Sera from 16 echinococcosis patients were analyzed with respect to poiyclonal B cell activation and autoantibody formation. At least 8 of the sera were from patients who were never in tropical countries and therefore their cases were not complicated by other parasitic diseases. In comparison with a group of 52 healthy controls, these patients had significant levels of antibodies to DNP and haemocyanin, indicators of polyclonal B cell activation. There were also significant differences between control and patient groups with respect to antibodies to dsDNA, histones, actin, vimentin, and desmin. This is the first report of autoantibodies in echinococcosi

Franklin, Richard M.

Mori, Hiroshi

Weiss, Niklaus

Wernli, Barbara

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978 TRANSACTIONS OF THE ROYAL SOCIETY or TROPICAL MEDICINE AND HYGIENE (1986) 81, 978-980 Autoantibodies in humans with cystic or alveolar echinococcosis HIROSHI MORP, BARBARA WERNLP, NIKLAUS WEISS* AND RICHARD M. FRANKLIN’ ‘Department of Structure Biology, Biocenter, University of Basel, 4056 Basel, Switzerland; 2Department of Medicine, Swiss Tropical Institute, 4051 Base& Switzerland Abstract Sera from 16 echinococcosis patients were analyzed with respect o polyclonal B cell activation and autoantibody formation. At least 8 of the sera were from patients who were never in tropical countries and therefore their cases were not complicated by other parasitic diseases. In comparison with a group of 52 healthy controls, these patients had significant levels of antibodies to DNP and haemocyanin, indicators of polyclonal B cell activation. There were also significant differences between control and patient groups with respect o antibodies to dsDNA, histones, actin, vimentin, and desmin. This is the first report of autoantibodies in echinococcosis. Introduction Polyclonal B cell activation accompanied by the appearance of autoantibodies is a recognized feature of several parasitic diseases such as visceral leish- maniasis (GALVAO~ASTRO et al., 1984!, Chagas disease (SCOTT & SNARY, 1982), and malaria (ADU et al., 1982; MORTAZAVI-MILANI et at., 1984). In Chagas dsease both humoral and cellular nnmunopathologic- al mechanisms have been implicated in the overall pathology of the chronic state (SCOTT & SNARY, 1982). The parasitic factor or factors involved in the complex immunopathologies are now under investiga- tion in several laboratories. We report here yet another parasitic disease, caused by larvae of mem- bers of the genus Echinoco<cw,. which is accomp+anied F~~.$~lonal B cell actlvauon and autoantlbody Materials and Methods Patients and controls Sera from a total of 16 patients (14 with E.granulosus, 2with E.muZtiloculuris) were analyzed. There were 7 male and 9 female patients ranging in age from 12 to 72 years. Of these, 8 had never been to the tropics, 2 were Turkish males working in Switzerland, and one had visited Tibet. Unfortu- nately no records of any possible visits to tropical countries were available for the re maining 5 patients. Nevertheless, there was no evidence that any patient was suffering from any other parasitic or other infectious disease at the time the serum samples were collected. All of the patients were positive for Epstein-Barr virus (EBV) as determined by viral capsid antigen (VCA) and Epstein-Barr nuclear antigen (EBNA) titres. The control sera were from a group of Caucasian ormal controls (CNC) selected from laboratorv personnel including 32 males and’ 20 females ranging in age from 20 to 54 vears. Of these. 4were EBV-negative. All were healthy at the-time of bleed&g and none had been in tropical countries for at least a year before bleeding. ELZSA procedure The protocol for ELISA tests was modified from that described by VAINIO et al. (1983). Microtest plates (A/S Nunc, Raskilde, Denmark) were coated with appropriate amounts of the purified antigens. Most of the antigens used have been described (VAINIO et al., 1983). Dinitropheny- lated-bovine serum albumin (DNP-BSA) was prepared in our own laboratory and its ELISA readings were compared with BSA alone. Haemocyanin was Limulus pol hemus haemocyanin (Sigma Chemical Co, St Louis, i&souri 63178, USA). Appropriate dilutions of sera were incubated for 2 hours at 37”, the second antibody was peroxidase- labelled goat anti-human Ig (Nordic Immunological Labor- atories, Tillburg, The Netherlands). Absorbances (A,& in the individual wells were read with an automatic ohotometer (Titertec Multiskan, E&b OY, Helsinki, Finland) inter- faced with an Apple IIc computer. Data files stored on floppy disks we&processed and analyzed statistically using our own package of statistical programs. Results The raw data presented in Table 1 are the mean values of A492 (+ SD) of the appropriate serum dilutions (alwavs the same dilution for the control and patient g&ups j, rounded off to two significant figures. Background A 492 has not been subtracted and is also shown in Table 1. The background in the absence of serum always has a considerably lower A 492 than the serum samples but the absolute value is quite high in the case of keratin. Although there is a clear trend to higher A 492 average values in the patient group, we cannot make any judgement concerning quantitative differences between control and patient groups with- out further statistical analyses. Analysis-using CNC standard deviation (SD) units (cf. VAINIO et al.. 1983 for a similar, but hot >denti&, analysis), ho&ever, indicates that the average CNC SD units are all greater than zero for the patient series and the average values for haemocyanin, dsDNA, histones, actin, keratin, laminin, and collagen type I are all greater than 1 (Table 1). The SDS for the CNC SD units do fluctuate very strongly, however. As we are dealing with small samples, we applied the Wilcoxon rank test to detect differences between the two groups. We ranked both A d0, and CNC SD units from lowest to highest and compared the hypotheses that the two groups (CNC and patients) were the same or diffe- rent. As a measure of the difference we used Z, and present the results in Table 2 along with the corresponding p-values. Differences between the two groups may be seen, with the patient group always having higher values (except for ssDNA, tubulin, laminin, and collagen type I, where no differences were detected). The differences for haemocvanin, dsDNA, histone, actin, and vimentin were s&r&cant at the ~<0*005 level; that for keratin at the ~cO.01 level; and those for i)NP and desmin at the p~O.05 level. The same results were obtained ranking A492 l-l. MORI et al. 979 Table l-ELISA tests for polyclonal B cell activation and autoantibodies in controls and echinococcosis patients %F Haemocvanin dsDNA d ssDNA Histone Actin Tubulin Desmin Vimentin Keratin Laminin Collagen Type CNc(Ad’ -26 IL *04 90 + -25 *99 zk -28 *75 + .19 *45 zlz *IO .92 k *17 .57 k *19 -43 t *14 .43 f -16 l-21 f *18 -38 f .18 .36 + .13 1.35 5 -52 *26 + -01 1.38 + 44 -24 f .Ol .88 + *52 ~24 + -03 *87 +- .30 *15 + .Ol 1.38 + .59 -19 + .Ol .60 XL -19 *14 5 *Ol *52 + .18 .15 rk *02 .56 I! *24 .I7 z!I *Ol 1.49 31 .42 .60 I!Z *03 .55 + .52 .15 It .Ol .53 k .30 *15 + .Ol Patients4 .77 + 1.19 1.80 31 2.09 1.41 31 1.60 .60 f 2.77 4.98 I? 2.98 2.71 + 3.47 *19 f 1.03 *60 k 1.28 *85 + 1.45 1.51 IL 2.26 1.12 f 3,55 1.29 3~ 2.31 1) Mean A4g2 + SD of the group of 52 Caucasian normal controls (CNC). Background values (in the absence of serum) are given in (3) but are not subtracted from (1). 2) Mean Ada2 + SD of the group of 16 echinococcosis patients. The comment concerning background values given under (1) is applicable here as well. 4) Mean CNC SD units t- SD of the 16 natients; CNC SD units = (mean A,gz patients - mean A~92 CNC)/(SD of A492 CW. Table 2-Quantitation of the Wilcoxon rank test zo (1) p 2.1687 <0.05 --.- ~sa;~Acyanin 3.4121 <0*005 3.36’76 -co*005 ssDNA Histone Actin *2241 * 4.4402 <o-o05 3.8169 <0*005 Tubulin Desmin Vimentin Keratin Laminin Collagen type I *8458 * 2.0530 <0.05 2.8410 <0*005 2.7181 <O*Ol 1.1494 * 1.6988 * (1) Z, = U-Un/o where U is the rank value of the group of 16 echinococcus patients and UR is the expected rank value assuming no difference in A492 between the CNC and patient groups; u is the standard deviation as defined by Wilcoxon. (2) p is the probability value (based on the unit normal distribution) for the difference hypothesis. * means there is no significant difference between the two groups. and CNC SD units. Correlation coefficients were then calculated for patient antibodies in those cases where significant differences could be found between the CNC and patient groups (Table 3). High correlations (p<O*Ol evel) were found for anti-DNP and all other antibodies except anti-DNA. Anti-DNA could be correlated only with anti-histone at the p<O*Ol level and anti&stone only with anti-DNP and anti-DNA. All other antibodies correlated with each other and with anti-DNP and anti-haemocyanin at the p<O*Ol confidence level. Discussion Little is known concerning immune mechanisms in cystic or alveolar echinococcosis and it was quite surprising to find evidence for antibodies to DNP and haemocyanin, indicators of polyclonal B cell activa- tion (GALVAO-CASTRO et al., 1984), in these patients. The polyclonal B cell activation could be correlated with antibodies to cytoskeletal proteins. Antibodies to nuclear antigens (dsDNA and histones) were also found but these were not well correlated with polyclonal B cell activation. Despite the spatial delimitation of the hyatid cyst, humoral and cellular immune reactions are associated Table 3-Correlation coefficients for & g2 in the group of echinococcosis patients Antigen DNP I-Ia-IrAc yanin Histone Actin Desmin Vimentin Keratin DNP Haemocyanin - .794** .,gp* 1. .608* .535* ,627** .37a* .674** .860** .806** .814** .712** .818** .755** .877** dsDNA .608* .535* - ::i:** .584: %02* ,558* Histom 627** .378* .715** .188 .503** .357** .297** Actin .674** .860** .504* .188 G5** .882** .909** Desmin .806** .814** .584* .503* .805** G2** .847** Vimentin .712** .818** .602* .357 .882** .872** G5** Keratin .755** .877** .558* .297 .909** .a47** .895** - ** p<O.Ol * p<o.os No asterisk indicates p>O.l (no correlation) 980 AUTOANTIBODIES IN ECHINOCOCCOSIS with such lesions. There is a granulomatous cell proliferation with many lymmphocytes, as well as eosinophils and mast cells, surrounding the parasitic vesicles (WILLIAMS, 1982; VUITTON et al., 1984). At the same time there is a suggestion of severe impairment of the immune system, at least in alveolar echinococcosis. In this disease the percentage and absolute number of B cells and total PBL are lower than in controls. Furthermore, despite a normal percentage of T cells, T cell function is impaired, as measured by the leucocyte migration test (VUITTON et al., 1984). High levels of serum Ig (VUITTON et al., 1984) might he one reflection of the polyclonal B cell activation we report here. Acknowledgements Miss U. Pira orovided technical assistance and Mrs M.-C. Gauler was our excellent word processor. This work was supported in part by research funds from the City of Base1 and by a grant from the Sandoz Stiftung. References Adu, G., Gwyn Williams, D., Quakyi, I. A., Voller, A., Anim-Addo, Y., Bruce-Tagoe, A. A., Johnson, G. D. & Holborow, E. J. (1982). Anti-sss DNA and antinuclear antibodies in human malaria. Clinical and Exwrimental Marzochi, M. C., Coutinhq, S. G. & Lambert, P. H. (1984). Polyclonal B cell activation, circulating munune complexes and autoimmunity in human american viscer- al leishmaniasis. Clinical and Experimental Immunology, 56, 58-66. Mortaxavi-Milani, S. M., Badakere, S. S. & Holborow, E. J. (1984). Antibody to intermediate filaments of the cytos- keleton in the sera of patients with acute malaria. Clinical and Experimental Immunology, 55, 177-182. Scott, M. T. & Snary, D. (1982). American Trypanoso- miasis (Chagas’ Disease). In: Zmmunology of Parasitic Infections. (2nd edit.). Cohen, S. & Warren, K. S. (editors). Oxford; Blackwell Scientific Publications, pp. 261-298. Vainio. E., Lenoir, G. M. & Franklin. R. M. (1983). Autoantibodies in three populations of Burkitt’s lynrpho- ma patients. Clinical and Experimental Immunology, 54, 387-396. -~ Vuitton, D. A., Lasstgue, A., Miguet, J. P., Hen+, P., Barak, T., Seilles, E. & Capron, A. (1984). Humoral and cellular immunity in patients with hepatic alveolar echinococcosis. A 2 year follow-up with and without flubendaxole treatment. Parasite Zmmunology, 6,329-340. Williams, J. F. (1982). Cestode Infections. In: Zmmunologv of Parasitic Infections. (2nd edit.). Cohen, S. & Warren, K. S. (editors). Oxford: Blackwell Scientific Publica- tions, pp. 676-714. Immunology, 49, 310-316. Galvao-Castro, B., Sa Ferreira, J. A., Marzochi, K. F., Accepted for publication 27th August, 1985. 

Autoantibodies in humans with cystic or alveolar echinococcosis

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