Recognition of Treponema pallidum Antigens by IgM and IgG Antibodies in Congenitally Infected Newborns and Their Mothers

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1 THE JOURNAL OF INFECTIOUS DISEASES VOL. 157, NO.5. MAY by The University of Chicago. All rights reserved /88/ $01.00 Recognition of Treponema pallidum Antigens by IgM and IgG Antibodies in Congenitally Infected Newborns and Their Mothers Simon R. M. Dobson, Larry H. Taber, and Robert E. Baughn From the Departments of Pediatrics, Microbiology and Immunology, and Dermatology, Baylor College of Medicine; and the Syphilis Research Laboratories, Veterans Administration Medical Center, Houston, Texas Immunoblotting techniques were used to examine the proteins of Treponema pallidum recognized by IgM and IgO antibodies in sera from infants with congenital syphilis and their mothers. Infected infants' serum IgM reactivity to treponemal antigens differed from that of control infants born to normal, serofast, and biologic false-positive mothers. Each of the infected infants' sera exhibited IgM reactions to the 47- and 37-kilodalton (kda) proteins of T. pallidum. Although rheumatoid factor was detected in the sera of half of the infected infants, removing this factor did not alter the pattern of IgM blots. IgO reactions in infants were almost exclusively of the IgOI and Ig03 subclasses and mirrored those of the mother, except for IgOI and IgG3 reactions to the 83-kDa treponemal protein, which were unique to infants' sera. Our results suggest that the findings of IgM antibody directed against the 47- or 37-kDa antigens of T. pallidum may help to diagnose congenital syphilis at birth. Congenital syphilis remains an important serious, but treatable, infection in newborns. Its diagnosis continues to be a problem, particularly in those infants born without overt symptoms to mothers with positive reagin and treponemal antibody tests and with no history of adequate treatment before or during pregnancy. The Venereal Disease Research Laboratory slide test (VDRL) and Fluorescent Treponemal Antibody Absorption Test (FTA-ABS) assess a combination of both IgG and IgM classes of antibodies [1], but positive tests in the newborn may only reflect placental transfer of IgG from the mother. IgM does not cross the placenta, and its production by the fetus suggests active infection [2]. Initial optimism was expressed when Scotti and Lo- Received for publication 17 August 1987and in revised form 10 December This study was supported by funds from the Veterans Administration and by grant R30/CCR l from the Public Health Service. Dr. Dobson is the recipient of a Research Training Fellowship in Sexually Transmitted Diseases from the American Social Health Association and a Wellcome Trust Research Travel Grant. We thank Dr. Carol J. Baker (Baylor College of Medicine) for helping to acquire the STD Research Fellowship for Dr. Dobson and for her critical review of the manuscript; we also thank Inez M. Campbell for assistance in preparing this manuscript. Please address requests for reprints to Dr. Robert E. Baughn, Syphilis Research Laboratory, Building 211, Room 230, Veterans Administration Medical Center, 2002Holcombe Boulevard, Houston, Texas gan [3]in 1968introduceda modification of the FTA ABS test to detect IgM to Treponema pallidum. This method was not borne out, however, by clinical testing in the newborn and gave a false-positive rate of and a false-negative rate of up to 35% [4]. Thus, the FTA-ABS IgM test has languished, as has further study of the humoral immune response in infants with congenital syphilis. Little is known about the antigens of T.pallidum against which fetal antibodies are formed. Information regarding these antigens would allow further studies as to their role in pathogenesis, their ability to confer resistance in both the cellular and humoral areas ofdefense, andtheir potential for use as diagnostic reagents. Furthermore, the presence of IgM directed againstmaternaligg in the sera ofcongenitally infected infants, including infants with syphilis, has been described [5], and this fact may obscure investigationofthe IgM response to T. pallidum antigens. In the present study, protein blotting techniques (western blots) were used to detect antigens of T.pallidum recognized by IgM, IgG, and IgG subclass antibodies in sera from congenitally infected infants when compared with those from infants born to serofast, biologic false-positive (BFP) and uninfected mothers. Attempts were made to determine whether congenitally infected infants produced antibodies to antigens that were not recognized by antibodies in their mothers. Additionally, we deter- 903

2 904 Dobson, Taber, and Baughn mined the presence of 7s and 19srheumatoid factors (RF) in the sera of the infected infants, and we used an immunoprecipitation technique to investigate the pattern of antigen recognition by IgM antibody before and after removing IgG. Patients and Methods Sera. The study group consisted of six infants with congenital syphilis and the mothers of five of these babies. Clinical and serological details are outlined in table 1. All the infants fulfilled criteria for diagnosis as suggestedby Rathbun [6]. Controls were sera from five infants born to mothers with negative rapid plasma reagin (RPR) tests and no history of syphilis and serum pairs from infants and mothers who were either BFP with positive RPR but negative MHATP (microhemagglutination antibody to T. pallidum; three pairs) or serofast, as determined by repeated, unchanging, positive RPR at low titers in the light of documented, adequate treatment for previous infection (five pairs). Sera were obtained by venipuncture within two days of birth or on the day ofhospital admission (patients 3 and 5). Exceptions were the infants born to uninfected mothers from whom cord blood was obtained at parturition. Immunologic reagents and preparation of 125 1_ labeled second antibodies. Unconjugated second antibodies to human IgM and IgG were obtained from Microbiological Associates (Bethesda, Md) or Cappel/Worthington Biochemicals (Cooper Biomedical, Malvern, Pa). Monoclonalantibodies to the human IgG subclasses were obtained from Chemicon International (El Segundo, Calif) and from Unipath (Bedford, England). IgG subclass reference preparations were obtained from the World Health Organizational/International Union of Immunologic Societies Immunoglobulin Subcommittee (Berne, Switzerland). After DEAE Affi-Gel blue (Bio-Rad, Richmond, Calif) chromatography to remove carrier bovine serum albumin (BSA), immunoglobulin fractions were labeled with (1 2 5 I]-Na as described [7]; unbound 125 1was removed by passing radiolabeled materials through PD-1O columns (Pharmacia Fine Chemicals, Piscataway, NJ). Before labeling, monoclonal antibodies were purified using Affi-Gel Protein A MAPS II (Bio-Rad). Western blot. Solubilized proteins of T. pallidum subspecies pallidum [8, 9] were subjected to discontinuous SDS-PAGE by using 3070 stacking gels and separating gels of 10% acrylamide [10] or % gradient gels [7]. Gels were fixed in 10% acetic acid, stained with coomassie brilliant blue, and destained; the molecular weights of individual proteins were then determined by comparing their relative electrophoretic mobilities with those ofstandard proteins (Bio-Rad). The molecular weights, expressed in kilodaltons (kda), in the present study conform to the consensus weights agreed upon by Norris et al. [11]. Resolved proteins from materials run on duplicate slab gels were electrophoretically transferred to nitrocellulose paper to further characterize host proteins and treponemal antigens by using a modification ofthe method described by Towbin et al. [12]. Proteins were transferred at 190 rna for 3 h in a buffer consisting of 25 mm Tris, 192 mm glycine, and methanol. Sheets werecut into separate strips corresponding to individual lanes, and the efficacy of the transfer was assessed by staining one of the strips with amido black. Other strips were placed in PBS (ph 7.4) containing 0.025% Tween 20 (PBS-Tween) for 1 h at room temperature (rv23 C) to block nonspecific protein binding sites [13]. All subsequent steps were also carried out at room temperature. Strips were then incubated overnight with sera at a 1:1000 dilution. After a series of three washes in PBS-Tween, strips were exposed to rv2 x 10 6 cpm of the appropriate second antibody for 2 h, washed with several changes of PBS-Tween to remove unbound label, rinsed in water, and air dried. Blots of IgG subclass reference preparations (myelomas) wereused to verify the specificity of each 125 1_ labeled monoclonal antibody used. The nitrocellulose papers wereautoradiographed for various times (12-48 h) with Kodak ARX5 film (Kodak, Rochester, NY). RIA for IgM and IgO RF. The ELISA methodologies for 19s IgM and 7s IgG RF described by Faith et al. [14] were modified for RIA. The reason for the modification was the technical constraint imposed by limited amounts of infants' sera, amounts that made it virtually impossible to perform ELISAs based on titration analysis starting with triplicate 1:12 dilutions of sera. Polyvinyl microtiter wells were coated with 100 tjl of human IgO (1 ug/ml. in 0.1 M carbonate, ph 9.6) or rabbit IgG (2 ug/rnl. in carbonate) to detect IgM and IgG RF, respectively. After overnight incubation at 4 C, wells for detecting IgM RF were washed three times with PBS-Tween, and 100-tJL aliquots of serial dilutions of test sera were added to triplicate wells. After overnight incubation at room temperature, wellsweresubsequently

3 IgM and IgG in Congenital Syphilis 905 washed three times with PBS-Tween and then exposed to cpm of125i-iabeledgoat antibody to human IgM for 2 h at room temperature. For IgG RF, sera were diluted 1:5 in pepsin-acetate buffer [15], neutralized with an equal volume ofdisodium hydrogen phosphate in PBS-Tween, then added in triplicate to the wells of plates coated with rabbit IgG, and incubated overnight at room temperature. These plates, after appropriate washings, were reacted with cpm per well of 125I-Iabeled Ftab'), fragments of goat antibody to human IgG for 2 h at room temperature. After exposure to the radiolabeled second antibodies, plates were washed five times with PBS Tween, and individual wells were cut out and counted in a gamma counter. RF-positive and RF-negative control sera were assayed with each run. Nonspecific binding in these RIAs was measured by counting the amount of 125I-Iabeledsecond antibody that bound to dilutions of serum samples that had been added to microtiter wells coated with only 1% BSA in lieu of antigen and was determined by the following formula: nonspecific binding = (BSA + serum) (BSA + BSA). Total binding was determined by subtracting antigen plus BSA from antigen plus serum. Net binding by each serum sample was determined by subtracting nonspecific binding from total binding. Removing RFby immunoprecipitation ofigg in sera. A modification of the method described by Cernyet al. [16] was used to remove RF. Forty microliters ofeach serum specimen to be precipitated was diluted 1:50 in borate buffer (ph 8.4; g of boric acid, of sodium tetraborate, and g of NaCI in 1 L of water) using glass tubes. The diluted serum (2 ml) was then added to 2 ml of a 6% polyethylene glycol (PEG) solution in borate buffer and 2 ml of goat antibody to human IgG Table 1. Characteristics of infected infants. Patient Age (d) Sex Signs and symptoms for a final dilution of 1:150. After incubation overnight at 4 C the mixture was centrifuged at 1700 g for 40 min. The supernatant was used in the western blot analysis, except for a 500-J.1L aliquot that was stored at - 70 C for later use in a repeat IgM RF RIA. Results 1 1 F Hepatosplenomegaly, osteitis 2 I F Rash, hepatosplenomegaly, osteitis 3 52 M Rash, rhinitis, hepatosplenomegaly 4 I M Hepatosplenomegaly 5 54 F Rash, rhinitis, osteitis 6 1 M None NOTE. RPR = rapid plasma reagin. NR = nonreactive; DO = reactive, undiluted. t Mother treated adequately one week before delivery. t Mother adequately treated one month before delivery. The IgM and IgG antibody reactions of infants' and maternal sera to T. pallidum proteins were studied using western blot techniques. With the exception of normal infants and their mothers, cumulative results obtained with sera from each group ofinfantmother pairs (infected, serofast, and BFP reactors) are shown in table 1. IgMreactions. No IgM antibody reactions (bands) with T.pallidum polypeptides on western blots were seen withthecord sera from the five normal infants. Two of the five infants born to serofast mothers yielded IgM reactions with the 190-, 83-, and 61-kDa treponemal proteins, whereas the other three gave no reactions. The sera of infants born to BFP mothers showed faint but detectable bands to several proteins. All reacted to 190-, 83-, and 61-kDa proteins and reacted variably to the 71-, 61-, 44.5-, and 41 kda treponemal antigens. The sera from the symptomatically infected infants (table 2; excluding infant 6 who was asymptomatic) reacted with the same proteins as did the control infants' sera; however, reactions were more intense and to a wider range ofadditional proteins (figure 1). Eight treponemal proteins were identifiable with IgM antibodies present in the sera ofthe infected, symptomatic infants; these proteins were not detected by any of the control sera. Not all of the infected infants' sera reacted with all RPR VORL Maternal sera Infants' sera Infants' CSF 1:256 1:256 1:2 1:256t 1:2 1:2 1:128 1:128 NR 1:64 1:32 1:4 1:64 1:8 NR 1:64t 1:16 DO

4 906 Dobson, Taber, and Baughn Table 2. Reactivity of IgM and IgG antibodies to T. pallidum antigens in the sera of infants and mothers. Congenitally Asymptomatically infected (5) infected (1) Serofast (5) BFP reactors (3) Molecular IgM IgO IgM IgO IgM IgO IgM IgO mass (kda) M M M M I M M M M M = Mother, I = infant. Except for the congenitally infected group, the numbers in parenthesis indicate the number NOTE. of infant-mother pairs studied; only four of the five mothers' sera in that group were available for study. Data are the number of positive reactions detected out of the total number tested. The absence of numbers indicates that no reactions were observed. these antigens. IgM reactions with the 47- and 37 kda proteins were positive with all infected infants' sera. The single serum not reactive with the 34.5-kDa, 30-kDa, 24-kDa, 17-kDa, and 15.5-kDa proteins was from a newborn whose mother had been treated with a single dose of penicillin just before delivery. IgM reactions to the 190-kDa and 47-kDa proteins were present in the sera of the asymptomatic infant; this infant was assumed to have been infected because his CSF serology was positive. The mother of this infant had been treated adequately one month before delivery. One infected infant was studied serially (figure 2), and the IgM bands disappeared between one and three months after treatment. In the congenital syphilis group, all maternal IgM responses were also present in sera from their infants. The infants, however, producedadditionaligm reactions to antigens to which the mothers elicited no IgM response; an example is shown in figure 1. In the BFP group the infants' IgM responses mirrored those of their mothers. IgG andigg subclass reactions. The cord sera from normal infants either yielded no IgG reaction to T.pallidum proteins or occasional faint reactions to the 83- and 71-kDa antigens. IgG reactive in the congenitally infected infant-mother pairs was pres- ent to a wide range of proteins (figure 1); reactions to at least 15 treponemal proteins with molecular masses ranging from 15.5 to 190 kda were noted. Although on an individual basis the-infected pairs yielded more-intense IgG bands to a wider spectrum of antigens than did the serofast and BFP pairs, no ~~~~~~ B M B M B M B M BM B M I Figure 1. IgM, IgG, and IgG subclass responses to T. pallidum antigens in the sera of a congenitally infected infant (B; patient 1) and her mother (M). - I II

5 IgM and IgG in Congenital Syphilis I 9 M Rea ct i vi t y Table 3. Detecting 19s IgM RF by RIA Day of Life Figure 2. IgM reactivity to T. pallidum antigens in sera obtained at successive time intervals from an infected infant (patient 4). reactions were exclusive to the infected group. Each of the 15 antigens was represented by IgO reactions in the sera of at least one of the pairs where the mother was either a BFP reactor or had had the disease in the past and had been treated before pregnancy. Infants' IgO reactions mirrored those oftheir mothers for the serofast and BFP groups. This also was true for the infected group, with one notable exception. Apart from the asymptomatic infant whose mother was adequately treated one month before delivery, intense IgO reactions with the 83-kDa protein were seen with all of the sera from the infected infants; IgO reactions with the 83-kDa T. pallidum antigen were absent in four of five mothers, and a very faint reaction was noted in sera from the fifth mother (table 1). Serum from the sixth mother in this group was unavailable for western blot analysis. IgO responses in the infected group were restricted almost exclusively to the IgOl and Ig03 subclasses. Subject 10 Normal control infants Infant I Infant 2 Infant 3 Infant 4 Infant 5 Infant 6 Positive control 1 Positive control 2 Positive control 3 CPM ± SE* 323 ± 146t 4026 ± ± ± ± ± ± ± ± ± 125 * SE = standard error of triplicate determinations. t This result is represented as ± SO. Weak Ig02 and Ig04 reactions to only the 47-kDa protein were present in these infants (figure 1). Again, the major difference noted between mother-infant pairs was the reactivity with the 83-kDa antigen. Four of five maternal serum samples failed to react with the 83-kDa polypeptide in western blot, using the four IgO subclass reagents for development (figure 1), whereas sera from their infants yielded intense IgOl and Ig03 reactions. RFRIA andigo immunoprecipitation. It is possible, particularly with IgM, that a true representation of the antibody-antigen reactions on immunoblots is not given. Competition by IgO for antigen sites could lead to an underestimation of IgM reactions [5, 17]. Conversely, if IgM antibody to IgO was present in the sera, thenan apparent IgM bandmight be a false representation of an underlying antigen-igg antibody reaction. Thus, an overestimation of IgM reactions might occur. All sera from infants and sera from their infected mothers were initially tested for both 19sand 7s RF. All sera tested for 7s RF were uniformly negative. Results for 19s RF are shown in table 3. Of the six sera from infected infants, three were found to contain 19sRF. Although the raw data (in cpm) observed in these three infants' sera were much lower than the values ofthree adult, positive controls run simultaneously, their values were significantly elevated (>2 SD above the mean of 10normal infants' sera). The three infants (2, 5, and 6) whose sera did not contain RF were those who had had prior exposure to penicillin (two exposed in utero). The third infant had had blood drawn after two days of treatment with penicillin. None of the infected mothers had RF, and none of the sera from infants of BFP or serofast mothers had detectable RF.

6 908 Dobson, Taber, and Baughn Figure 3. IgM and IgG responses to T. pallidum antigens in the sera of an infected infant (patient 3). Lane 1, IgM response using whole sera for development; lane 2, IgM response after attempts to remove IgG (see Results); lane 3, IgG response using whole serum for development; and lane 4, response with affinity-purified IgG. Discussion in each of the infected infants' sera. Three other reactions, to the 34.5-, 17-, and 15.5-kDa antigens, were detected with all sera, except for that from the infant whose mother had received a single dose of penicillin six days before delivery. The serum IgM response of the infant assumed to be infected, where the mother was treated one month before delivery, was limited to only the 47-kDa T.pallidum protein. Together, these findings appear consistent with the observation that the fetus is capable of producing IgM antibodies to a variety ofinfectious agents [18] and with the concept that IgM reactivity usually peaks at the height of clinical disease. The wide variety ofthe IgM response in our patients is also consistent with that described for adults with secondary syphilis [17]. The number of IgM-specific reactions in the infected infants also exceeded the more-limited responses mounted by their respective mothers who, on the basis of medical history, were in the early-latent stage of disease. The results obtained with maternal sera generally agree with those of adults with early-latent disease [19]. In adults, treatment of syphilis in the secondary or latent stages has been reported to have little effect on the number of IgM reactions found on western blot for up to one year [20]. In this regard, it is interesting that in the infected infants the IgM response did not seem to be as long lasting. This result was demonstrated in the single infant studied serially, ill. whom loss of IgM reactivity with treponemal antigens was noted one to three months after treatment. The induction of RF in response to several infectious agents including syphilis is well described [21-24]. Binding ofigm RF to syphilitic IgG is held responsible for the problems encountered in using the antibody to human IgM conjugate in sensitive assays such as immunofluorescence assays and ELISAs because RF interferes with the detectionof specific IgM indicative of active disease [5, 16, 25]. We found IgM RF in half of the sera from our infected infants. Removing IgG, the substrate for RF, from the infants' sera did not affect the number of IgM-T. pallidum antigen reactions on western blot. Although the reaction with the 37-kDa antigen was less intense, it was still present. Repeat blots for IgG antibody reactions suggested that IgG had beeneffectively removed apart from the faint retention of the 47-, 17-, and 15.5-kDa bands. The quality ofthe IgM reactions was, however.vlargely unchanged. Thus, it appears that the IgM reactions noted in our western blots were genuinely directed towards T. pal IgM 1 2 IgG 3 4 Sera from two infants with RF were subjected to IgG immunoprecipitation to attempt to remove the substrate for RF. Repeat RIAs for RF after immunoprecipitation were negative. The immunoblots were repeated (figure 3), and although most of the IgG reactions were no longer present, faint reactions to the 47-, 17-, and 15.5-kDa proteins were still evident. The IgM reactions were largely unchanged, except for the reaction with the 37-kDa protein, which was diminished in intensity but still detectable. No additional reactions were noted. The results described above indicate that infants with congenital syphilis respond to several previously described T. pallidum antigens [11] by developing IgM antibodies. Sera from these infants contained IgM antibodies capable of reacting with 15 distinct treponemal polypeptides on western blots. Eight of these reactions wereexclusiveto infected infants' sera and were not present on blots reacted with sera from control infants. However,only two ofthese IgM reactions, to the 47- and 37-kDa proteins, were detected

7 IgM and IgG in Congenital Syphilis 909 lidum antigens and not through an IgG intermediary. No additional reactions on blots were revealed by removing IgG from the infants' sera. Although competitive inhibition for antigen binding sites has been reported [5, 24], no evidence was obtained to suggest that such reactions occurred with the sera of congenitally infected infants. Although we found that removing RF made little difference to the western blot analyses, we confirmed that RF is often present in sera from infants with congenital syphilis. Fetal IgM may be directed toward maternaligg [5], and inflammatoryresponses in aborted fetuses with syphilis, absent in the early first trimester, are possibly detected only beyond 15 w ofgestation [26]. Conceivably, fetal IgM RF may be induced by maternal IgG complexed with treponemal antigens in infected infants; immunization with immunecomplexes are an efficient means of inducing RF in experimental animals [27, 28]. While the role of maternal antibody in the pathogenesis of this disease remains unclear, the situation may be far more complicated. Studies of human secondary syphilis and experimental animal models suggest that the autoimmune phenomena associated with this infection cannot be attributed solely to RF. Antibodies have been demonstrated to fibronectin [7,29,30], to laminin and collagen [31], and to muscle creatine kinase [30, 32, 33]. Consequently, the role, if any, of passively acquired maternal autoantibodies in the pathogenesis of congenital syphilis is even less clear. Although cells capable of producing IgG appear in the fetus as early as 12.5w ofgestation, most immunoglobulin ofthis class at birth, as judged by Gm allotyping, is maternal in origin [34]. Furthermore, each of the four IgG subclasses are known to cross the placenta [35]. Thus, it was not surprising that, in most instances, IgG reactivity of an infant's sera mirrored that of the mother. This was also the case for the serofast and BFP serum pairs. The IgG responses to T. pallidum observed on western blots with sera from infected infants and their mothers werealmost entirely ofthe IgGl and IgG3 subclasses. Only limited IgG2 and IgG4 reactions were detected. Adults with secondary syphilis show a similar subclass restriction [7]. Although intense IgGI and IgG3 reactions to the 83-kDa protein were noted in sera from infected infants, similar reactions in their respective mothers' sera were absent. We have no explanationfor this perplexing observationbecause of the fact that patients with secondary syphilis rou- tinely exhibit both IgM and IgG antibodies to this antigen [7]. All of the other 14 IgG antibody-antigen reactions on blots with infants' sera werematched by their mothers. Differences in antibody recognition of T. pallidum antigens have been demonstrated not only between infected and noninfected, seropositive newborns, but also between the newborns with congenital syphilis and their mothers. IgM antibody to the 47- and 37 kda proteins was found only in sera from infected infants. Detecting these antibodies at the time of birthmight form the basis ofa more reliable test for congenital syphilis. Other investigators [19] have also suggested that a 48-kDa molecule may be among the most promising candidates for improved diagnostic reagents. Because only overtly infected babies were evaluated in our study, the clinical value of such a reagent would depend upon its sensitivity for sera from infants asymptomatically infected, who, if untreated, would develop symptomatic infection and possible permanent sequelae. References 1. Julian AJ, Logan LC, Norins LC. Early syphilis: immunoglobulins reactive in immunofluorescenceand other serologic tests. J Immunol 1969;102: Alford CA Jr. Immunoglobulin determinations in the diagnosis of fetal infection. Pediatr Clin North Am 1971; 18: Scotti AT, Logan L. A specific IgM antibody test in neonatal congenital syphilis. J Pediatr 1968;73: Kaufman RE, Olansky DC, Wiesner PJ. The FTA-ABS(IgM) test for neonatal congenital syphilis: a critical review. Journal of the American Venereal Disease Association 1974; 1: Reimer CB, Black CM, Phillips DJ, Logan LC, Hunter EF, Pender BJ, McGrew BE. The specificity of fetal IgM: antibody or anti-antibody? Ann NY Acad Sci 1975;254: Rathbun KC. Congenital syphilis: a proposal for improved surveillance, diagnosis, and treatment. Sex Transm Dis 1983;10: Baughn RE, Mcneely MC, Jorizzo JL, Musher DM. Characterization of the antigenic determinants and host components in immune complexes from patients with secondary syphilis. J Immunol 1986;136: Baughn RE, Musher DM. Isolation and preliminary characterization of circulating immune complexes from rabbits with experimental syphilis. Infect Immun 1983;42: Baughn RE, Adams CB, Musher DM. Circulating immune complexes in experimental syphilis: identification of treponema.lantigens and specific antibodies to treponemal antigens in isolated complexes. Infect Immun 1983;42: Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680-5

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