WART-VIRUS ANTIBODIES AND THE PROGNOSIS OF WART DISEASE

1330

WART-VIRUS ANTIBODIES AND THE PROGNOSIS OF WART DISEASE SEPPO PYRHÖNEN

KARI PENTTINEN

Laboratory of Viral Immunopathology, Department of Virology, University of Helsinki, 00290 Helsinki 29, Finland Wart-virus antibodies from 182 patients with warts were studied by the microimmunodiffusion method. 57% of the patients had measurable antibodies, the titres varying from 1 to 512. The longer the duration of the disease or the larger the number of tumours, the lower the serum antibody levels were. Complement-fixation antibodies were of the IgG type only, and were demonstrable in only 12% of the cases (titres 4-512). If the antibodies were IgG type, there was a good chance of healing. Summary

Introduction

HUMAN wart disease is a benign skin tumour, which usually affects children or young adults1 and, uncommonly, older age-groups. This association with age suggests the development of some protective mechanism or altered manifestation among older people. There are observations suggesting wart virus to be a causal factor in some cases of mucosal papillomatosis, which may further suggest a connection with Electron microscopy malignant mucosal diseases. reveals the presence of papovaviruses in oral mucosal2 condyloma acuminatum or genital warts,3laryngeal papillomas,4 and papillomatous tumours of brain chorionic villi. The association of virus particles with malignant changes in the skin in a case of epidermodysplasia verruciformis is reported by Ruiter and van Mullem,6 and there is a report of papovavirus particles in certain cells cultured from nephroblastoma.7 Research into wart disease has been limited by lack of any system for propagating the virus; however, this virus disease has value as a model for viral oncogenicity and connected immunological processes in man. Circulating wart-virus antibodies have been detected with four different techniques: direct visualisation of virus-antibody complexes under the electron microscope, complement-fixation (c.F.) test, immunodiffusion (I.D.) in gels, and passive hsemagglutination. Using Lid. and electron microscopy Almeida and Goffe8 found that of 42 patients with warts, 45 % had circulating wart-virus antibodies. In another studythey showed that sera of wart patients usually contained IgM antibodies and only sometimes IgG antibodies, and this was the explanation suggested for the recurrence of warts. The use of the c.F. technique with genital warts was reported by Madema10 and Almeida et al.11 Almeida et al. 11reported a one-way crossreactivity between genital warts and skin wart antibodies. Genner 12 found that, before treatment, only 6-3% of the wart patients had c.F. antibodies, but after therapy antibody production was stimulated so that 20% of the patients had c.F. antibodies. The same response was reported briefly by Alexander. 13A study by Ogilvie 14 demonstrated wart-virus antibodies using c.F., precipitation, and passive-haemagglutination tech-

a relation between the appearance of antiviral antibodies and the regression of warts; people whose warts underwent spontaneous resolution had also developed antibodies. Before that finding, antiviral antibodies had not been considered to be connected with the cure of warts. The aim of our study was to shed some further light on the immunology of wart disease.

niques. She found

Patients and Methods 182 sera for the study were obtained from patients (123 sera) attending a special clinic in the Helsinki University skin unit, from medical students (32), and from Finnish Servicemen (27). All of them had active wart disease that had been present for differing periods, and one serum specimen was taken from each of the 182 patients. The ages of the patients ranged from six to fiftyWhen the sample was taken the duration of seven years.

the disease and the number of warts were recorded. No distinction was made between warts at different sites, Wart-virus antigen was prepared from surgically removed warts. Wart tissue (1 g.) was minced with scissors and ground with sand in 10 ml. phosphate-buffered saline solution, the resulting suspension was clarified twice at 600 g for thirty minutes. The supernatant was used as antigen in subsequent tests. To isolate the antigen 1 >0 ml. sample was centrifuged at 9340 g in rotor SW-40 for thirty minutes at 4°C and then the supernatant was spun at 114,000 g for four hours. The pellet of the last centrifugation was suspended in 0-2 ml. of phosphate-buffered saline (P.B.S.) and centrifuged in cxsium-chloride gradient. The fractions were collected and examined under the electron microscope with negative-staining technique. Wart viruses were detected in fractions with density 1’31-1-37. The virus-containing fractions were tested against the unpurified antigen used in all the tests. No differences between the precipitation lines were detected. A normalskin control antigen was prepared in the same way as the wart-virus antigen; no precipitation lines were detected with anti-wart serum. Lid. tests were carried out in a 0-9% agarose phosphatebuffered medium, pH 7-4, using the micro-modification of gel double diffusion described by Watsworth,15 modified by Krause and Raunio 16 and Salmi.17 The sera were titrated by diluting them twofold in P.B.S. and layering into the peripheral wells of diffusion plates around the antigencontaining central well. The precipitates were allowed to form at room temperature, the slides being incubated in a humidified chamber for three and a half days. The final reading was taken under standardised light conditions after staining with amido-black.18 The greatest dilution at which a visible precipitation line was detected was registered as the titre of a serum, and reciprocals of dilutions were recorded. At least two measurements were made for each serum, the variation in titres being more than twofold in only 7 cases. If the difference was twofold a third measurement was made to determine the titre. In the 7 cases where the titre variation was more than twofold, numerous titrations were made and the median value was accepted as the " right " titre. C.F. tests were done using the standard technique by Sever.19 4 units of antigen and 2 full units of complement were used. Optimal concentration of antigen was determined by checkerboard titration against wart-virus antiserum, and the complement was titrated in the presence of antigen at this concentration. Measurements were performed on plastic plates using 0-1 unit volumes. For immunoglobulin determinations samples of 0-1 ml. were layered on linear 12-5-37% w/v sucrose gradients and centrifuged in a Spinco SW 50-1 rotor for seventeen hours

1331 It seems that the peak antibody titre is usually reached early in the disease; when the disease becomes chronic, the number of tumours increases and the antibody

Fig. I-Antibody titres and duration of

titre falls. The wart-virus antibodies from 172 patients were also measuied by c.F. technique. 12% (20/172) had measurable wart-virus c.F. antibodies. The titres varied from 4 to 512. The correlation (for 12 sera) of c.F. titres with the I.D. titres and the immunoglobulin class is presented in fig. 3. The scanty data suggest that if the c.F. titre is not measurable and the Lid, titre is 8 or more the immunoglobulin type is IgM. With the c.F. technique it seems to be possible to

warts.

only human wart IgG antibodies, but by Lid. both IgM and IgG antibodies measure

The fractions were collected dropat 84,000 g at 4°C. wise from below. For sedimentation coefficient determination reference markers (serum containing IgM 19S and IgG 7S rubella haemagglutination-inhibiting antibodies) were sedimented in companion gradients. The wart sera fractions were examined directly by LD. tests because sucrose in the amount present did not interfere.

Results

The mean number of tumours recorded tended to increase with the duration of the disease up to about two years, but quite large individual variations, between 1 and 50 tumours, were found among patients with the same disease duration. 57% (102/182) had antibodies measurable by I.D. technique, the titres varying from 1 to 512. Of 32 control sera from children aged six months to one year only 1 had measurable I.D.-antibodies (titre 2), but it is not known whether this child had warts. I.D. titres of 16 or more were found in 33% (28/86) of the patients who had warts for a year or less but in only 4% (4/96) among those with warts for more than a year (fig. 1). This difference is highly significant

(P<0001). Fig. 2 shows the correlation between I.D. titres and the number of tumours. 22 % (30/139) of the patients with ten warts or less had titres Ø: 16 but only 5 % (2/43) of those with more warts had similar titres. The difference between the groups is significant (P<0-02).

Fig. 2-Antibody titres and number of warts.

be measured. The prognostic value of antibody data for the healing of warts, after therapy and within two-month follow-up period, is presented in the table. If wart-virus antibodies were measurable by Lid. in low concencan

CORRELATION OF THE WART-VIRUS ANTIBODIES AT THE TIME OF THERAPY AND THE PROGNOSIS OF THE DISEASE WITHIN THE 2-MONTH FOLLOW-UP PERIOD

trations at the time of therapy or if they were of IgM type only, albeit of high titre, healing occurred in 46% (24/52) of the patients; in cases when antibodies were not measurable, the prognosis seems to be of the same order or better, healing occurred in 64%(30/47). The

Fig. 3-C.F. and LD. titres

of wart-virus IgG and

IgM antibodies.

1332

highest probability for the

cure

in

were

situation when there

of warts seemed

to

be

measurable c.F. antiwere cured. The differences in rate of cure between the first two and the third group is statistically significant (p<0’01). a

bodies, 94% (15/16) of patients

Discussion

An inverse correlation

was

found between the wart wart tumours, as

antibody level and the duration of

well as the number of tumours. Principally two different mechanisms could be involved: (1) when the number of warts is high, antibody consumption is increased and titres fall, and (2) when antibody production is decreased the wart number will increase. It is interesting to compare these results with the study of Barrera-Oro et aI.,20 in which there seems to be a correlation of the virus particles in wart tissue with the duration of the disease, warts six to twelve months old containing the highest concentration of particles. The timing of the peak antibody titres in our series accords with these results and with the finding that production of wart antibodies in guineapigs is dependent on the amount of wart antigen .21 No measurable antibody response is obtained with a small antigen quantity by the immunodiffusion method, but by slightly increasing the amount of antigen inoculated the IgM response is obtained. If a large dose of antigen is used, the IgM response is followed by IgG antibody form-

ation. In Ogilvie’s study,14

virus

IgM antibodies of rabbit sera were able to fix complement; we did not find this for human wart IgM antibodies. Only the wart IgG antibodies of human sera were able to fix complement. The finding that IgG antibodies are a favourable prognostic indicator is interesting: production of IgG antibodies by vaccination would perhaps be of value. wart

How the cell-bound immune reactions are connected with the appearance of humoral antibodies is not known; the understanding of immune reactions during human wart disease might provide some valuable knowledge of the immunology of tumour diseases. This survey was supported in part by grants from the Sigrid Juselius Foundation and Cancer Foundation. Requests for reprints should be addressed to S. P. REFERENCES

Barr, A., Coles, R. B. Br. J. Hosp. Med. 1970, 3, 831. Frithiof, L., Wersäll, J. Arch. ges. Virusforsch. 1967, 21, 31. Dunn, A. E. G., Ogilvie, M. M. J. ultrastruct. Res. 1968, 22, 282. Boyle, W. F., McCoy, W. G., Fogarty, W. A. Ann. Otol. Rhinol. Laryngol. 1971, 80, 693. 5. Bastian, F. O. Lab. Invest. 1971, 25, 169. 6. Ruiter, M., van Mullem, P. J. invest. Derm. 1966, 47, 247. 7. Smith, J. W., Pinkel, D., Dabrowski, S. Cancer, 1969, 24, 527. 8. Almeida, J. D., Goffe, A. P. Lancet, 1965, ii, 1205. 9. Goffe, A. P., Almeida, J. D., Brown, F. ibid. 1966, ii, 607. 10. Maderna, C. Rif. med. 1935, 51, 93. 11. Almeida, J. D., Oriel, J. D., Stannard, L. M. Microbios. 1969, 3, 225. 12. Genner, J. Acta derm.-vener. Stockh. 1971, 51, 365. 13. Alexander, S. Br. J. Derm. 1966, 78, 598. 14. Ogilvie, M. M. J. Hyg., Camb. 1970, 68, 479. 15. Watsworth, C. Int. Archs Allergy, 1957, 10, 355. 16. Krause, U., Raunio, V. Acta path. microbial. scand. 1967, 71, 328. 17. Salmi, A. ibid. 1969, 76, 271. 18. Crowle, A. J. Immunodiffusion; p. 304. New York, 1961. 19. Sever, J. L. J. Immun. 1962, 88, 320. 20. Barrera-Oro, J. G., Smith, K. O., Melnick, J. L. J. natn. Cancer Inst. 1962, 29, 583. 21. Furminger, I. G. S. Progr. immunobiol. Standard. 1970, 4, 166. 1. 2. 3. 4.

DOES MATERNAL SMOKING DURING PREGNANCY HAVE A LONG-TERM EFFECT ON THE CHILD ?

JANET B. HARDY

E. DAVID MELLITS

Departments of Pediatrics and Biostatistics, Johns Hopkins Medical Institutions, Baltimore, Maryland, U.S.A. Summary

Possible long-term effects of maternal

smoking on physical growth and intellectual development through the first seven years of life were investigated, in a matched-pair study, by comparisons between children of women who smoked substantially throughout pregnancy and children whose mothers did not smoke. There was no significant difference between the groups of mothers with respect to nine other major variables known to affect birthweight. The babies of smokers showed significant intrauterine growth retardation. They were 250 g. lighter and shorter at birth than the babies of nonsmokers; they were still shorter at one year. At four and seven years there was no significant difference in either physical measurements or intellectual functioning. Thus, in those children who survived the perinatal period harmful long-term effects of smoking were not identified. Introduction

INCREASING concern for the prevention of long-term neurological and intellectual handicaps in children has focused attention on the identification of factors, biological and/or environmental, which may adversely affect fetal growth. In general, the lowest risks of neonatal death and the greatest likelihood of optimal physical and intellectual development are experienced by children weighing 3000 g. (6-21 lb.) or more at birth. The chance of an adverse outcome increases as birthweight decreases below this level. Maternal cigarette smoking during pregnancy is one of many factors known to retard fetal growth. Because it is a potentially controllable environmental factor, and because smoking is common among childbearing women in Western cultures, the possible adverse effects upon the fetus are of some consequence. According to the 1971 report of the Surgeon General, U.S. Public Health Service,t a third of American

aged 15-44 are cigarette smokers. 54% of the White women and 42% of the 20,167 Black 19,048 women enrolled in the " core " sample of the Collaborative Perinatal Study (C.P.S.)2 of the National Institute of Neurological Diseases and Stroke (N.I.N.D.S.) in which information about smoking was prospectively collected at each prenatal visit, smoked one or more cigarettes per day at the time of registration for prenatal care. Rather similar frequencies were observed in the British Perinatal Study.3,4 The relation between smoking and birth-weight has been reviewed and summarised.l,4 The infants of mothers who smoke during pregnancy weigh, on the average, 170-180 g. less at birth than the infants of non-smokers. The lower weight reflects retardation of fetal growth because the gestation period of women who smoke is only very slightly reduced.5 The effect of maternal cigarette smoking on birth-

women