Antibody-based enzyme-linked immunosorbent assay for determination of anti-pgl-i specific circulating immune complex in leprosy patients

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1 Lepr Rev (1999) 7, Antibdy-based enzyme-linked immunsrbent assay fr determinatin f anti-pgl-i specific circulating immune cmplex in leprsy patients J. TOMIMORI-YAMASHITA*, P. CRUAUD**, O. ROTTA*, & P. H. LAGRANGE*** * Department f Dermatlgy, Federal University f sa Paul, Brazil ** Service de Micrbilgie, Hpital Jean Verdier, Bndy, France *** Service de Micrbilgie, Hpital Saint Luis, Paris, France Accepted fr publicatin 5 April 1999 Summary A serlgical study was perfrmed in 122 individuals: 75 leprsy patients and 47 healthy cntrls. The ELISA test was perfrmed fr IgG and IgM using the glyclipid PGL-I antigen frm Mycbacterium leprae. Circulating immune cmplexes (CIC) were islated by PEG 6 precipitatin methd and after dissciatin with an acid slutin, the IgG and IgM specific against PGL-I were tested with the ELISA test. The multibacillary patients had high levels f antibdies, cmpared with paucibacillary patients and cntrls. The antibdies islated frm the CIC presented a similar spectrum spectral distributin as the serlgy. A psitive crrelatin between the levels f free and CIC bund antibdies was bserved. In cntrast with tuberculsis patients, specific antibdies present in CIC were nt respnsible fr false-negative results fund in sme multibacillary patients' serlgy, since n r very lw levels f specific antibdies were fund in PEG precipitated serum f these patients. N relatin was bserved with specific antibdy levels detected in CIC during leprsy reactins. Intrductin When the bdy establishes cntact with a freign substance, an immune respnse may ccur. Tw types f specific respnse can be bserved: the cell-mediated and the humral immune respnses. Mycbacteria can stimulate antibdy prductin directed specifically against mycbacterial antigens. A specific antigen PGL-I (phenlic glyclipid I) frm Mycbacterium Zeprae has been described by Brennan and Barrw, l and has been used fr serdiagnsis Part f this study has been presented at the 57th Meeting-The Sciety fr Investigative Dennatlgy and Eurpean Sciety fr Dennatlgical Research (May 1996, Washingtn) Crrespndence t: Dr Jane Tmimri-Yamashita, Department f Dennatlgy, Federal University f Sa Paul, Rua Btucatu 74, CEP 423-9, Sa PaullSP, Brazil. Telefax: +55(11) jane.denn@ riginet.cm.br /99/ $1. Lepra 261

2 262 T. Tmimri-Yamashita et ai. in leprsy. High levels f immunglbulin G and M (lgg and IgM) against PGL-I have been detected in multibacillary patients, and sme authrs reprted higher IgM than IgG? - 4 Hwever, a small prprtin f multibacillary patients (BL and LL) have lw levels f anti-pgl-i antibdies, as d mst paucibacillary (BT and IT) patients. In tuberculsis, sme authrs recently fund that the negativity f serlgical tests in patients with cnfirmed tuberculsis was due t specific immune cmplexes in the serum. 5-8 We pstulated that specific antibdies present within circulating immune cmplexes (CIC) wuld nt be detected by the enzyme-linked irnmunsrbent assay (ELISA) test fr PGL-I, and this culd explain the false-negative reactins fund in sme paucibacillary and multibacillary patients sera befre and during treatment. Elevated CIC levels have been reprted in leprsy patients 9,!O and depsitin r in situ frmatin is thught t be a precipitating factr fr the develpment f the erythema ndsum leprsum (ENL)Y It is argued that sme CIC are nt eliminated and persist in the circulatin, causing a hypersensitivity reactin, resulting in immune cmplexe (IC) disease, especially when they are depsited in bld vessel walls and peripheral tissues causing inflarnmatin. 1 2 The plyethylene glycl (PEG) precipitatin test fr CIC was perfrmed t shw the presence f specific anti-pgl-i antibdy in the precipitates, and t verify if there is any relatinship between the specific CIC levels and the presence f leprsy reactin (reversal reactin r erythema ndsum leprsum), as the real rle f the specific CIC in the leprsy reactin is nt certain. Materials and methds SERA Seventy-five sera frm patients with leprsy [13 leprmatus leprmatus (LL), 26 brderline leprmatus (BL), 3 brderline bderline (BB), 16 brderline tuberculid (BT), 14 tuberculid tuberculid (IT) and 3 indeterminate] were btained frm the Department f Dermatlgy, UNIFESP-Escla Paulista de Medicina, Sa Paul, Brazil. Their ages ranged frm 14 t 63 years, and 39 female and 36 male patients were included. The diagnsis was made by clinical, bacterilgical (slit skin smears), histpathlgical and immunlgical (Mitsuda reactin) features. The cases were classified accrding t the Ridley-Jpling classificatin. 13 Operatinal classificatin f leprsy (Wrld Health Organizatin, 1988) patients accrding t their type f treatment was gathered: LL and BL patients as multibacillary leprsy and BT and IT as paucibacillary leprsy. This classificatin was used in this reprt because the humral respnse seems t crrbrate such peratinal view. All sera were cllected frm patients befre treatment, except frm five wh were treated fr less than 1 year. Thirteen patients had type 1 reactin (reversal) and fur patients type 2 reactin (ENL). Tw t fur samples f serial sera were cllected frm seven patients with leprsy reactin (n = 19 sera). The interval between each cllected sample was 15 days; these patients have been treated with prednislne and/r thalidmide. In additin, sera frm 47 nrmal bld dnrs frm the same hspital in Sa Paul, Brazil were included as cntrls. Their ages ranged frm 18 t 57 years; 16 females and 31 males. ANTIGEN The natural PGL-I f Mycbacterium Zeprae was used. The methds fr islatin, purificatin and characterizatin were reprted previusly.!

3 ELISA f r anti-pgl-/ 263 ELISA PROCEDURE An enzyme-linked immunsrbent assay (ELISA) was perfrmed, using the methd described by Cruaud et al.14 fr glyclipid mycbacterial antigens. Plystyrene micrtitre plates purchased frm COSTAR (USA) were cated with PGL-I (25 ng/well). Fr cating, the indicated amunts f antigen in f n-hexane were placed in the wells, and the slutins were left t dry vernight at 37 C. T ascertain the absence f nnspecific adsrptin, ne well treated with f n-hexane withut antigen was included fr each test. The strage f sera were at -2 C, until the serlgy was perfrmed. The sera were diluted 1125 in PBS (phsphate buffer saline) cntaining 5% f prcine gelatin (Sigma, USA). After saturatin by PBS cntaining 5% f prcine gelatin (vernight at 4 C), the plates were washed with PBS withut Tween (Micr plate washer LP 35, Diagnstics Pasteur, France) and f diluted sera was incubated at 37 C during 9 r 18 min fr IgG r IgM determinatin, respectively. After washings, the cnjugates with apprpriate dilutin were allwed t react fr 2 h at 37 C; the cnjugates were gat antihuman IgM (anti p.)lbeta-galactsidase (Bisys, France). After washing, the apprpriate substrate, 2-nitrphenyl-beta-D-galactpyranside (Merck, Germany) was added, and the plates were incubated at 37 C fr 3 min. T crrelate the data, three knwn tested sera having lw, medium and high levels f antibdies were used as standards. They were included in each plate, and PBS was used as the cntrl (zer pint activity). The clr develped was read at 45 nm, using an autmatic spectrphtmeter (LP 4, Sanfi Diagnstics Pasteur, France). After reading, fr each plate, a curve was drawn using the zer and standard values after calculatin f the slpe and the crrelatin cefficient (cc). If these data were nt satisfactry (slpe t lw, cc belw 98%...), the plate was rejected and the assay was repeated. The values f tested sera were crrected as fllws: first, the difference between absrbance f serum and the nnspecific absrptin was calculated. Then, the data were calculated t establish the crrected Li45 values by using the curve f the standards. PEG PRECIPITATION The PEG 6 (plyethylene glycl 6, Prlab, Paris, France) precipitatin test fr CIC (circulating immune cmplexes) precipitatin was perfrmed accrding t the methd described previusly by Luzir et al.,15 develped frm the riginal technique by Ohlsn and Zetterstrand. 16 The sera were diluted 111 with PBS (ph 7.4) withut Tween. T f diluted serum, f 5% PEG 6 in 15 mm NaCl, 2 mm ptassium phsphate buffer (ph 7-4) was added. The mixture was incubated vernight at 4 C. The precipitates were cllected by centrifugatin (155 g, 2 min, 4 C) (CR 4. 11, Juan, France), washed twice with 2 5% PEG 6 in buffer described belw and then were disslved in f 15 mm NaCl, 2 mm ptassium phsphate buffer (ph 7 5) 1 mm ethylene diamine tetraacetic acid [EDT A (Sigma, France)] by incubatin fr 3 min at 37 C. T f the slubilized CIC slutin, f cld 2 M HC1-glycine (PH 2 8) was added and incubated at 4 C fr 15 min. This slutin was neutralized with f 1 M K2HP4 (ph 9 ) and then diluted 112 by adding f PBS cntaining 5% f prcine gelatin withut Tween. This slutin was tested fr ELISA within 3 minutes. The main difference between the CIC precipitatin test and the current ELISA serlgy was the dilutin factr fr CIC 112, and fr the sera, 1125.

4 264 T. Tmimri-Yamashita et al. SUPERNATANT ANALYSIS After the vernight serum incubatin at 4 C with 2 5% PEG slutin and first centrifugatin, 4 III f the supernatant (crrespnding vlume f 1 III f serum) were diluted in 2 5 ml f PBS with 5% f prcine gelatin, and distributed in the ELISA plates. The final dilutin crrespnded t an equivalent 1125 serum dilutin. Fr sme sera (n = 24), a duble PEG precipitatin was perfrmed. The first supernatants were re-precipitated, repeating the same technique fr PEG precipitatin. The precipitate was analysed t verify if sme CIC remained in the supernatant after this first PEG precipitatin. The secnd supernatant btained frm the re-precipitatin was tested fr ELISA in fllwing prceeding: 16 III f the secnd supernatant was diluted in 2 34 ml PBS cntaining 5% prcine gelatin (a final serum dilutin f 1125), and tested fr ELISA. It allwed us t determine whether the antibdy activity remained after tw PEG precipitatins. STA TISTICAL ANALYSIS The ANOVA (Analysis f Variance) and Kruskal-Wallis nn-parametric test were perfrmed fr statistical analysis f the antibdies levels in respect t the different frms f leprsy and the cntrl grup. A bx plt graphic was develped t represent the 1, 25, 5 (median), 75 and 9 percentiles f serlgy repartitin results f each leprsy frms and cntrl grup, analyzing the IgG and IgM anti-pgl-i levels. The scattergraphs were used t represent the crrelatin f antibdies levels in leprsy patients and fr all these statistical analysis the crrelatin cefficient was calculated. The cntingency tables were analysed by the X2 test, using a significant P-value f <5%. Results INDIVIDUALS AND MEAN ANTIBODY LEVELS AGAINST PGL-I IN SERUM The initial assessment was perfrmed by ELISA, using PGL-I as antigen, n all whle serum befre PEG precipitatin. As expected, higher IgG and IgM antibdies levels were bserved (Figure 1) in the serum f LL and BB leprsy patients as cmpared with thse f BT and TT patients. A significant difference (P < '5) in IgG and IgM levels was fund between healthy cntrls and multibacillary (LL + BL) patients. Mean levels were als significantly higher (P < '5) in multibacillary (LL + BL) patients than in paucibacillary patients (BT + TT). The crrected 1145 ptical density value crrespnding t the 95 percentiles f all healthy subjects was taken as the nrmal upper limit fr each immunglbulin class (Table 1). Using this definitin, we btained a specificity f 95% fr IgG and IgM classes. The sensitivity was 74 % and 57 1 % fr IgG and IgM classes, respectively fr the leprsy patients cnsidered as a whle grup. Hwever, when the leprsy grup was subclassified int tw grups, the multibacillary and the paucibacillary, the sensitivity was 97'4% and 92 3% fr IgG and IgM classes in the multibacillary grup and 45 2% and 19 3% fr IgG and IgM classes fr the paucibacillary grup. LL and BL leprsy patients were significantly (P < '1) mre psitive than BT and TT patients fr bth immunglbulin classes. The presence f specific IgG and IgM antibdy against PGL-I in dissciated CIC was tested in the same sera after the PEG precipitatin.

5 12 1 ELISA f r anti-pgl-i 265 A 8!J. 45 nm x 13 6 IgG anti PGL I LL BL BB BT TT Ind M- Ind M+ C 1 B 8 b. 45 nm X 13 6 IgM anti PGL-I 4 LL BL BB BT TT Ind M- Ind M+ c Figure 1. Distributin f antibdies levels anti-pgl-i IgG (A) and IgM (B) in whle sera accrding t the different frms f leprsy: 13 leprmatus leprmatus (LL), 26 brderline leprmatus (BL), 3 brderline brderline (BB), 16 brderline tuberculid (BT), 14 tuberculid tuberculid (TI), ne indeterminate with negative Mitsuda's reactin (Ind M-), 2 indeterminate with psitive Mitsuda's reactin (Ind M+) patients and 47 healthy cntrls (C). DEMONSTRATION OF SPECIFIC ANTIBODY AGAINST PGL-I IN IMMUNE COMPLEXES All cllected sera were subjected t PEG precipitatin, the CIC were dissciated and the liberated IgG and IgM immunglbulin classes were assayed by ELISA using the PGL-I antigen. Figure 2 illustrates the distributin f the individual IgG and IgM antibdy levels after PEG precipitatin in respect t the different frms f leprsy and cntrl grup. As

6 266 T. Tmimri-Yamashita et al. Table 1. Repartitin f psitive results fr each grup f leprsy patients. The cut-ff pint is defined as the crrected Ll.45 ptical density value crrespnding t the 95 percentiles f all healthy subjects is taken as the nrmal upper limit in each immunglbulin class. Using this definitin, the specificity is 95% Free immunglbulins CIC cmplexed immunglbulins Bth free and CIC cmplexed Immunglbulins Grups ps. (%) ps. (%) ps. (%) ps. (%) ps. (%) ps. (%) ps. (%) ps. (%) ps. (%) LL (n = 13) BL (n = 26) BB (n = 3) BT (n = 16) IT (n = 14) Ind. Mit + (n = 2) Ind. Mit - (n = I) 13 (1) 12 (92'3) 13 ( 1) II (84,6) 1 (76'9) 25 (96'2) 24 (92'3) 26 (96'2) 2 (74') 23 (85'2) 3 ( 1) I (33'3) 3 (1) 2 (66,7) I (33'3) 8 (5') 4 (25,) 8 (5') 2 ( 12'5) 2 (12,5) 6 (42'9) 2 (13-3) 6 (4') 2 ( 13-3) 2 (13-3) I (5') () I (33'3) () I (33'3) I (1) I (1) I (1) () I ( 1) II (84'6) 13 (1) 12 (92'3) 13 ( 1) 23 (85'2) 26 (96'3) 25 (92'6) 26 (96'3) 2 (66,7) 3 (1) I (33'3) 3 ( 1) 4 (25,) 1 (62'5) 6 (37'5) 1 (62,5) 2 ( 13-3) 6 (4') 3 (2') 6 (4') I (33'3) I (33'3) I (33'3) 2 (66'7) I ( 1) I (1) I (1) 1(1) already shwn in Figure 1, higher IgG and IgM antibdy levels were bserved in ele f BL + LL frms as cmpared with BT, TT and cntrls. The spectral distributins appeared t be similar, when the antibdy titers were cmpared with thse btained with the whle serum befre ele precipitatin. The difference between the BL + LL and the BT + TT frm was mre evident fr IgM. RELATION BETWEEN ANTI-PGL-I FREE IMMUNOGLOBULINS AND SPECIFIC ANTIBODIES CONT AINED IN CIC As shwn in Figure 3, individual sera with high titers f free IgM antibdies als had high titers f the same antibdy islated frm ele. Althugh the dilutin was nt the same fr serlgy and eie precipitatin, a psitive crrelatin was bserved in patients between the free antibdies and thse islated frm ele (r = '885; P < '1). Cmparing leprsy patients with and withut reactin, n significant difference n IgM islated frm ele was nticed. SUPERNATANT ANALYSIS The analysis f the remained individual supernatants tested by the same ELISA methd shwed similar results in cmparisn with serlgy (r = '983, P < '1). N difference f free immunglbulins levels was bserved befre and after PEG precipitatin n the studied sera (Figure 4) indicating that free immunglbulins were nt cprecipitated by PEG. When the supernatant was re-precipitated by PEG, using the same technique, the precipitate btained had very lw r n antibdy activity. The secnd supernatant had almst the same activity when cmpared with the first supernatant r serlgy (data nt shwn). OCCURRENCE OF CIC AND FREE ANTIBODIES IN TYPE I AND TYPE 2 REACTIONS A difference between patients with r withut reactin (type I and 2) in relatin t the specific antibdies levels, was nt bserved in all tested sera. The same analysis was als

7 EUSA f r anti-pgl-/ A nmx1 3 IgG anti PGL-I D n Ind M- Ind M+ C L--r _ r_----r_-- - LL BL BB BT " a B nm x 1 3 IgM anti PGL-I LL BL BB BT n Ind M- Ind M+ C Figure 2. Distributin f dissciated anti-pgl-i IgG (A) and IgM (B) frm plyethylene glycl 6 precipitated circulating immune cmplexes accrding t the different frms f leprsy: 13 leprmatus leprmatus (LL), 26 brderline leprmatus (BL), 3 brderline brderline (BB), 16 brderline tuberculid (BT), 14 tuberculid tuberculid (TT), ne indeterminate with negative Mitsuda's reactin (Ind M-), 2 indeterminate with psitive Mitsuda's reactin (ind M+) patients and 47 healthy cntrls (C). valid fr antibdies islated frm ele. N significant difference (x 2 = 171, P > 5) was bserved cmparing patients with r withut leprsy reactins fr anti-pgl-i antibdies islated frm eie (Table 2). The cut-ff-pint fr this analysis was '253 (.::l45) (mean value plus three standard deviatins f btained titres in the cntrl grup). Using this analysis separately fr reactin type 1 and 2, we fund the same results. There are n significant differences in patients with reactin type 1 (x 2 = '263, P > '5) r in patients with reactin type 2 (X 2 = 17, P > 5), cmpared t patients withut leprsy reactin, althugh the number f patients with leprsy reactin was small. Nine patients with BT frm had reactin type 1 and amng them, eight patients had negative IgM titres f anti-pgl-i islated frm eie. Fur patients with BL frm had reactin type 2, and 3 f them had psitive titres fr IgM anti-pgl-i islated frm eie.

8 268 T. Tmimri-Yamashita et al r 1 r1' 1 1 cp IgM anti PGL-I in sera Figure 3. Scattergraphs shwing individual paired IgM levels against PGL-I btained by ELISA (.i45 nrn x 1 3 ) in sera and circulating immune cmplexes f leprsy patients. Open circles fr patients withut reactin and clsed circles fr patients with reactins (r = '885, P :5 '1) ; ;; 6 E..... ill 4 2 sera Figure 4. Scattergraphs shwing individual paired IgM levels against PGL-I btained by ELISA (45 nm x 1 3 ) in whle sera and in plyethylene glycl 6 treated supernatants (r = '983, P :5 '1).

9 Table 2. Cmparisn f psitive r negative results f anti-pgl-i IgM antibdies islated frm circulating immune cmplexes in leprsy patients witb r witbut reactins (type I r type 2) EUSA fr anti-pgl-i 269 Psitive Negative Ttal Withut reactin Reactin type I r type 2 Ttal i = '171, P > 5. Serial sera were cllected during leprsy reactin, but there was n scillatin f the serlgy titres (data nt shwn). Discussin The specific PGL-I antigen frm Mycbacterium leprae had been largely used fr serlgical studies in leprsy diagnsis, althugh it is nt used rutinely. As shwn in this reprt, almst all multibacillary leprsy patients (LL and BL) have high levels f IgG and IgM against PGL_ In the present wrk, leprsy sera fr PGL-I serlgy were tested by ELISA and the results are in agreement with ur previus wrk. 14, 1? High antibdy titres were bserved in the multibacillary grup (LL and BL) cmpared with lw titres in the paucibacillary grup (BT and TT) and cntrls. The specific IgM antibdies against PGL-I had higher titres while IgG presented lw titres in the sera and in the PEG 6 precipitates frm CIC. In the present study, almst all the patients studied had nt received specific leprsy treatment and, the attentin was fcused n IgM, as the majrity f the studies in leprsy cncern this immunglbulin. The IgG and IgM antibdy against PGL-I islated frm CIC by PEG precipitatin test had the same spectral distributin bserved in serlgical studies by ELISA, that extends ur previus bservatin. I? The present data cmparing the frequency f specific antibdy present in the whle serum with that in dissciated CIC, d nt cnfirm ur hypthesis since leprsy patients with lw antibdy levels in serum als have lw antibdy titres measured by ELISA in CIC. Other factrs fr the false-negativity in the multibacillary patients must be investigated, such as cncmitant acquired immunsuppressin. Amng the three typical multibacillary leprsy patients, whse serlgical and CIC analysis were negative, ne f them was c-infected with human immundeficiency virus (HIV). Anther explanatin fr the lw antibdy levels is that perhaps ser-psitivity is related t high systemic and skin bacterial lad. Perhaps exclusive high cutaneus bacterial lad des nt prduce high levels f antibdies. We als bserved tw patients with a negative bacterial index and high psitivity fr serlgy, the histpathlgy and Mitsuda reactin, suggesting the multibacillary frm. In these cases, perhaps the systemic bacterial lad crrelated with the high antibdy titres, but nt with the skin bacterial lad. The levels f specific free antibdies and islated antibdies frm the CIC were crrelated, and the presence f CIC did nt mdify these antibdies levels as detected by

10 27 T. Tmimri-Yamashita et al. ELISA using the glyclipid PGL-I antigen. The CIC precipitatin methd by PEG des nt increase the sensitivity f the ELISA test (Table 1). This is in sharp cntrast with the results described by Bhattacharya et al.,1 8 studying CIC by PEG precipitatin methd in tuberculsis. They presented data cmparing the frequency f antibdy with that f immune cmplexes. Circulating immune cmplexes were less frequently bserved in their cntrl subjects. Hwever, the data presented by Bhattacharya et al. 1 8 did nt give any assessment f the antigenic cmpnents f Mycbacterium tuberculsis in the immune cmplexes. The CIC islatin by the 2 5% PEG precipitatin is very efficient. The supernatant after the first precipitatin had similar antibdy levels as was fund in serlgy analysis, s PEG used in this cnditin did nt precipitate the free immunglbulins present in the sera. Submitting this supernatant t anther re-precipitatin by PEG, very lw r n antibdy was fund in the secnd precipitate, it demnstrated that all CIC have been effectively detected in the first precipitatin. The supernatant resulted after the secnd precipitatin shwed almst the same antibdy against PGL-1. Analysing the levels f specific antibdies in sera and in CIC, we did nt find any difference between patients with r withut reactin (reversal reactin and ENL). Sme authrs have demnstrated that levels f free specific antibdies were nt increased in reactinal frms. 1 9, 2 Separate analysis f different leprsy reactins did nt shw any difference in patients with reactin type 1 r 2, cmparing t patients withut leprsy reactin. Patients with reactin type 1 presented a spectral results accrding t leprsy frm. Paucilbacillary patients (BT) presented lw levels f antibdies in sera and in CIC and; multibacillary patients (BL), high anti-pgl-i titres. These findings shw that high levels f specific antibdy, free r binding t CIC, is nt related t leprsy reactin type 1, but t leprsy frm. Unfrtunately, this study did nt include a large number f patients with reactin type 2, as we analysed patients befre the intrductin f specific treatment. Mst patients have ENL during the treatment, and we culd nt include patients with a lng perid f treatment, as these patients wuld have lw levels f anti-pgl-1. In ur previus wrk, 1 7 we have nt fund statistically significant difference, cmparing patients with and withut type 2 reactin, during specific treatment. Sme authrs reprted that there is a decrease f antibdies during ENL and an increase after this reactin. This antibdy level variatin culd be due t residual depsitin f antibdies r immune cmplexes?, 2 1 In this reprt, we were testing nly the specific CIC cntaining PGL-I antigens and anti PGL-I antibdies. Such specific CIC did nt seem t be invlved in leprsy reactin. Other antigen r antibdy culd be invlved, since Ramanathan et al. 1 O reprted different results. These authrs measured CIC by PEG precipitatin, and fund a high titre f ttal CIC precipitates in all the patients wh presented with a leprsy reactin. They als fund that althugh BT and LL patients had elevated CIC titres, this increase was much less, cmpared t BT with reactin and ENL, respectively. A deficiency in any f the cmplement cmpnents culd be respnsible fr the lack f disslutin f immune aggregates in plasma, and thus further precipitate them in varius tissues during the inflammatry prcesses. 11, 22 Accrding t ur findings, we can cnclude that leprsy patients, mainly BL + LL frms, prduce specific CIC and their presence in peripheral circulatin are nt related t leprsy reactins. Hwever, we are nt able t cnclude if the peripheral specific CIC reflect part f the immune cmplexes that are depsited in the tissues and if they have an imprtant immunlgical rle.

11 ELISA fr anti-pgl-/ 27 1 Acknwledgements This study was supprted by CNPq (Cnse1h Nacinal de Desenvlviment Cientific e Tecn16gic), Brazil, which prvided a schlarship fr the first authr; and partially by an ANRS (RJN 12), France. References I Brennan PJ, Barrw WW. Evidence fr species-specific lipid antigens in Mycbacterium leprae. Int J Lepr Other Mycbact Dis, 198; 48: Ch S-N, Yanagihara DL, Hunter SW, Gelber RH, Brennan PJ. Serlgical specificity f phenlic glyclipid I frm Mycbacterium leprae and use in serdiagnsis f leprsy. Infect Immun, 1983; 41: Levis WR, Meeker HC, Schuller-Levis G, Sersen E, Schwerer R. IgM and IgG antibdies t phenlic glyclipid I frm Mycbacterium leprae in leprsy: insight int patient mnitring, erythema ndsum leprsum, and bacillary persistence. J Invest Dermatl, 1986; 86: Miller RA, Grder D, Harnisch JP. Antibdies t phenlic glyclipid-i during lng term therapy: serial measurements in individual patients. Int J Lepr Other Mycbact Dis, 1987; 55: Brstff J, Lenzini L, Rttli L. Immune cmplexes in the spectrum f tuberculsis. Tubercle, 1981; 62: Carr RI, Chakrabarty AK, Brunda MJ, Davidsn PT, Damle PB, Hardtke MA, Gilbride KJ, Minden P. Immune cmplexes and antibdies t BCG in sera frm patients with mycbacterial infectins. Clin Exp Immunl, 198; 39: Grange JM. The humral immune respnses in tuberculsis: its nature, bilgical rle and diagnstic usefulness. Adv Tuberc Res, 1984; 21: Simmney N, Mlina JM, Mlimard M, Oksenhendler E, Lagrange PH. Circulating immune cmplexes in human tuberculsis sera: demnstratin f specific antibdies against Mycbacterium tuberculsis glyclipid (DA T, PGLTb l, LOS) antigens in islated circulating immunes cmplexes. Eur J Clin Invest, 1997; 27: Bjrvatn B, Barnetsn RS, Krnvall G, Zubler RH, Lambert PH. Immune cmplexes and cmplement hyperctablism in patients with leprsy. Clin Exp Immunl, 1976; 26: t Ramanathan VD, Parkash, Ramu G, Parker D, Curtis J, Sengupta U, Turk JL. Islatin and analysis f circulating immune cmplexes in leprsy. Clin Immunl Immunpathl, 1984; 32: II Sehgal VN, Sharma V, Sharma VK. Cmprehensive evaluatin f cmplement cmpnents in the curse f type I (lepra) and type II (ENL) reactins. Int J Dermatl, 1989; 28: Daha MR. Measurement and rle f immune cmplexes in disease. J Int Fed Clin Chem, 1993; 5: Ridley DS, Jpling WH A classificatin f leprsy fr research purpses. Lepr Rev, 1962; 33: Cruaud P, Yamashita JT, Casabna NM, Papa F, David HL. Evaluatin f a nvei 2,3-diacyl-trehalse-2' -sulphate (SL IV) antigen fr case finding and diagnsis f leprsy and tuberculsis. Res Micrbil, 199; 141: Luzir H, Temynck T, Grgi Y, Ayed K, Avrameas S. Enzyme immunassay analysis f antibdy specificities present in the circulating immune cmplexes f selected pathlgical sera. J Immunl Methds, 1988; 114: Ohlsn S, Zetterstrand K. Detectin f circulating immune cmplexes by PEG precipitatin cmbined with ELISA. J Immunl Methds, 1985; 77: Yamashita JT, Cruaud P, Papa F, Rtta, David HL. Circulating immune cmplexes in leprsy sera: demnstratin f antibdies against mycbacterial glyclipidic antigens in islated immune cmplexes. Inl J Lepr Other Mycbact Dis, 1993; 61: Bhattacharya A, Ranadive SN, Kale M, Bhattacharya S. Antibdy-based enzyme-linked immunsrbent assay fr determinatin f immune cmplexes in clinical tuberculsis. Am Rev Respir Dis, 1986; 134: Schwerer B, Meeker HC, Sersen G, Levis WR. IgM antibdies against phenlic glyclipid I frm Mycbacterium leprae in leprsy sera: relatinship t bacterial index and erythema ndsum leprsum. Acta Leprl (Geneve), 1984; 2: Lyns NF, Shannn EJ, Ellis BPB, Naafs B. Assciatin f IgG and IgM antibdies t phenlic glyclipid-i antigen f Mycbacterium leprae with disease parameters in multi bacillary leprsy patients. Lepr Rev, 1988; 59: Andreli A, Brett SJ, Draper P, Payne SN, Rk GA W. Changes in circulating antibdy levels t the majr phenlic glyclipid during erythema ndsum leprsum in leprsy patients. Int J Lepr Other Mycbact Dis, 1985; 53: Chakrabarty AK, Kashyap A, Sehgal VN, Saha K. Slubilizatin f prefrmed immune cmplexes in sera f patients with type 1 and type 2 lepra reactins. Int J Lepr Other Mycbact Dis, 1988; 56:

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