoropopliteal occlusions, with and without proximal disease. The waveforms were analysed for Laplace transform and pulsatility index values. The omega 0 coefficients of the Laplace transform analysis at femoral and ankle levels were compared in each subject, as the omega 0 gradient (femoral/ankle omega 0): and pulsatility index damping factor (femoral/ankle P1) was also calculated. The omega 0 gradient was shown to detect femoropopliteal occlusion in the presence of multisegmental arterial disease and to give some indication of its haemodynamic significance. The diagnostic accuracy of the omega 0 gradient was superior to that of pulsatility index damping factor. When combined with its existing ability to detect aortoiliac stenosis, this new application of the Laplace transform method offers the possibility both of a system for complete localisation of significant arterial lesions, and potential for follow-up of vascular surgical procedures in the lower limb, from two simple Doppler recordings.

Angioplasty gives good results in critical lower limb ischaemia. A 5-year follow-up in patients with known ankle pressure and diabetic status having femoropopliteal dilations.

137 consecutive patients with known ankle pressures and diabetic status had attempted femoro-popliteal dilatation for lower limb ischaemia in an English provincial teaching hospital. All except one were followed until failure or death to assess survival and amputation rates. Non-diabetic patients with critical limb ischaemia had a 5 year survival rate of 62.2% (SE 17.1) compared to 50.5% (SE 7.0) for claudicants, with no significant difference on logrank testing. Diabetics had a relative risk of amputation of 11.2 compared to nondiabetics. Patients with pre-treatment ankle pressures of 50 mm or less had a relative risk of amputation of 2.6 compared to those with higher resting pressures. It is concluded that angioplasty should be the treatment of first choice in critical lower limb ischaemia whenever it is technically possible. Including patients with rest pain in the critical ischaemia group does not significantly affect cumulative patency rates.

Complications in percutaneous transluminal angioplasty: relationships with patient age.

Five hundred consecutive attempted lower limb angioplasties for ischaemic disease (370 patients, mean age 65.6 years, range 33-91 years) were reviewed. Significant complications occurred in 44 cases (8.8%). Nine patients (1.8%) underwent emergency surgery related to a complication. A further 12 patients (2.4%) underwent elective surgery related to a complication. In addition, four patients died within 30 days of the procedure; one following surgery performed because of a complication of angioplasty, one following a myocardial infarction, one following severe bleeding associated with subsequent thrombolytic therapy and one during emergency surgery related to a complication of angioplasty. A correlation was found between complication rate and age. This relationship was independent of the approach to, the position of, and the severity of the treated lesion. Elderly patients are at increased risk of complication in lower limb angioplasty.

Detection of arterial stenosis: increased accuracy using biplanar angiography and Doppler signal analysis.

Femoral artery Doppler signals were analysed from 68 patients with lower limb ischaemia undergoing biplanar angiography. These studies (pulsatility index, P.I. and LaPlace transform damping, LTD) were compared with clinical examination using this biplanar angiographic standard. Accuracy of femoral pulse palpation in predicting upstream stenosis was 67%. Biplanar angiography unmasked single plane stenosis underestimates in 21% of patients. In a subgroup of 39 lower limbs with aorto-iliac stenoses between 26 and 85% femoral LTD was the best predictor, being 84% accurate: whereas PI did not improve upon clinical examination. The need for biplanar angiography is underlined and the place of Doppler signal analysis in the assessment of arterial stenosis is confirmed.

A 5-year review of carotid endarterectomy in a vascular unit using a computerised audit system.

BIPAS, a computerised vascular audit has been used to analyse the results of 203 carotid endarterectomies performed over a 5-year period in a vascular unit. In addition, all but two patients have been followed up with regular duplex scans. The indications and surgical techniques have remained similar over the study period though preoperative carotid arteriography is no longer considered essential and intraoperative monitoring with transcranial Doppler insonation is becoming routine. There were six perioperative deaths and 20 postoperative neurological defects. However, only three survivors had any long-term disability. It was not possible to identify any particular patients at high risk of perioperative stroke, although simultaneous major surgery and significant bilateral carotid endarterectomy seemed to be more hazardous. Routine follow-up using duplex scanning identified patients with late occlusion (5%) and restenosis (8%), but only three patients (1.5%) suffered a late stroke. Once the perioperative hazards of death and permanent stroke (4.4% in this series) have been overcome, carotid endarterectomy provides good protection against subsequent stroke.

Computerised audit of carotid endarterectomy: audit loopholes closed?

The number of carotid endarterectomies being performed in the UK is increasing. The role of carotid endarterectomy (CEA) in the prevention of stroke depends on the procedure being associated with as few operative strokes as possible. Good clinical practice, with minimum morbidity, depends upon the integration of recent advances. Continuing audit has been used to examine changes in surgical practice and in case mix. There was a combined death and permanent stroke rate of 3.6% after 333 CEAs in a 6-year period (1990-1995) compared with 4.4% in 203 CEAs in an earlier 5-year audit period (1985-1989). The impact of an increase in the number of operations performed after recovered strokes, those performed by trainees and the use of prosthetic patches on the results of CEA has been assessed. Specific areas to be targeted in future audits are identified.

A cluster of keratin-associated proteins on mouse chromosome 10 in the region of conserved linkage with human chromosome 21.

A gene cluster of three to five high-cysteine keratin-associated proteins (KAPs) has been identified on mouse Chromosome 10 (MMU10) in the region of conserved linkage with human chromosome 21 (HSA21). One of these genes, Krtap12-1, has been sequenced in its entirety and shown to be an intronless gene encoding a predicted 130-amino-acid protein. Krtap12-1 is most closely related to two previously identified KAP4 genes, but variation in sequence and cysteine content suggests that it represents a new KAP family. Krtap12-1 is expressed in the skin of a 3-day-old mouse. The corresponding region of HSA21, between ITGB2 (integrin beta2) and PFKL (the liver isoform of phosphofructokinase), has proven refractory to cloning, and thus mapping of this region at high resolution has been problematic. Based on the KAP gene cluster position in mouse, evidence has been found for an orthologous human KAP cluster on HSA21q22.3, reinforcing the observation that comparative genomics can play an essential and practical role in determining mammalian genome organization.

The efficacy of graduated compression stockings in the prevention of deep vein thrombosis after major gynaecological surgery.

The efficacy of graduated compression stockings in the prevention of deep vein thrombosis (DVT) after major gynaecological surgery was investigated in a controlled randomized prospective trial in 196 patients who were greater than 35 years of age. The stockings were worn by 104 of the 196 patients throughout their stay in hospital, the other 92 patients did not wear the stockings (control group). All the patients were scanned for DVT postoperatively with the 125I-labelled fibrinogen test. None of the 104 patients who wore the stockings developed a thromboembolism, but four of the 92 control patients who did not wear the stockings had DVT. This difference between the two groups was statistically significant.

Physical mapping of the evolutionary boundary between human chromosomes 21 and 22 on mouse chromosome 10.

Adjacent regions of mouse Chromosome 10 (MMU10) show conserved synteny with human chromosome 22 (HSA22) and the telomeric region of HSA21. Physical mapping on MMU10 using YAC fragmentation and PAC contig analyses demonstrates that Prmt2 has a position consistent with its human homolog, HRMT1L1, being telomeric to S100B on HSA21. This result establishes Prmt2 as the new proximal boundary of the region of conserved synteny between MMU10 and HSA21 and predicts that it is the most telomeric gene known on HSA21. Physical mapping refines the positions and order of HSA22 homologs Mmp11, Mif, and Ddt, demonstrates the orientation of S100b on the mouse chromosome, and localizes the junction of conserved synteny between HSA21 and HSA22 on MMU10. Comparative mapping in this region is important for defining gene structure and dosage imbalance in Down syndrome (DS), for developing animal models of DS, and for understanding processes of chromosome evolution.

The clinician and the vascular laboratory in the diagnosis of aortoiliac stenosis.

Two hundred and twenty-one lower limbs in 116 arteriopaths were examined for evidence of aortoiliac stenosis, by analyses of common femoral artery Doppler waveforms for pulsatility index (PI) Laplace delta, and principal components, pulse volume recordings (PVR) and thigh pressure measurements. Clinical assessment of the aortoiliac segment was also recorded in 124 limbs. The accuracy of prediction of aortoiliac stenosis, measured on biprojectional arteriograms, was calculated for the different methods, using multiple regression analysis. Considering all grades of stenosis together, clinical judgement was the best predictor of severity (R2 - the correlation coefficient squared = 0.66): addition of non-invasive tests increased the accuracy of prediction to R2 = 0.70. For stenoses greater than 50 per cent PVR amplitude was the best non-invasive test (R2 = 0.39), but was no better than clinical assessment (R2 = 0.40). However, for stenoses less than 50 per cent clinical judgement was poor (R2 = 0.13), and derived most benefit from addition of Laplace delta (R2 = 0.20) or PVR upstroke time (R2 = 0.26). In addition, common femoral PI and Laplace delta were compared in the arteriopaths, and in 146 limbs of normal control subjects, by calculation of sensitivity and specificity. PI was superior to Laplace delta for detecting severe stenoses and occlusions, but Laplace delta was more accurate in the detection of lesser degrees of disease. These results suggest that conventional clinical assessment is the best method for detecting aortoiliac stenoses greater than 50 per cent, but for less severe disease addition of vascular laboratory tests is helpful - especially PVR and Laplace waveform analysis.

Overexpression of S100beta in transgenic mice does not protect from serotonergic denervation induced by 5,7-dihydroxytryptamine.

Transgenic mice overexpressing S100beta were used to examine whether the chronic elevation of this protein alters the response to selective partial serotonergic lesions produced by bilateral intracerebroventricular injections of 5,7-dihydroxytryptamine (5,7-DHT). Basal levels of S100beta mRNA examined by in situ hybridization were two- to threefold higher throughout the brain in transgenic than in control mice, whereas 5-HT levels in forebrain were similar in both. After the 5,7-DHT-induced lesions, no differences were found in the S100beta mRNA levels in either normal or transgenic mice. At 5 and 60 days after the lesion, forebrain 5-HT levels were reduced by 56% and 35%, respectively, in control mice and by 51% and 35%, respectively, in the transgenic mice. Analysis of the 5-HT immunostaining showed a marked decrease of the immunoreactivity in various brain regions, which was comparable at the two intervals postlesion. One exception was the medial hypothalamus, where an almost complete disappearance of 5-HT immunoreactivity was observed in the medial region at 5 days after lesion, followed by a marked reinnervation 60 days later. These hypothalamic changes were seen in both controls and S100beta-overexpressing transgenic mice. Quantitative analysis of the density of 5-HT transporter sites using [(3)H]citalopram binding, a marker of serotonergic terminals, showed a marked decrease in different brain regions at both 5 and 60 days after 5,7-DHT injections. No difference in basal and postlesion levels of [(3)H]citalopram binding was seen between transgenic and control mice. In conclusion, this study demonstrates that constitutive overexpression of S100beta in transgenic mice does not modify serotonin levels during development, nor does it protect the serotonergic neurons from selective neurotoxicity or modify the serotonergic sprouting induced by partial lesion.

The role of balloon angioplasty in the management of lower limb ischaemia.

Three hundred and twenty-three angioplasties (159 iliac; 164 femoro-popliteal) were performed for lower limb ischaemia on 253 consecutive patients from 1980-86. The mean resting ankle/brachial pressure index (ABPI) was increased at one month in open angioplasties as follows: Femoro-popliteal 0.56-0.82; iliac with open superficial femoral artery (SFA) 0.7-0.95; iliac with occluded SFA 0.52-0.63. Successful angioplasty virtually abolished the post-exercise fall in ABPI except for patients having iliac dilations when multisegment disease was present. At 5 years, cumulative patency was 72% for iliac angioplasty and 53% for femoro-popliteal angioplasty. The results of angioplasty were compared with operative arterial reconstructions during 1985. Eighty-eight (91%) of 96 surgical bypasses were alive and patent at hospital discharge and 44 (71%) of 62 angioplasties were patent at 1 month. There were many fewer complications with angioplasty which involved a much shorter hospital stay (2 days vs 16 days).

Role of duplex scanning in the selection of patients for carotid endarterectomy.

A retrospective review of 63 patients undergoing duplex scanning and angiography for suspected carotid artery disease was performed to evaluate the need for routine angiography before carotid endarterectomy. A consultant surgeon (M.H.) made a simulated management decision on the basis of a clinical summary and a duplex scan report. Twenty-four patients were selected for surgery without angiography; duplex scanning had a sensitivity of 100 per cent and a specificity of 90 per cent in the detection of internal carotid artery stenosis. In two cases duplex scanning misdiagnosed a total occlusion as a critical stenosis. Eighteen patients failed to meet the criteria for surgery and were referred for angiography. Twenty-one patients were selected for conservative treatment on the basis of the duplex scan report. Combining the surgical and conservative groups (45 patients), duplex scanning had a sensitivity of 96 per cent and specificity of 95 per cent for the detection of stenosis greater than 50 per cent. In the identification of a total occlusion, duplex scanning had a poor sensitivity of 50 per cent. These results suggest that routine angiography before carotid endarterectomy is unnecessary in selected patients but that a suspected occlusion should be confirmed by angiography.

Fate of patients undergoing transluminal angioplasty for lower-limb ischemia.

A prospective study of 370 patients who underwent 500 percutaneous transluminal angioplasties (PTAs) for lower-limb ischemia over a 7-year period was performed. A 97% follow-up rate was achieved. The first PTA was successful in 188 patients (51%). Of the failures, 31% were failed attempts at dilation and 73% occurred within 1 month of intervention. Of the patients with failed PTA, 39% underwent bypass surgery and 24% underwent amputation. The 30-day mortality rate was 3%, with 1% of the deaths attributed to PTA. The survival rate at 5 years for the successes was double that for the failures (P less than .0005). The best results were in femoropopliteal stenoses with two or three patent calf vessels (cumulative patency rate, 78% at 3 years) and the worst in femoropopliteal occlusions with one or no patent calf vessels (cumulative patency rate, 25% at 3 years). Log rank tests on the life-table data were used to show factors favoring a good outcome. It is concluded that PTA is the treatment of first choice in suitable patients and, although the failure of intervention in critical ischemia has a significant risk, it is a valuable addition to the therapeutic options in patients with little chance of surgical treatment.

Percutaneous transluminal angioplasty for lower limb ischaemia.

Forty successful percutaneous transluminal angioplasties (PTA) were performed in the iliac and femoropopliteal segments of 33 patients with lower limb ischaemia. There was immediate symptomatic relief in 37 limbs (92 per cent) although 7 relapsed and 5 patients required reconstructive arterial surgery within a month of PTA. Objective testing showed that the longer term relapse rate (median follow-up 12 months) was low (10 per cent). Despite a significant incidence of early complications and relapse, PTA provided a good long term result in the majority of patients treated.