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Anti-endothelial antibodies and coronary artery disease after cardiac transplantation

Accelerated coronary artery disease is the most serious complication after cardiac transplantation. The disease has a multifactorial aetiology, with little agreement about the relative importance of the various risk factors. We have investigated the frequency of antiendothelial antibodies against human umbilical vein endothelial cells by one-dimensional sodium

dodecyl sulphate polyacrylamide gel electrophoresis and western blotting. Peptide-specific antiendothelial antibodies were found in 15/21 heart transplant recipients with accelerated coronary artery disease, and 1/20 transplant patients who had not developed the disease. Positive immunofluorescence of patients’ serum on frozen sections of coronary vessels confirmed the endothelial specificity of antibodies. These results provide evidence of an immune aetiology for transplant-associated coronary artery disease and could have important implications for its diagnosis and therapy. Introduction Accelerated or transplant-associated coronary artery disease (CAD) is the most serious complication after cardiac transplantation,! affecting 6% of our patients at 1 year and progressing to 17% after 3 years. Other centres have reported an incidence of 18% at 1 year and 44% at 3 years.2 Although there are clearly several possible risk factors (numbers of acute rejection episodes, type of immunosuppression, serum lipid concentrations, viral infection), there is little agreement about their relative

importance.3 The integrity of the endothelium is recognised as being a crucial factor in maintaining normal vessel function; endothelial injury is probably the earliest event that initiates all forms of arteriosclerosis.4 Although there is consensus that in transplant-associated CAD, initial damage to the endothelium is mediated by immune mechanisms, there has been no direct evidence for an immune response against graft or vessel components. Anti-endothelial antibodies can be highly destructive—eg, they cause rapid rejection of xenografted organs.5 Here we use a technique of onedimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting to show the occurrence of peptide-specific anti-endothelial antibodies in heart transplant recipients with accelerated coronary artery disease; these are rare in transplant patients who have not developed the disease. Patients and methods Patients All (41) patients had received orthotopic heart transplants between March, 1987, and July, 1989. They were selected in chronological order of transplantation by the following criteria:

survival for at least 2 years; availability of angiographic data at 1 and 2 years after transplantation; and availability of serum samples collected before transplantation and at the time of angiography. Patients were divided into those who had no evidence of CAD at 1 or

2 years (20 patients;

17 male, 3 female; mean age 49, range 16-60)

and those who had evidence of CAD at 2 years (21 patients; 19 male, 2 female; mean age 42, range 22-66). CAD was found in 12 patients at 1 year and in all 21 at 2 years. The original type of heart disease in the patients who did not develop CAD was ischaemic heart disease (6), ischaemic cardiomyopathy (1), dilated cardiomyopathy (6), postpartum cardiomyopathy (1), congenital heart disease (3), valvular disease (1), and viral myocarditis (2). In patients who developed CAD, the original heart disease was ischaemic heart disease (12), ischaemic cardiomyopathy (3), dilated cardiomyopathy (3), postpartum cardiomyopathy (1), congenital heart disease (1), and valvular disease (1). Serum was taken before transplantation and at 1 and/or 2 years post-transplant at the time of the patient’s annual assessment. The presence of accelerated CAD was diagnosed by angiography at annual assessment and was defined as at least 25% luminal stenosis of one or more coronary arteries or loss of small intramyocardial branches.

Cell culture Human umbilical cords were harvested at delivery and processed within 72 h. Isolation and culture of endothelial cells were based on the method of Jaffe et al.6 Cells were cultured in M199 containing 20% v/v fetal calf serum and 10 ng/ml endothelial cell growth factor. Cells were used for experiments at either second or third passage. Cells of the human epithelial cell line A549 (Flow Laboratories, High Wycombe, UK) were cultured in Dulbecco’s modification of Eagle’s medium containing 10% v/v fetal calf serum. Endothelial and epithelial cells were stimulated with 200 U/mly-interferon (Genzyme, West Malling, UK) for 4 days.

Total cell protein samples Confluent cell layers were washed with phosphate-bufferedsaline (PBS), lysed by the addition of 200 gel of 1% w/v sodium dodecyl sulphate (SDS), and harvested by scraping. To digest DNA and RNA, the cellular material was incubated on ice with 20 µl of a solution containing 1 mg/ml DNAase I (Boehringer Mannheim, Lewes, UK), 500 µg/ml RNAase A (Boehringer Mannheim), 05 mol/1 "tris"-HCI, pH 70, and 50 mmol/1 MgC12 for 30 min. Protein samples were stored frozen at - 80°C.

SDS-PAGE and western blotting Protein concentration of samples was measured by the dyebinding procedure of Bradford.7 Samples (25 µg) were solubilised by heating at 100°C for 2 min in 50 pl sample buffer.8 Cell proteins were separated on 7 cm long 10%T SDS-PAGE gels, with 3%T stacking gel, at 60 mA per gel for about 2-5 h at 15°C until the bromophenol blue tracking dye reached the end of the gel. Before electroblotting, gels were equilibrated for 30 min in transfer buffer (20 mmol/1 "tris" base, 150 mmol/1 glycine). Proteins were electrophoretically transferred onto nitrocellulose (Hybond C Super, Amersham, Aylesbury, UK) at 500 mA for 60 min.

Anti-endothelial antibodies Nitrocellulose strips with the separated endothelial or epithelial cell proteins were incubated for 1 h with 3% w/v non-fat dried milk ADDRESS: Department of Cardiothoracic Surgery, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK (M. J. Dunn, PhD, S. J. Crisp, BSc, M. L. Rose, PhD, P. M. Taylor, PhD, Prof M. H. Yacoub, FRCS). Correspondence to Dr Michael J. Dunn.

Fig 1-Western blots probed with 2-year serum samples from patients with CAD and developed with anti-IgM antibodies. 62and60kDa bands are indicated. (a) Unstimulated (left track) and gamma-interferon stimulated (right track) human umbilical vein endothelial cells. This serum is only reactive with the doublet. (b) Unstimulated (left track) and gamma-interferon stimulated (right track) human umbilical vein endothelial cells. This serum is reactive with other bands in addition to the doublet. (c) Human umbilical vein endothelial cells (left track) and A549 epithelial cells (right track). No reactivity with proteins of 62 and 60 kDa is observed in the A549 cells. (d) Umbilical vein endothelial cells probed with pooled normal human serum showing no bands of reactivity. The scale at the right indicates molecular weight x 103 kDa. in PBS containing 0-05% w/v Tween 20 (PBS-Tween) to block non-specific protein binding sites. Strips were then incubated with patient’s serum (pretransplant, 1 year and 2 year post-transplant samples), diluted to 1 in 200 in blocking solution, and agitated

vigorously for 1 h. Pooled normal human serum was used as a negative control. After thorough washing in PBS-Tween, strips were incubated for a further 1 h in either peroxidase-conjugated rabbit anti-human IgG (Dako) or peroxidase-conjugated rabbit anti-human IgM (Dako) at a dilution of 1 in 1000. The strips were then washed four times in 10 ml and twice in 200 ml PBS. Extensive washing at this stage was essential to minimise background activity. Protein bands to which circulating antibodies had bound were visualised with an enhanced chemiluminescence (ECL) detection system (Amersham International, Aylesbury, UK),9 which is much more sensitive than the system based on diaminobenzidine, which we have previously described. 10 Blots were incubated with the ECL detection reagent, according to the manufacturer’s instructions, for 1 min and exposed to Hyperfilm ECL for 30 s to 1 min. Films were developed with an automated radiograph developer.

Immunofluorescence Normal and diseased coronary arteries

were

removed from the

explanted heart at the time of transplantation, frozen immediately, and stored in liquid nitrogen until use. Normal atrium was obtained from the donor organ as "atrial trimmings" and stored in liquid nitrogen. Patients’ serum or control serum (from pooled normal

donors) was diluted from 1 in 2 to 1 in 20 and added to 6 um cryostat sections of normal or diseased coronary artery or normal atrium. Serum was incubated on the tissue for 30 min at room temperature. Sections were washed with PBS and incubated with fluoresceinconjugated rabbit anti-human IgG or IgM (Dako) for 30 min. Sections were washed again with PBS, mounted in Uvinert aqueous mountant (Merck, Poole, UK), and examined with a Zeiss Axiophot incident-light fluorescence microscope with a filter specific for fluorescein isothiocyanate (FITC).

Fig 2-Western blots of human umbilical vein endothelial cell proteins probed with 2-year serum samples from patients without CAD and developed with anti-IgM antibodies.

(a) Example of serum showing no bands of reactivity. (b) Serum sample showing bands of reactivity, but the 62 and 60 kDa doublet is (c) Umbilical vein endothelial cells probed with pooled normal human serum showing no bands of reactivity. The scale at the right indicates molecular weight x 103 kDa. not observed.

Human umbilical vein endothelial cells were cultured on glass coverslips and then fixed with 2% paraformaldehyde. Coverslips were washed in PBS, then incubated with patients’ serum (diluted 1 in 10) or pooled normal human serum (diluted 1 in 5) for 1 h at 4°C. Coverslips were washed with PBS and incubated with fluoresceinconjugated rabbit anti-human IgM for 1 h at 4°C. Specimens were washed again with PBS, mounted, and examined as described above.

Results Western blotting Patients with CAD-20/21 samples had bands of reactivity against proteins of cultured endothelial cells (fig la, lb). In 15 of these samples, reactivity with a doublet of protein bands of 62 and 60 kDa was observed. In 4 cases, reactivity was only against the 62 and 50 kDa doublet (fig la), and in the other 11cases additional bands of reactivity were also observed (fig lb). In 10 cases, the reactivity was IgM only, in 1 case IgG only, and in the other 4 cases both IgM and IgG. These antibodies were present in 3 patients before transplantation and were IgM only. This doublet of reactivity was not found when patients’ serum was tested against proteins from cultured A549 epithelial cells (fig lc, right-hand track). No differences were observed in endothelial cells stimulated with gamma-interferon (fig 1). No bands of reactivity were observed when pooled normal human serum was tested against endothelial cell proteins (fig ld). Patients without CAD-9120 post-transplant samples had reactivity against proteins from cultured endothelial cells (fig 2b). In the other 11 patients, no bands of reactivity were detected (fig 2a). In only one sample was there activity

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Fig 4-Reactivity with human atrium.

(a) Frozen section of human atrium incubated with a monoclonal antibody agamsttheCD31 endothelial cell marker Note the network of staining of capillaries (b) Similar section incubated with a patient’s serum showing strong IgM reactivity with endothelial cell proteins by western blotting Reactivity is confined largely to nuclear membranes with very little binding to capillaries when

developed with FITC anti-human IgM (figs 3a, 3b). Activity was clearly against endothelial cells lining the coronary artery; the media was also brighter than in the

Fig 3-IgM reactivity

in coronary artery

specimens.

control sections without human serum. Pooled normal human serum together with serum samples from patients without CAD did not show immunofluorescent staining of endothelial cells in coronary arteries (figs 3c, 3d) and neither did serum with anti-endothelial activity that lacked the specificity for the peptide doublet. Sera with IgG reactivity did not show more fluorescence in coronary arteries than the

Cryostat sections of normal (a, c) and diseased (b, d) coronary artery incubated with a patient’s serum showing !gM reactivity with the 62 and 60 kDa doublet by western blotting (a, b) or pooled normal human serum (c, d). Note the positive fluorescent labelling (arrows) of the endothelial cell layer lining the artery in (a) and (b). No staining was observed with the pooled normal human serum (c, d)

against protein bands of 62 and 60 kDa. Anti-endothelial reactivity was detected in pretransplant sera in only 1 patient. No bands of reactivity were observed when pooled normal human serum was tested against endothelial cell proteins (fig 2c). Immunofluorescence To test whether the antibodies react with intact endothelial cells from coronary arteries, patients’ serum was added to frozen sections of normal and diseased coronary arteries and binding was detected by addition of fluorescent labelled anti-human immunoglobulin. Serum from 5 patients with CAD was selected on the basis of having strong bands of IgM (in 3 cases), IgG (in 1 case), or IgM and IgG (in 1 case) reactivity against the endothelial proteins of 60 and 62 kDa by western blotting. In addition, serum with IgG reactivity against endothelial cell proteins, but not the 60 and 62 kDa doublet, was tested, from 3 patients without CAD. Thus, 9 sera were tested along with pooled normal serum as a control. All 4 sera with IgM reactivity showed strong fluorescence of endothelial cells in normal and diseased coronary artery

Fig 5-Human umbilical vein endothelial cells.

as were sera

(a) Incubated with a patient’s serum showing IgM reactivity with the 62 and 60 kDa doublet by western blotting. Strong punctate staining of the cell surface is observed (b) Incubated with pooled normal human serum. Only weak staining of the cell surface is apparent.

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control slide without serum. However, background staining of FITC anti-human IgG was high in coronary arteries. To test whether patients’ sera with strong IgM activity against coronary endothelial cells also had reactivity against microvascular endothelial cells, sera were tested against frozen sections of human atrium. The positive control was binding of a monoclonal antibody against the CD31 pan-endothelial cell marker (fig 4a), which showed a characteristic network of capillaries throughout the tissue. By contrast, 3 patients’ sera (2 IgM, 1 IgG) did react with the heart, but reactivity was confined largely to nuclear membranes and there was very little binding to capillary endothelial cells (fig 4b). To test whether patients’ sera with strong IgM activity by western blotting against the 60 and 62 kDa proteins were reactive with cell surface antigens of endothelial cells, sera were tested against cultured endothelial cells fixed with paraformaldehyde. Such fixation does not allow access of antibodies to the cytoplasmic compartment, so that any positive staining must be associated with the cell surface. Strong punctate staining of the endothelial cell surface was observed with sera that were positive for the 60 and 62 kDa doublet by western blotting (fig 5a). By contrast, both pooled normal human serum and sera from patients without CAD showed very weak staining of the cell surface (fig 5b).

Discussion Our results show anti-endothelial antibodies in patients with accelerated coronary artery disease after cardiac transplantation and provide evidence of immune involvement in this disease. Several bands of reactivity were found by western blotting against proteins of human umbilical vein endothelial cells, but a doublet of bands at 60 and 62 kDa showed a strong association with CAD (15/21 patients with disease and in only 1 transplant patient without CAD). Although the mean age of the patients is higher in the group who developed CAD, and there were more patients with an original diagnosis of ischaemic heart disease, the observation that anti-endothelial antibodies were only found in 3 patients before transplantation (2 with ischaemic heart disease and 1 with dilated cardiomyopathy) makes it unlikely that these factors affected the production of antibodies. Our data suggest that development of antibodies is associated with development of accelerated coronary artery disease. The naturally occurring human anti-endothelial antibodies, which cause rapid destruction of porcine xenografts, are of IgM class.s Of 15 patients with antibodies against the 60/62 kDa doublet, in 10 the antibodies were IgM only and in 4 they were IgM and IgG. Western blotting of porcine arterial cells has shown that the molecules involved are glycoproteins of 115, 125, and 135 kDa," and the specificity may well reside within sugar residues. Thus, they are different molecules from those that seem to be recognised on allotransplanted endothelium. From the immunofluorescent staining studies of cultured endothelial cells fixed with paraformaldehyde, it is clear that patients’ sera that are positive for the 60 and 62 kDa doublet by western blotting are reactive with antigens present on the surface of the endothelial cell. Although the molecules involved seem to be highly immunogenic peptides or glycopeptides, they do not correspond in molecular weight to any of the known molecules expressed by the endothelium. Thus, immunocytochemical staining of both normal and diseased coronary arteries in situ has shown that endothelial cells express immunogenic molecules of the

class I

(HLA-A, B, C) and class II (HLA-DR) major histocompatibility complex. 12 In addition, coronary endothelial cells express the well-diaracterised 13 accessory adhesion molecules, PECAM, ICAM-1, and VCAM, 12 which facilitate leucocyte adhesion and emigration. The 60/62 kDa doublet is present on normal endothelial cells, since blots of cytokine-activated cells did not produce different results. Direct microsequencing of protein bands from western blots is under way to identify the 62 and 60 kDa proteins. The strong immunofluorescent staining of frozen sections of coronary artery endotheliuim with sera from 4 patients showed that the activity was against coronary endothelium and not only umbilical vein cells. Our results to date suggest that reactivity is against epicardial coronary artery endothelial cells and not microvasculature. In particular, the observation that only sera with activity against the 60 and 62 kDa bands reacted with coronary endothelial cells suggests that these peptides may be specific for large vessels. Immunocytochemical studies have shown heterogeneous expression of endothelial antigens between large vessels and microvasculature. 12 This finding could explain the distribution of the disease, which seems to be limited to the epicardial and larger myocardial vessels and spares or

capillaries. Clinically accelerated coronary artery disease is difficult to diagnose. The denervated heart prevents anginal symptoms and the diffuse concentric distribution of the lesions can obscure angiographic evidence of stenosis. T cells have been described immunocytochemically beneath the endothelium in atherosclerotic plaques from patients with accelerated coronary artery disease.14 It is likely, however, that T cells invade the endothelium at an early stage of the disease long before there is angiographic evidence of abnormalities. This method of detecting antibodies can be modified to an enzyme-linked immunosorbent assay technique15 that can process large numbers of samples. Patients should be monitored at regular intervals after transplantation to provide information about the natural history of CAD, which may be of prognostic and therapeutic value. This work is supported by the British Heart Foundation and S. J. C. is in receipt of a BHF Studentship. We would like to thank Mrs C. Lovegrove for assistance with preparing the photographic illustrations. We are grateful to the staff of the Princess Alexandra Hospital, Harlow, for supplying us with umbilical cords.

REFERENCES 1. Banner NR, Fitzgerald M, Khaghani A, et al. Cardiac transplantation at Harefield Hospital. In: Terasaki P, ed. Clinical transplants. Los Angeles: UCLA Tissue Typing Laboratory, 1987: 17-26. 2. Uretsky BF, Murali S, Reddy PS, et al. Development of coronary artery disease in cardiac transplant patients receiving immunosuppressive therapy with cyclosporine and prednisone. Circulation 1987; 76: 827-34. 3. Gao S, Hunt SA, Schroeder JS. Accelerated transplant coronary artery disease. In: Yacoub MH, Loop FD, eds. Seminars in thoracic and cardiovascular surgery. Philadelphia: Saunders, 1990: 241-49. 4. Ross R. The pathogenesis of atherosclerosis: an update. N Engl J Med

1986; 314: 488-500. 5. Auchincloss J Jr. Xenografting: a review. Transplant Rev 1990; 4: 14-27. 6. Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. J Clin Invest 1973; 52: 2745-56. 7. Bradford MB. A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 1976; 72: 248-54. 8. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227: 680-85. 9. Simmonds J, Price R, Corbett J, Dunn MJ. Enhanced chemiluminescence detection of Western blotted proteins from twodimensional SDS PAGE. In: Dunn MJ, ed. 2-D PAGE ’91: proceedings of the international meeting on two-dimensional

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electrophoresis. London: National Heart and Lung Institute, 1991: 46-48. 10. Dunn MJ, Rose ML, Latif N, et al. Demonstration by Western blotting of antiheart antibodies before and after cardiac transplantation. Transplantation 1991; 51: 806-12. 11. Platt JL, Lindman BJ, Chen H, Spitalnik SL, Bach FH. Endothelial cell antigens recognised by xenoreactive human natural antibodies. Transplantation 1990; 50: 817-22. 12. Page C, Rose ML, Yacoub MH, Pigott R. Antigenic heterogeneity of vascular endothelium. Am J Pathol (in press). SHORT REPORTS

Prediction of normal-tissue tolerance to radiotherapy from invitro cellular radiation sensitivity

The success of radiotherapy depends on the total radiation dose, which is limited by the tolerance of surrounding normal tissues. Since there is substantial normal-tissue variation among patients in radiosensitivity, we have tested the hypothesis that in-vitro cellular radiosensitivity is correlated with in-vivo normal-tissue responses. We exposed skin fibroblast cell lines from six radiation-treated patients to various doses of radiation and measured the proportions surviving. There was a strong relation between fibroblast sensitivity in vitro and normaltissue reactions, especially acute effects. Assessment of radiosensitivity could lead to improved tumour cure rates by enabling radiation doses to be tailored to the individual.

is the most important non-surgical in cancer and success depends mainly on the total radiation dose. This dose is limited by the tolerance of normal tissues surrounding the tumour. There is wide variation among patients in normal-tissue tolerance and hence in reactions to the same curative dose.12 Severe damage to normal tissue can cause substantial morbidity and may even be life-threatening. Selection of appropriate radiation dose is based on a balance between keeping the rate of severe normal-tissue complications acceptably low and increasing the probability of local control. Much of the variation in normal-tissue reactions is due to differences in sensitivity, rather than other factors such as uncontrolled differences in delivered dose.2-4 Identification of individuals with the most sensitive normal tissues might allow the dose to be increased in the majority of patients, which in turn might increase local control and cure.5,6 Improved local control might also reduce the incidence of metastatic disease.7 We report the preliminary results of a study that aims to develop a predictive assay of normal-tissue response. We tested the hypothesis that in-vitro cellular radiosensitivity is correlated with in-vivo normal-tissue response .8 We studied fibroblasts, which have a wide range of in-vitro radiosensitivity,9 and are the only normal cell type amenable to mass culture.

Radiotherapy

treatment

13.

Springer TA. Adhesion receptors of the immune system. Nature 1990; 346: 425-34.

RN, Hughes CCW, Schoen FJ, et al. Human coronary transplantation associated arteriosclerosis: evidence for a chronic immune reaction to activated graft endothelial cells. Am J Pathol 1991;

14. Salomon

138: 791-98. 15. Heurkens AHM, Gorter A, de Vreede TM, et al. Methods for the detection of anti-endothelial antibodies by enzyme-linked immunosorbent assay. J Immunol Methods 1991; 141: 33-39.

The subjects were selected from those in the Gothenburg fractionation trials2-4 who were receiving radiotherapy after mastectomy for early breast cancer. All had parasternal nodal irradiation, with direct anterior fields; skin doses were measured and skin changes afterwards were observed. Reflectance spectrophotometry was used to measure peak acute skin reactions (erythema) at the time of radiotherapy, and late changes were quantified in terms of the degree of telangiectasia. We selected patients with a range of late normal-tissue reactions from no telangiectasia (grade 0) to confluent telangiectasia (grade 5; group CRE IVb2). The whole group received the same treatment regimen in 1978-79, so follow-up is longer than 10 years, which is necessary in view of the progression with time of late skin changes. 3,4 At first, ten fibroblast strains were established from six patients with the full range of skin changes; duplicates were produced for four. Clonogenic assays were used to assess intrinsic cellular radiosensitivity. Fibroblasts were grown to confluence in Dulbecco’s modified Eagle’s minimum essential medium plus 10% fetal calf serum, then plated out 4 h before irradiation with Cobalt-60 y-rays at low dose rate (LDR; 0.01 Gy/min) and high dose rate (HDR; 1-2 Gy/min). LDR has the practical advantage of increasing small differences between cell survival curves, and a theoretical advantage of more closely simulating clinical fractionated radiotherapy, at least for tumours.1o A range of doses was chosen to produce a maximum of 3 orders of magnitude of cell kill (0-8 Gy at HDR and 0-12 Gy at LDR). Survival curves were fitted by non-linear regression with the linear quadratic model. All experiments were carried out without knowledge of the clinical response or of which strains were duplicates.

Features of the survival curves for the ten strains at LDR summarised in the table. The dose required to reduce the surviving fraction to 0.01 (DO.01) ranged from 6.43 to 8.12 Gy. All the strains showed almost linear survival curves, shown by the low values of 0. In the figure, the ranking of the strains’ sensitivity at LDR is related to rank of acute or late normal-tissue reactions. There was a correlation between in-vitro fibroblast cellular sensitivity and in-vivo tissue response, stronger for acute than for late reactions. Statistical analysis is difficult with so few subjects. However, there is a striking relation between in-vitro are

CELL SURVIVAL DATA FOR TEN FIBROBLAST STRAINS AT LDR I

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*From semi-logarithmic survival curve, surviving fraction=exp (-CLD-&bgr;D2), where D= dose. SF,= surviving fraction after dose of 2 Gy tMean mactivation dose= area under survival curve on linear scale. tDose of radiation to reduce SF to 0 01.

Anti-endothelial antibodies and coronary artery disease after cardiac transplantation.

Accelerated coronary artery disease is the most serious complication after cardiac transplantation. The disease has a multifactorial aetiology, with l...
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