roxr~, vol. 30, r°. 9. ~. Iln-1IZ1 .199z . Printed in Gtat Britain.
ao41-0lol~t u.ao + .ao
® 1992 PerQamon Proa Ltd
SHORT COMMUNICATIONS IgG ANTIBODIES TO LOXOSCELES SP. SPIDER VENOM IN HUMAN ENVENOMING K. C. BA1ts~Ito, 1 J. L. C. CAxnoso, 2 V. R. D.
EICKST'I~T'
and I. MOTA l
'Centro de Pesquisa e Formaçâo em Imunolo~a Prof. Otto G . Hier, Instituto Botenfan, Av. Vital Brazil, 1500, 05504 Si;o Paulo, Brazil ; 'Hospital Vital Brazil; and'Seçâo de ArtropGdos Peçonhentos, Instituto Botenfan, Säo Paulo, Brazil (Received 5 Dectmber 1991 ; accepted 18 March 1992)
K. C. BARBARO, J. L. C. CAxnoso, V. R. D. E1Cxs~nT and I. MOTH. IgG antibodies to Loxosceles sp. spider venom in human envenoming. Toxicon 30, 1117-1121, 1992 .-The presence and specificity of IgG antibodies produced by patients with loxoscelism were studied. The loxoscelism diagnosis was supported mainly by clinical parameters . A search for IgG antibodies antiLoxosceles gaucho venom in patients with lozoscelism submitted to serumtherapy showed antibodies in four out of 20 patients. The IgG antibodies were detected as early as 9 days and as late as 120 days after bite. The highest IgG antibody titer was 1 :640 and the lowest was 1 :80. Immunoblotting tests showed that human anti-L. gaucho IgG antibodies recognize preferentially the components responsible for the dermonecrotic and lethal activities of the venom. A comparison of the clinical picture, the level of serum IgG antibodies and the dose of antivenom administered suggest that there is no relationship between these parameters . THE sP>D$x Loxosceles gaucho is the most common species of this genus in the state of Sâo Paulo, Brazil, and is responsible for the majority of accidents in this region (CAxnoso et al., 1988). Venom of Loxosceles spiders induces necrotic skin reactions and sometimes hemolytic effects in bitten patients . We report here the results of a study on human IgG antibodies to L. gaucho venom and its presumed relationship to the patients' clinical picture. The study was performed in 20 patients, with envenoming confirmed by clinical examination, treated with serumtherapy. Serum samples were obtained by centrifugation after blood coagulation and kept frozen at -20°C until used. Two sertun samples were collected from each patient one on the admission day and another on a later day. As antigen we used a pool of venom, obtained as previously described (BARBARO et al., 1992). The anti-L . gaucho venom antibody content of the patients' sera was determined by ELISA according to the method of T~AxsTOx et al. (1977) . The antibody specificity was detected by the Western blotting technique. Venom (300 hg) was first fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions (818% acrylamide resolutions gels), using the method of LAEMMI,I (1970) . The separated venom components were transferred to nitrocellulose membranes that were incubated with the patients' serum diluted 1 :20 as described by Towsnv et al. (1979) . As shown in Table 1, the time elapsed between the bite and the admittance of the patient to the hospital 1117
1118
Short Communications
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Short Communications
1119
semer Conrrwxuva ~rtrnan~s To Loxosceles gaucho velvox. Venom (300 ~was fractionated by SDS-PAGE (8-I8%)end revealed by Western blotting using patients' aman with IgG antibodies to the venom at a dilution of 1 :20. (1) Normal human sera; (2) patient serum ao. 16 ; (3) patient serum no. 1; (4) patient serum no . 18; (5) patient serum no . 2. Numbers at the right correspond to position of mol. wt markers. Mouse IgG (150,000), BSA (67,000), hen egg albumin (43,000), trypsinogen (24,000) and lysozyme (14,000) (Sigma Chemical Co, St. Louis, MO, U.S .A.) were used as mol. wt markers. Fia. 1.
IMNUNOBL0ITINO WITH PAT~NIS'
varied from 4 hr to 144 hr. The diagnosis of loxoscelism was supported by clinical signs such as swelling, infiltration area, erythema, ecchymosis, blister, ischaemia, exanthema and necrosis . However, it must be pointed out that these signs may also be present in patients bitten by other species of the genus Loxosceles (D11,LAxA et al., 1964 ; R>~s et al., 1985 ; Yourro and Pnv, 1988). Two out of 20 of these patients brought the spider, that was classified as Loxosceles sp. and L. gaucho . The majority of the patients were submitted to serumtherapy in the Vital Brazil hospital at the Butantan Institute. The range of antivenom serum administered i.v. in the patients was 2-10 vials (each vial contains 5 ml), and only one patient received the antiserum by the s.c. route . The horse anti-L. gaucho
1120
Short Communications
venom serum was prepared by Butantan Institute as described by FbRLANSrin et al. (1962) . The clinical picture was evaluated at different times after the bite. Necrosis was detected in nine patients, scar in eight, endurated plaque in one patient, and two patients were completely recovered by the time they were re-examined. IgG antibodies to anti-L. gaucho venom were detected in four out of the 20 patients' sera. Antibodies were detected in sera collected from different patients on days 9, 10, 67 and 120 after the bite. The highest antibody titer was 1 :640 (67 days after the bite) and the lowest was 1:80 (10 days after the bite). Figure 1 shows the results of the immunoblotting tests performed with the positive sera. The antibodies from these sera recognized venom components around the 35,000 mol. wt region . These components are responsible for the dermonecrotic and lethal activities of the venom (BARBARO et al., 1992). The intensity of staining of the components revealed in the blotting was correlated with the antibody titer. Patients' serum no. 16, which had an ELISA titer of 640, stained strongly (Fig. 1, strip 2), whereas patients' serum no. 2, with an ELISA titer of 80, stained only weakly (Fig. 1, strip ~. This suggests that the venom components present around the 35,000 mol. wt region are the most immunogenie venom component in humans. It is possible that most of .our patients have been bitten by L. gaucho since this species of the genus Loxosceles is responsible for the large majority of accidents in our region (CARDO80 et al., 1988). However, we cannot exclude the involvement of more rare species as L. laeta and L. intet~nedia since the venom of these spiders present antigenic cross-reactivity (to be published) . Furthermore, it is possible that some of the patients may have been bitten by a non-Loxosceles species. However, this chance is very small since the other spiders found in the state of Sâo Paulo do not induce the dermonecrotic lesion typical of Loxosceles venom (JORGE et al., 1990; R>BEmo et al., 1990 ; JORGE et al., 1991). The observation that only a few patients showed detectable antibodies may be due to serumtherapy . This may have reduced or even abolished the antibody response due to sequestration of the immunogenic material in the circulation or to feedback inhibition induced by the horse antivenom or by the suppressor effect of the venom-antivenom complexes. These results agree with those of Do1~ulvGOS et al. (1990), who reported a low specific antibody response to Bothrops jararaca snake venom in patients treated with serumtherapy . In spite of the serumtherapy, in all the cases where the patients produced antibodies to L. gaucho venom, their sera reacted with the dermonecrotic and lethal components of the venom. A comparison of the clinical picture, the level of serum IgG antibodies and the dose of antivenom administered to patients with loxoscelism suggests that there is no relationship between these parameters. Further studies using a larger number of patients are required to validate this suggestion. Acknowledgerrunts-This paper was supported by Fundaçito de Amparo à Pesquisa do Estado de Sâo Paulo (FAPESP grant 90/1879-2). K. C. B~tts~to was recipient of a FAPESP fellowship (89/1189-9). The suthore are indebted to Mr Ceaws A. JARS for excellent technical assistance . REFERENCES
HeaaNeo, K. C., Cexnaso, J. L. C., EICIC3rFDT, V. R. D. and More, I (1992) Dermonecrotic and lethal components of Loxosceles gaudes spider venom. Toxicon 30, 331-338. Cennoeo, J. L. C., Fwuvce, F. O. S., E~cgst~r, V. R. D., Boxo~, I. and Noave~xe, M. T. (1988) Loxosoelismo: estudo de 242 casus (1980-1984). Rcv. Soc, bras. Toxtcol. 1, 580. Du.uxe, C. J., Je~v, G. T., Horn~rcvrr, W. M. and HeYDEN, C. R. (1964) North American Loxosceliem . 1AMA 188, 153-156. Dowxoas, M. O., CNenoso, J. L., Mouse De Sn.ve, A. M. and More, I. (1990) The humoral immune responses of patients bitten by snake Bothrops jararaca (jararaca) . Toxicon 28, 723-726.
Short Communications
112 1
Fuxt.nrtrrrro, R. S., SeivTas, N. P. and Noves, J. (1962) Preparaçâo, purificaç$o e doseamento do sôro antiloxoscélico . Ci~ncia e Cultwa 14, 254. JOeaE, M. T., Lutes, S. M., VoN Eicesreor, V. R. D. and Rtameo, L. A. (1990) Acidentes por aranhas Clriracanthiwn : aspectos epidemiol6gicoa e elinioos . Mem. Inst. Butantan 52, Supplement, 81-$2. Joßoe, M. T., VoN EtcK .~or, V. R. D., KNVSee, I. and Rreettto, L. A. (1991) Curso sobre acidentes tom animais peçonhentos. Arq bras Med. 66, 45p6ß. i e,za~a , U. K. (1970) C,7eavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 68085. Ris, R. S., Ar .~vaExN, D. P., Lnvcx, J. H. and Ktxa, L. E. (1985) Brown recluse spider bites. A comparison of early surgical excision versus dapsone and delayed surgical excision. Ann. Surg. 202, 65963. Rn~neo, L. A., Joaoe, M. T., Pnssco, R. V. and N>stnotce, S. A. (1990) Wolf spider bites in Süo Paulo, Brawl: a clinical and epideatiological study of 515 cases. Toxicon 2g. 715-717. T~e¢srorr, R. D. G., Ltovn-Jots, M. J. and RPrn, H. A. (1977) Micro-ELISA for detecting and assaying snake venom and anti-venom antibody. Lancet ü, 639~r41. Towarrt, H., Srw»t.ua, T. and Gotenox, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications . Proc . natn. Acad. Sci. U.S.A . 76, 4350354. Youxa, V. L. and Pnv, P. (1988) The brown recluse spider bite. Arm. Plant. Surg. 211, 44752.
ao41-0lol~t u.ao + .ao
® 1992 PerQamon Proa Ltd
SHORT COMMUNICATIONS IgG ANTIBODIES TO LOXOSCELES SP. SPIDER VENOM IN HUMAN ENVENOMING K. C. BA1ts~Ito, 1 J. L. C. CAxnoso, 2 V. R. D.
EICKST'I~T'
and I. MOTA l
'Centro de Pesquisa e Formaçâo em Imunolo~a Prof. Otto G . Hier, Instituto Botenfan, Av. Vital Brazil, 1500, 05504 Si;o Paulo, Brazil ; 'Hospital Vital Brazil; and'Seçâo de ArtropGdos Peçonhentos, Instituto Botenfan, Säo Paulo, Brazil (Received 5 Dectmber 1991 ; accepted 18 March 1992)
K. C. BARBARO, J. L. C. CAxnoso, V. R. D. E1Cxs~nT and I. MOTH. IgG antibodies to Loxosceles sp. spider venom in human envenoming. Toxicon 30, 1117-1121, 1992 .-The presence and specificity of IgG antibodies produced by patients with loxoscelism were studied. The loxoscelism diagnosis was supported mainly by clinical parameters . A search for IgG antibodies antiLoxosceles gaucho venom in patients with lozoscelism submitted to serumtherapy showed antibodies in four out of 20 patients. The IgG antibodies were detected as early as 9 days and as late as 120 days after bite. The highest IgG antibody titer was 1 :640 and the lowest was 1 :80. Immunoblotting tests showed that human anti-L. gaucho IgG antibodies recognize preferentially the components responsible for the dermonecrotic and lethal activities of the venom. A comparison of the clinical picture, the level of serum IgG antibodies and the dose of antivenom administered suggest that there is no relationship between these parameters . THE sP>D$x Loxosceles gaucho is the most common species of this genus in the state of Sâo Paulo, Brazil, and is responsible for the majority of accidents in this region (CAxnoso et al., 1988). Venom of Loxosceles spiders induces necrotic skin reactions and sometimes hemolytic effects in bitten patients . We report here the results of a study on human IgG antibodies to L. gaucho venom and its presumed relationship to the patients' clinical picture. The study was performed in 20 patients, with envenoming confirmed by clinical examination, treated with serumtherapy. Serum samples were obtained by centrifugation after blood coagulation and kept frozen at -20°C until used. Two sertun samples were collected from each patient one on the admission day and another on a later day. As antigen we used a pool of venom, obtained as previously described (BARBARO et al., 1992). The anti-L . gaucho venom antibody content of the patients' sera was determined by ELISA according to the method of T~AxsTOx et al. (1977) . The antibody specificity was detected by the Western blotting technique. Venom (300 hg) was first fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions (818% acrylamide resolutions gels), using the method of LAEMMI,I (1970) . The separated venom components were transferred to nitrocellulose membranes that were incubated with the patients' serum diluted 1 :20 as described by Towsnv et al. (1979) . As shown in Table 1, the time elapsed between the bite and the admittance of the patient to the hospital 1117
1118
Short Communications
w
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Short Communications
1119
semer Conrrwxuva ~rtrnan~s To Loxosceles gaucho velvox. Venom (300 ~was fractionated by SDS-PAGE (8-I8%)end revealed by Western blotting using patients' aman with IgG antibodies to the venom at a dilution of 1 :20. (1) Normal human sera; (2) patient serum ao. 16 ; (3) patient serum no. 1; (4) patient serum no . 18; (5) patient serum no . 2. Numbers at the right correspond to position of mol. wt markers. Mouse IgG (150,000), BSA (67,000), hen egg albumin (43,000), trypsinogen (24,000) and lysozyme (14,000) (Sigma Chemical Co, St. Louis, MO, U.S .A.) were used as mol. wt markers. Fia. 1.
IMNUNOBL0ITINO WITH PAT~NIS'
varied from 4 hr to 144 hr. The diagnosis of loxoscelism was supported by clinical signs such as swelling, infiltration area, erythema, ecchymosis, blister, ischaemia, exanthema and necrosis . However, it must be pointed out that these signs may also be present in patients bitten by other species of the genus Loxosceles (D11,LAxA et al., 1964 ; R>~s et al., 1985 ; Yourro and Pnv, 1988). Two out of 20 of these patients brought the spider, that was classified as Loxosceles sp. and L. gaucho . The majority of the patients were submitted to serumtherapy in the Vital Brazil hospital at the Butantan Institute. The range of antivenom serum administered i.v. in the patients was 2-10 vials (each vial contains 5 ml), and only one patient received the antiserum by the s.c. route . The horse anti-L. gaucho
1120
Short Communications
venom serum was prepared by Butantan Institute as described by FbRLANSrin et al. (1962) . The clinical picture was evaluated at different times after the bite. Necrosis was detected in nine patients, scar in eight, endurated plaque in one patient, and two patients were completely recovered by the time they were re-examined. IgG antibodies to anti-L. gaucho venom were detected in four out of the 20 patients' sera. Antibodies were detected in sera collected from different patients on days 9, 10, 67 and 120 after the bite. The highest antibody titer was 1 :640 (67 days after the bite) and the lowest was 1:80 (10 days after the bite). Figure 1 shows the results of the immunoblotting tests performed with the positive sera. The antibodies from these sera recognized venom components around the 35,000 mol. wt region . These components are responsible for the dermonecrotic and lethal activities of the venom (BARBARO et al., 1992). The intensity of staining of the components revealed in the blotting was correlated with the antibody titer. Patients' serum no. 16, which had an ELISA titer of 640, stained strongly (Fig. 1, strip 2), whereas patients' serum no. 2, with an ELISA titer of 80, stained only weakly (Fig. 1, strip ~. This suggests that the venom components present around the 35,000 mol. wt region are the most immunogenie venom component in humans. It is possible that most of .our patients have been bitten by L. gaucho since this species of the genus Loxosceles is responsible for the large majority of accidents in our region (CARDO80 et al., 1988). However, we cannot exclude the involvement of more rare species as L. laeta and L. intet~nedia since the venom of these spiders present antigenic cross-reactivity (to be published) . Furthermore, it is possible that some of the patients may have been bitten by a non-Loxosceles species. However, this chance is very small since the other spiders found in the state of Sâo Paulo do not induce the dermonecrotic lesion typical of Loxosceles venom (JORGE et al., 1990; R>BEmo et al., 1990 ; JORGE et al., 1991). The observation that only a few patients showed detectable antibodies may be due to serumtherapy . This may have reduced or even abolished the antibody response due to sequestration of the immunogenic material in the circulation or to feedback inhibition induced by the horse antivenom or by the suppressor effect of the venom-antivenom complexes. These results agree with those of Do1~ulvGOS et al. (1990), who reported a low specific antibody response to Bothrops jararaca snake venom in patients treated with serumtherapy . In spite of the serumtherapy, in all the cases where the patients produced antibodies to L. gaucho venom, their sera reacted with the dermonecrotic and lethal components of the venom. A comparison of the clinical picture, the level of serum IgG antibodies and the dose of antivenom administered to patients with loxoscelism suggests that there is no relationship between these parameters. Further studies using a larger number of patients are required to validate this suggestion. Acknowledgerrunts-This paper was supported by Fundaçito de Amparo à Pesquisa do Estado de Sâo Paulo (FAPESP grant 90/1879-2). K. C. B~tts~to was recipient of a FAPESP fellowship (89/1189-9). The suthore are indebted to Mr Ceaws A. JARS for excellent technical assistance . REFERENCES
HeaaNeo, K. C., Cexnaso, J. L. C., EICIC3rFDT, V. R. D. and More, I (1992) Dermonecrotic and lethal components of Loxosceles gaudes spider venom. Toxicon 30, 331-338. Cennoeo, J. L. C., Fwuvce, F. O. S., E~cgst~r, V. R. D., Boxo~, I. and Noave~xe, M. T. (1988) Loxosoelismo: estudo de 242 casus (1980-1984). Rcv. Soc, bras. Toxtcol. 1, 580. Du.uxe, C. J., Je~v, G. T., Horn~rcvrr, W. M. and HeYDEN, C. R. (1964) North American Loxosceliem . 1AMA 188, 153-156. Dowxoas, M. O., CNenoso, J. L., Mouse De Sn.ve, A. M. and More, I. (1990) The humoral immune responses of patients bitten by snake Bothrops jararaca (jararaca) . Toxicon 28, 723-726.
Short Communications
112 1
Fuxt.nrtrrrro, R. S., SeivTas, N. P. and Noves, J. (1962) Preparaçâo, purificaç$o e doseamento do sôro antiloxoscélico . Ci~ncia e Cultwa 14, 254. JOeaE, M. T., Lutes, S. M., VoN Eicesreor, V. R. D. and Rtameo, L. A. (1990) Acidentes por aranhas Clriracanthiwn : aspectos epidemiol6gicoa e elinioos . Mem. Inst. Butantan 52, Supplement, 81-$2. Joßoe, M. T., VoN EtcK .~or, V. R. D., KNVSee, I. and Rreettto, L. A. (1991) Curso sobre acidentes tom animais peçonhentos. Arq bras Med. 66, 45p6ß. i e,za~a , U. K. (1970) C,7eavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 68085. Ris, R. S., Ar .~vaExN, D. P., Lnvcx, J. H. and Ktxa, L. E. (1985) Brown recluse spider bites. A comparison of early surgical excision versus dapsone and delayed surgical excision. Ann. Surg. 202, 65963. Rn~neo, L. A., Joaoe, M. T., Pnssco, R. V. and N>stnotce, S. A. (1990) Wolf spider bites in Süo Paulo, Brawl: a clinical and epideatiological study of 515 cases. Toxicon 2g. 715-717. T~e¢srorr, R. D. G., Ltovn-Jots, M. J. and RPrn, H. A. (1977) Micro-ELISA for detecting and assaying snake venom and anti-venom antibody. Lancet ü, 639~r41. Towarrt, H., Srw»t.ua, T. and Gotenox, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications . Proc . natn. Acad. Sci. U.S.A . 76, 4350354. Youxa, V. L. and Pnv, P. (1988) The brown recluse spider bite. Arm. Plant. Surg. 211, 44752.
