23

distal vessels, such that a reduced pulse can be restoredto normal by a new distal obstruction;6,7 and the clinicalverdict is influenced also by vessel wall calcification oraneurysmal dilation, obesity, thrills, and cardiac

arrhythmias. Great experience and cunning is requiredto gauge the collective effect of serial stenoses andcollaterals, as well as the contribution of contralateralvessels.

Clinical examination can be supplemented byphysiological tests,5,8 of which the simplest is the ankle-brachial systolic pressure index (ASPI)." The ASPI candistinguish normal limbs (>1) from claudicant limbs

(0.5-0’ 9) and those with rest pain (<0’ 5). It is afunctional guide to ischaemia and its overall severity,but pressures higher up the leg are needed to localisediseased segments. For example, a sharp gradientbetween high thigh and calf/ankle pressure indicatespopliteal artery stenosis. Plethysmography shouldlocalise disease in a similar manner, but in practice ismost useful for comparative studies (eg, intraoperativemonitoring of arterial reconstruction’ 0). Aorto-iliacdisease is hard to distinguish preoperatively fromfemoro-distal disease mainly because of thecontribution of stenosis of the profunda artery,proximal to which measurements are impossible. Inclaudicants, stress tests (exercise tests, post-occlusionreactive hyperaemia, and toe pulse reappearance times)tend to correlate better with disease severity than thoseperformed at rest.ll,12When operation is contemplated, most surgeons still

insist on angiography. This traditional technique isrisky in diseased vessels and can give misleadingresults: uniplanar arteriography is notorious for under-estimating stenoses and even biplanar views can missserious obstructions.l’,13 The advent of digitalsubtraction (intravenous) angiographyl4 has provided amethod that is at least safer; and duplex ultrasoundscanning allows non-invasive measurement of function(flow). At one time these techniques seemed to be indirect competition, but their advantages and

disadvantages have proved to be different; in

combination, they will usually supply the informationrequired. Conventional angiography, however, stillhas pride of place, particularly when done in

conjunction with flow or pressure measurements. 15

6. Baisdell FW, Gauder PJ. Paradoxical variation of the femoral pulse in occlusion of theiliac artery. Surgery 1961; 50: 529-32.

7. Keitzer WF, Fry WJ, Kraft RO, De Weese MS. Haemodynamic mechanism for pulsechanges seen in occlusive vascular disease. Surgery 1965; 57: 163-74.

8. Fronek A, Coel M, Bernstein EF. The pulse reappearance time-an index of overallblood flow impairment in the ischaemic extremity. Surgery 1977; 81: 376-81.

9. Dean RA, Yao JSR, Stanton PE, Bergan JS. Prognostic indications in femoro-poplitealreconstructions. Arch Surg 1975; 110: 1287-93.

10 Darling RC, Raines JK, Brener BJ, Austen WG. Quantitative segmented pulse volumerecorder. a clinical tool. Surgery 1972; 72 873-77.

11 Moore WS, Hall AD. Unrecognised aorto-iliac stenosis: a physiologic approach to thediagnosis. Arch Surg 1971; 103: 633-38.

12. Laing SP, Greenhalgh RM. Standard exercise test to assess peripheral arterial disease.Br Med J 1980, 280: 13-16.

13 Berne FA, Lawrence WP, Carlton WH Roentgenographic measurement of arterialnarrowing. A J R 1970; 110: 757-59.

14. Schneidau A. Digital subtraction angiography in ischaemic cerebrovascular disease. BrJ Hosp Med 1984; 32: 176-81.

15. Udoff EJ, Barth KH, Barrington DP, Kaufman SL, White RI. Haemodynamicsignificance of iliac artery stenosis: pressure measurements during angiography.Radiology 1979; 132: 289-93.

Multisegment large vessel ischaemia remains verydifficult to quantify despite advances in dopplerultrasound blood velocity techniques. 16 Doppler signalanalysis has great potential in this area but, as yet, nosingle method of mathematical manipulation ofwaveform data stands out. Site and severity of stenosescan be predicted by these methods but more clinicalevaluation and comparison with other physiologicaltests is needed. At present the techniques are hamperedby unsatisfactory signal processing: once thesedifficulties have been overcome their clinical useshould increase. Ultimately, when doubt remains as towhether aorto-iliac or distal surgery is required, thenexploration at the groin, with direct flow assessment, isthe final arbiter. -

In extracranial carotid artery evaluation duplexdoppler imaging has already won a secure place.Perhaps this kind of assessment at the femoralbifurcation and further down the leg will supersede thebattery of tests now in use. Ultrasound imaging iscertainly the choice in aneurysmal disease; and forthose 20% of patients with concomitant stenoses, flowmeasurement by ultrasound should be helpful beforerecourse to angiography. Its clinical use is alreadybeing reported." Non-invasive tests can provideimportant objective measurements in the

management, as well as the diagnosis, of vasculardisease. Arterial grafts may be imaged and their

patency assessed. The most appropriate amputationlevel can be determined. Measurement of skin blood-flow by radioisotope clearance has awaited develop-ment of labelled material with true mono-exponentialclearance (and so a direct indicator of flow); this is nowprovided by iodoantipyrine solution, and early clinicalstudies have produced excellent results.18 The hope isthat these new techniques will allow earlier treatment,with more salvage and less amputation.

RADIOSENSITIVITY AND THE CLINICIAN

THE existence of differences in cellular radiosensitivity inindividuals with different disorders has importantimplications for the clinician, from the points of view of bothdiagnosis and therapy. The autosomal recessive disorderataxia-telangiectasia (A-T) was the first identified instance ofa syndrome associated with cellular radiosensitivity. Threereports of patients with A-T who died as a consequence ofexposure to conventional therapeutic doses of radiation ledTaylor and co-workers’ to investigate the effects of ionisingradiation on cultured skin fibroblasts from A-T patients.They found that these-cells were about three times moresensitive to the lethal effects of ionising radiation than were

16. Baird RN, Bird DR, Clifford PC, et al. Upstream stenosis: its diagnosis by dopplersignals from the femoral artery. Arch Surg 1960; 115: 1316-22.

17. Lee RE, Aldoori MI, Horrocks M, Baird RN. A comparison of in situ and reversedfemoro-popliteal grafts using doppler ultrasound. Br J Surg (in press).

18. McCollum PT, Spence VA, Walker WF Antipyrine clearance from the skin of the footand lower leg in critical ischaemia. clinical implications. Proc Biol Eng Soc 1985,1-13

1. Taylor AMR, Harnden DG, Arlett CF, Harcourt SA, Lehmann AR, Stevens S,Bridges BA Ataxia telangiectasia: a human mutation with abnormal radiationsensitivity Nature 1975, 258: 427-29.

24

normal cells. This hypersensitivity has been found incultured fibroblasts and lymphoid cells of all A-T patientsstudied so far.2 It clearly indicates that radiotherapy must notbe used to treat the lymphoid and other tumours which are acommon feature of this disorder. Furthermore, it can providean extra diagnostic feature of A-T in cases with doubtfulclinical features. The biochemical3 and cytogenetic4anomalies associated with the radiosensitivity allow prenataldiagnosis in fetuses at risk for A-T.

In A-T the cellular radiosensitivity is unequivocal. A-T is,however, rare in the population, estimates of its frequencyvarying from one in 40 000 to one in 100 000. Over the pastdecade cellular radiosensitivity has also been identified inseveral other disorders, but in these the radiosensitivity is lessclear cut. Either varying results have been obtained indifferent laboratories, or not all individuals afflicted with thedisorders are radiosensitive. One important group in thiscategory comprises the symptom-free A-T heterozygotes,present at a frequency of about 1% in the population, and forwhom epidemiological evidences (which is still controversial)suggests an increased risk of malignant disease. If they areindeed at increased risk, identification of such individualswould be of some importance. Several laboratories

throughout the world have shown that cells from some A-Theterozygotes are radiosensitive.6,7 This degree of

hypersensitivity is, however, much less striking than that inthe affected homozygotes, and some A-T heterozygotes showno abnormality. As yet, therefore, cellular radiosensitivitycannot be used for identification of A-T heterozygotes.Several laboratories are, however, trying to improve thesensitivity and resolution of assays for radiosensitivity, and itis possible that a means of identifying A-T heterozygotes willbe devised.There has been talk of possible radiosensitivity in several

other disorders. Cellular radiosensitivity has been identifiedin some laboratories, but not always confirmed in others, forFriedreich’s ataxia,8,9 familial retinoblastoma,’ multiplesclerosis,14 Rothmund-Thomson syndrome,13,15 tuberous

2. Lehmann AR. The cellular and molecular responses of ataxia-telangtectasta cells toDNA damage In: -Bridges BA, Harnden DG, eds. Ataxia-telangiectasia&mdash;a cellularand molecular link between cancer, neuropathology and immune deficiency.London: John Wiley and Sons, 1982: 83-101.

3. Jaspers NGJ, Scheres JMJC, de Wit J, Bootsma D Ataxia-telangiectasia; a rapiddiagnostic test. Lancet 1981; ii: 473.

4 Giannelli F, Avery JA, Pembrey ME, Blunt S. Prenatal exclusion of ataxia-

telangiectasia In: Bridges BA, Harnden DG, eds. Ataxia-telangiectasia&mdash;a cellularand molecular link between cancer, neuropathology and immune deficiency.London: John Wiley and Sons, 1982: 393-400.

5. Swift M, Chase C Cancer and cardiac deaths in obligatory ataxia-telangiectasiaheterozygotes. Lancet 1983, i: 1049-50

6. Chen PL, Lavin ME, Kidson C, Moss D. Identification of ataxia telangiectasiaheterozygotes, a cancer prone population Nature 1978; 274: 484-86.

7 Paterson MC, Anderson AK, Smith BP, Smith PJ Enhanced radiosensitivity ofcultured fibroblasts from ataxia telangiectasia heterozygotes manifested by defectivecolony-forming ability and reduced DNA repair replication after hypoxic gamma-irradiation. Cancer Res 1979; 39: 3725-34.

8. Lewis PD, Corr JB, Arlett CF, Harcourt SA. Increased sensitivity to gamma irradiationof skin fibroblasts in Friedreich’s ataxia. Lancet 1979, ii: 474-75

9. Chamberlain S, Lewis PD. Studies of cellular hypersensitivity to ionising radiation inFriedreich’s ataxia. J Neurol Neurosurg Psychiatry 1982; 45: 1136-38.

10. Weichselbaum RR, Nove J, Little JB X-ray sensitivity of diploid fibroblasts frompatients with hereditary or sporadic retinoblastoma. Proc Natl Acad Sci USA 1978;75: 3962-64.

11. Arlett CF, Priestley A. Defective recovery from potentially lethal damage in somehuman fibroblast cell strains. Int J Radiat Biol 1983; 43: 157-67.

12. Ejima Y, Sasaki MS, Utsumi H, Kaneko A, Tanooka H. Radiosensitivity of fibroblastsfrom patients with retinoblastoma and chromosome-13 anomalies. Mutation Res

1982; 103: 177-84.13. Paterson MC, Bech-Hansen NT, Smith PJ. Heritable radiosensitive and DNA repair-

deficient disorders in man. In: Seeberg E, Kleppe K, eds Chromosome damage andrepair. New York Plenum Press, 1981 335-54.

14. Gipps E, Kidson C. Ionising radiation sensitivity in multiple sclerosis. Lancet 1981; i:947.

15. Smith PJ, Paterson MC. Enhanced radiosensitivity and defective DNA repair incultured fibroblasts derived from Rothmund Thomson syndrome patients.Mutation Res 1982; 94: 213-28.

sclerosis, 16 and Huntington’s disease (HD).13,17,18 With HDthere were indications that cellular radiosensitivity mightallow identification of carriers, raising hopes that the diseasemight be virtually eradicated within one or two generations.These hopes have not been fulfilled: although cellular

radiosensitivity was present in some HD patients, 13,17,19normal responses were found in others.19

In all these studies the investigators used long-termcultures of fibroblast or lymphoblastoid cell lines derivedfrom individuals with genetic disorders. It is likely, therefore,that the observed abnormal responses to radiation were adirect result of the genetic defects in the affected individuals.Lately, radiotherapy has been used effectively in treatmentfor rheumatoid arthritiS20,2 and polymyositis22,23-diseasesthat are not inherited in any simple mendelian manner. Thepossibility of radiosensitivity in association with theseautoimmune diseases is clearly important. Harris and co-workers24 therefore set out to develop rapid, reproduciblescreening methods and they offer two tests, both using short-term cultured lymphocytes. The first test involves inhibitionby radiation of lymphocyte proliferation induced byconcanavalin A. The second measures the ability of the cellsto restore nuclear structures after radiation damage. The twotests are simple and quick and gave concordant results. Withthe first test lymphocytes from several patients withrheumatoid arthritis, and from all those with systemic lupuserythematosus or polymyositis, were more radiosensitivethan those from normal controls. The sensitive cells alsoseemed to have severely reduced ability to restore the nuclearstructures following radiation damage. -

Taken at face value, these tests seem to offer a rapid meansof assessing radiosensitivity in individual patients, and thefindings should be taken into account if radiotherapy is

contemplated; but interpretation of the data is not

straightforward. Harris and co-workers used peripherallymphocytes, these cells being readily obtainable from thepatients. Lymphocytes, however, are a bewilderinglyheterogeneous mixture of subpopulations, not just ofB and Tcells but also of their various subsets; and we know that B andT cells have different radiosensitivities,25 as do theirsubsets.26 Moreover, the distributions of these various

subpopulations are likely to be severely altered in

16. Scudiero DA, Moshell AN, Scarpinato RG, et al. Lymphoblastoid lines and skinfibroblasts from patients with tuberous sclerosis are abnormally sensitive to ionizingradiation and to a radiomimetic chemical. J Invest Dermatol 1981; 78: 234-38.

17 Moshell AN, Barrett SF, Tarone RE, Robbins JH. Radiosensitivity in Huntington’sdisease; implications for pathogenesis and presymptomatic diagnosis. Lancet 1980,i 9-11

18 Chen P, Kidson C, Imray FP Huntington’s disease: implications of associated cellularradiosensitivity. Clin Genet 1981; 20: 331-36.

19. Arlett CF. Presymptomatic diagnosis of Huntington’s disease. Lancet 1980, 1: 540.20. Kotzin BL, Strober S, Engleman EG, et al. Treatment of intractable rheumatoid

arthritis with total lymphoid irradiation. N Engl J Med 1981; 305: 969-76.21. Trentham DE, Belli J, Anderson RJ, et al. Clinical and immunological effects of

fractionated total lymphoid irradiation in refractory rheumatoid arthritis. N Engl JMed 1981; 305: 976-82

22 Engel WK, Lichter AS, Galdi AP. Polymyositis remarkable response to total bodyirradiation. Lancet 1981; i: 658

23. Hubbard WN, Walport MJ, Halnan KE, Beaney RP, Hughes GRV. Remission frompolymyositis after total body irradiation. Br Med J 1982; 284: 1915-16.

24 Harris G, Cramp WA, Edwards JC, George AM, Sabovljev SA, Hart L, Hughes GRV,Denman AM, Yatvin MV. Radiosensitivity of peripheral blood lymphocytes inautoimmune disease. Int J Radiat Biol 1985; 47: 689-99.

25. Prosser JS. Survival of human T and B lymphocytes after X-irradiation. Int J RadiatBiol 1976; 30: 459-65.

26 Schwartz JL, Darr JC, Gaulden ME. Survival and PHA-stimulation of gamma-irradiated human peripheral blood T lymphocyte subpopulations Mutation Res1983, 107: 413-25

27. Morimoto C, Reinhertz EC, Schlossman SF, Schur PH, Mills JA, Steinberg AD.Alterations in immunoregulatory T cell subsets in active systemic lupuserythematosus. J Clin Invest 1980, 66: 1171-74.

28. Veys EM, Hermanns P, Schindler J, et al. Evaluation of T cell subsets with monoclonalantibodies in patients with rheumatoid arthritis. J Rheumatol 1982; 9: 25-29.

25

autoimmune disorders.27,28 Thus it is conceivable that the

apparent radiosensitivity in these autoimmune disorders infact reflects the presence of a larger population of a moreradiosensitive subset of lymphocytes in these disorders-ie, itmay result merely from an alteration in the lymphocytepopulation distribution. Harris and co-workers regard thisexplanation as unlikely. A further difficulty with the use oflymphocytes concerns their long lifespan in the bloodstream.Some properties of lymphocytes, such as chromosome

aberrations, reflect accumulated exposure to damagingagents over a long period. It is possible, therefore, that theabnormal radiation responses of lymphocytes from the

patients studied by Harris et al could be as much a reflectionof the "environmental history" of the patients as a reflectionof a genuine radiosensitivity associated with the disorders.For these reasons the data of Harris and co-workers should be

interpreted with some caution. If, however, these workers areable to validate their tests and demonstrate that they really doidentify radiosensitive individuals, they will have providedradiotherapists with a very useful aid to the planning oftreatment.

CALCITONIN GENE-RELATED PEPTIDE

CHARACTERISATION of peptide-hormone precursors andthe genes encoding for them has been greatly facilitated byrecombinant DNA technology; in this respect, the calcitoningene has proved exceptionally interesting. Cloning andanalysis of recombinant DNA from messenger RNAextracted from human C-cell tumours first showed that thehuman calcitonin’ was derived from a large-molecular-’ weight precursor protein,2 but the calcitonin precursor hasproved not to be the only precursor polypeptide encoded bythe calcitonin gene. The existence of another precursor cameto light during studies of a serially transplanted rat medullarythyroid carcinoma cell-line,3 when spontaneous and

permanent switching from high to low calcitonin productionwas observed, coinciding with the production of a differentmessenger RNA. Nucleotide sequence analysis showed thatthis represented an alternative processing of the primaryRNA transcript from the calcitonin gene. The 37-aminoacidpeptide predicted from the alternative message was namedcalcitonin gene-related peptide (CGRP). CGRP was isolatedfrom human C-cell tumours and its aminoacid sequence andstructure were established by Morris and his colleagues4 bymeans offast-atom-bombardment mass spectrometry. Whilstthe aminoacid sequence of CGRP differs from that of

calcitonin, it has certain structural similarities-eg, an aminoterminal 2-7 disulphide bridge and a carboxy terminalamide. In man, the situation has become even more complexwith the reports5,6 of a second calcitonin gene encoding for asecond CGRP whose predicted sequence has 3 aminoacidsubstitutions.6

1 Stevenson JC The structure and function of calcitonin. J Invest Cell Pathol 1980; 3:187-93

2. Craig RK, Hall L, Edbrooke MR, Allison J, MacIntyre I. Partial nucleotide sequenceof human calcitonin precursor mRNA identifies flanking cryptic peptides. Nature1982, 295: 345-47.

3. Amara SG, Jonas V, Rosenfeld MG, Ong ES, Evans RM. Alternative RNA processingin calcitonin gene expression generates mRNAs encoding different polypeptideproducts. Nature 1982; 298: 240-44.

4 Morris HR, Panico M, Etienne T, Tippins J, Girgis SI, Maclntyre I. Isolation andcharacterisation of human calcitonin gene-related peptide. Nature 1984, 308:746-48

5. Edbrooke MR, Parker D, McVey JH, et al. Expression of the human calcitonin/CGRPgene in lung and thyroid carcinoma EMBO J 1985; 4: 715-24..

6 Steenbergh PH, Hoppener JWM, Zandberg J, Lips CJM, Jansz HS. A second humancalcitonin/CGRP gene. FEBS Lett 1985; 183: 403-07.

Where is it found? Rosenfeld and colleagues initiallyreported that CGRP was present mainly in nervous tissue butwas absent from the thyroid;’ it is now clear, however, thatCGRP occurs in thyroid tissue in both animals and man.8,9Its major distribution is in the central nervous system wherespecific protein-binding sites have been shownlO and it is

especially abundant in the spinal cordS, 10, 1 where it co-

localises with substance P in the posterior horn and primarysensory neurons.12 In the brain it is distributed in the

olfactory and gustatory systems, and in the hypothalamic andlimbic regions.’,13 CGRP is found in the trigeminal ganglion’ 7and its sensory neurons (again co-localised with substancep14,lS), and in the pituitary glands Immunohistochemicalstudies have also demonstrated CGRP peripherally in nervefibres in the heart, lung, and gastrointestinal tract, oftenassociated with the smooth muscle of blood vessels.7,16Immunoreactive CGRP has been found in the plasma ofsome patients with medullary thyroid carcinoma4 and clearlycirculates in normal man, as shown by the report from DrGirgis and her colleagues on p 14 of this issue. Indeed, theircomparison of circulating levels of CGRP and calcitoninsuggests that CGRP is normally the major product of thecalcitonin gene in man.

Synthesis of both rat and human CGRP has permittedstudies of the pharmacology of these peptides. One of the firstactions to be discovered was stimulation of sympatheticoutflow. Thus, intracerebroventricular injection of CGRPincreased circulating noradrenaline levels, with

accompanying tachycardia and raised blood pressure, whilstperipheral injection caused tachycardia with hypotension.17When human CGRP is injected intradermally it causesintense microvascular dilatation.’8 Intravenousadministration of human CGRP into volunteers led to

striking peripheral vasodilatation with hypotension,tachycardia, and increased catecholamine secretion,19showing it to be the most potent vasoactive peptide yetdiscovered. In animal studies, positive inotropic and

chronotropic effects have been observed in the isolated

7. Rosenfeld MG, Mermod J-J, Amara SG, et al. Production of a novel neuropeptideencoded by the calcitonin gene via tissue-specific RNA processing Nature 1983;304: 129-35.

8. MacIntyre I. The calcitonin gene peptide family and the central nervous system. In:Labrie F, Proulx L, eds Endocrinology. Proceedings of the 7th InternationalCongress of Endocrinology Amsterdam: Excerpta Medica, 1984 930-33

9. Tschopp FA, Tobler PH, Fischer JA. Calcitonin gene-related peptide and calcitonin inthe human thyroid, pituitary and brain. Mol Cell Endocrinol 1984; 36: 53-57.

10. Tschopp FA, Henke H, Petermann JB, et al. Calcitonin gene-related peptide and itsbinding sites in the human central nervous system and pituitary Proc Natl Acad SciUSA 1985; 82: 248-52

11 Gibson SJ, Polak JM, Bloom SR, et al. Calcitonin gene-related peptideimmunoreactivity in the spinal cord of man and of eight other species. J Neuroscience1984, 4: 3101-11.

12. Lundberg JM, Franco-Cereceda A, Hua X, Hokfelt T, Fischer JA. Coexistence ofsubstance P and calcitonin gene-related peptide-like immunoreactivities in sensorynerves in relation to cardiovascular and bronchoconstrictor effects of capsaicin. EurJ Pharmacol 1985; 108: 315-19.

13 Takami K, Kawai Y, Shiosaka, et al. Immunohistochemical evidence for thecoexistence of calcitonin gene-related peptide and choline acetyltransferase-likeimmunoreactivity in neurons of the rat hypoglossal, facial and ambiguus nuclei.Brain Res 1985; 328: 386-89

14. Lee Y, Kawai Y, Shiosaka S, et al. Coexistence of calcitonin gene-related peptide andsubstance P-like peptide in single cells of the trigeminal ganglion of the rat:

Immunohistochemical analysis. Brain Res 1985; 330: 194-96.15 Terenghi G, Polak JM, Ghatel MA, et al. Distribution and origin of calcitonin gene-

related peptide (CGRP) immunoreactivity in the sensory innervation of themammalian eye. J Comp Neurol 1985; 233: 506-16.

16. Mulderry PK, Ghatei MA, Rodrigo J, et al. Calcitonin gene-related peptide in

cardiovascular tissues of the rat Neuroscience 1985; 14: 947-5417. Fisher LA, Kikkawa DO, Rivier JE, et al Stimulation of noradrenergic sympathetic

outflow by calcitonin gene-related peptide. Nature 1983; 305: 534-36.18. Brain SD, Williams TJ, Tippins JR, Morris HR, Maclntyre I. Calcitonin gene-related

peptide is a potent vasodilator. Nature 1985; 313: 54-5619. Struthers AD, Brown MJ, Beacham JL, Morris HR, Maclntyre I, Stevenson JC. The

acute effect of human calcitonin gene-related peptide in man. J Endocrinol 1985; 104(suppl): 129