Functional analysis of IgA antibodies specific for a conserved epitope within the M protein of group A streptococci from Australian Aboriginal endemic communities
Evelyn R. Brandt,
Wendy A. Hayman,
Bart Currie1,
Jonathan Carapetis1,
David C. Jackson2,
Kim-Anh Do3 and
Michael F. Good
Molecular Immunology Laboratory and CRC for Vaccine Technology, Queensland Institute of Medical Research, 300 Herston Road, Brisbane, Queensland 4029, Australia
1 Menzies School of Health Research, Casuarina, NT 0810, Australia
2 Department of Microbiology, University of Melbourne, Parkville, Victoria 3052, Australia
3 Centre for Statistics, University of Queensland, St Lucia, Queensland 4067, Australia
Correspondence to:
M. F. Good
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Abstract
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The mucosa is one of the initial sites of group A streptococcal (GAS) infection and salivary IgA (sIgA) is thought to be critical to immunity. However, the target epitopes of sIgA and the function of sIgA in GAS immunity, in particular the role of accessory cells and complement, is largely unknown. We studied the aquisition and the function of sIgA specific for a conserved region epitope, p145 (sequence: LRRDLDASREAKKQVEKALE) of the M protein. Peptide 145-specific sIgA is highly prevalent within an Aboriginal population living in an area endemic for GAS and acquisition of p145-specific sIgA increases with age, consistent with a role for such antibodies in immunity to GAS. Human sIgA and IgG specific for p145 were affinity purified and shown to opsonize M5 GAS in vitro. Opsonization could be specifically inhibited by the addition of free p145 to the antibodies during assay. Opsonization of GAS was totally dependent on the presence of both complement and polymorphonuclear leukocytes, and, moreover, affinity-purified p145-specific sIgA was shown to fix complement in the presence of M5 GAS. These data show that mucosal IgA to this conserved region peptide within the M protein has an important role in human immunity against GAS and may be useful in a broad-based cross-protective anti-streptococcal vaccine.
Keywords: rheumatic fever, vaccine
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Introduction
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Group A streptococci (GAS) are responsible for pharyngitis and impetigo, and nasopharyngeal infection can result in rheumatic fever (RF). The M protein is the major virulence factor of GAS, represented by >80 different serotypes (1). Protective immunity to GAS was thought to be primarily directed against the antigenically variable N-terminal region of the M protein since type-specific serum antibodies are capable of opsonizing the bacteria in the presence of polymorphonuclear leukocytes (PMN). Efforts to develop a vaccine against GAS have been hampered by the diversity of streptococcal serotypes. Therefore we and others (29) have searched for protective epitopes within the conserved region of the M protein. Antisera raised in mice to a defined epitope within the conserved region (referred to as p145) can opsonize GAS (6,7). IgG antibodies to this epitope were detectable in individuals living in areas highly endemic for GAS and the acquisition of p145-specific IgG antibodies was shown to be age related, suggesting that immunity acquired with age may, at least in part, be related to p145-specific antibodies (8). Furthermore, affinity-purified p145-specific human IgG could opsonize GAS (8,9), directly implicating the importance of these antibodies in immunity to GAS.
However, GAS first interacts with the body at the nasopharyneal mucosa or skin and IgA is the major Ig present in the mucosa. Although it is well known that IgA can block adherence of the bacteria to epithelial cells, neutralize bacterial toxins and specifically aggregate bacteria (10), its potential role and mechanism in immunity to GAS is not well understood. Previous studies have shown that mice immunized intra-nasally with conserved epitopes of the M protein conjugated to the B subunit of cholera toxin had a significant reduction in GAS colonization after challenge with M6 or M24 GAS (25). Although the presence of salivary IgA to these conserved region peptides was detected, the ability of these antibodies to opsonize GAS was not demonstrated.
In this study we examine the role of the conserved region epitope, p145, in mucosal immunity against GAS. We show that p145 is recognized by human salivary IgA (sIgA) in an age-stratified Australian Aboriginal population from an area of high GAS endemicity. Furthermore, both p145-specific human sIgA and serum IgG can directly opsonize the bacteria, and this opsonization is dependent on the presence of complement and PMN.
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Methods
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Patients
Sera and saliva were obtained from a total of 109 Australian Aboriginals (age 11+) from Northern Territory with established rheumatic heart disease (RHD) (n = 51), acute rheumatic fever (ARF) with or without carditis (n = 24), other non-rheumatic heart disease (OHD) (n = 7) and control subjects (n = 27), with no clinical history of heart disease or ARF. A total of 19 children aged 110 years were also included. All subjects were assessed by specialist physicians and patients with RHD met the Jones criteria for diagnosis (11). All patients with RHD were receiving penicillin prophylaxis.
Peptides
Synthetic peptides were synthesized as described (12) and purified by HPLC. The peptide of interest, p145, has the 20 amino acid sequence Leu-Arg-Arg-Asp-Leu-Asp-Ala-Ser-Arg-Glu-Ala-Lys-Lys-Gln-Val-Glu-Lys-Ala-Leu-Glu (6). The non-specific control peptide, an unrelated schistosoma tegument antigen, has the amino acid sequence Glu-Gly-Lys-Val-Ser-Thr-Leu-Pro-Leu-Asp-Ile-Gln-Ile-Ile-Ala-Ala-Thr-Met-Ser-Lys (13). The type-specific peptide representing the N-terminus of M5 (p160) has been previously described (6).
Collection and detection of antibodies
Saliva samples were frozen at 70°C within 2 h of collection. Samples were thawed before use and heated at 56°C for 30 min to inactivate proteases, and centrifuged at 1300 g for 15 min to remove mucin and debris (14). An ELISA was used to measure human serum antibodies and human sIgA to the peptides as previously described (6,8), with the addition that standard curves of optical density versus known concentrations of human IgG and human IgA were used to calculate antibody concentration (14). Where a value of titer is used to measure quantity of antibody, it is defined as the mean + 3 SD of the blank (no antibody) wells (6).
Purification of antibodies
Purification of human p145-specific IgG was performed as described (8). Total human IgA was purified from saliva by passing over an anti-human IgAagarose column (Sigma, St Louis, MO) and elution procedures were essentially as described (8). To isolate p145-specific antibodies, an immunoabsorbent was prepared by synthesizing p145 on Novasyn TG resin (Calbiochem-Novabiochem, Switzerland) without first adding a linker to the support (8). For purification of p145-specific sIgA, total sIgA were passed over the column, washed with PBS, pH 8.0, and eluted with 3 M citric acid, pH 3.0. For buffer exchange to PBS, pH 7.4, the eluate was passed over a disposable PD-10 Sephadex G-25 M column according to the manufacturer's instructions (Pharmacia, Uppsala, Sweden) and concentrated to 1/10 of the original volume using a centricon-10 unit (Amicon, Beverly, MA).
Opsonization assay
Human anti-p145 sIgA and human anti-p145 IgG were assayed for their ability to opsonize M5 GAS and the ability of peptides to block opsonization using a peptide inhibition assay as described in Brandt et al. (8,9). Human donor blood (Caucasian) used in the opsonization assay, as source of PMN and complement, was screened prior to use in opsonization assays to ensure it (i) did not opsonize the strain of GAS in question, (ii) had no detectable antibodies to p145 and (iii) had no antibodies to N-terminal sequences of the M protein, as described previously (8,9). Donor blood and bacteria controls were included with each experiment. Donor blood was classified as supporting the growth of the bacteria if the bacterial population increased >32 times the inoculum size (6,8,9,15,16).
PMN opsonization assay
PMN were prepared by one-step centrifugation from non-opsonic human blood using Polymorphprep (Nycomed Pharma, Norway). Briefly, 5 ml of heparinized blood was layered over 3.5 ml of Polymorphprep and centrifuged at 500 g for 30 min at room temperature. PMN were isolated from the lower leukocyte band, washed in RPMI and resuspended in RPMI at a final concentration of 106 cells/ml.
Peptide inhibition opsonization assays were performed as described previously (8,9) with the exception that antibody in the presence or absence of peptide was added to 50 µl of PMN (106 cell/ml) and 10% human non-opsonic blood group AB sera (complement source), and made up to a final volume of 0.4 ml with RPMI. The mixture was incubated at 37°C with end-to-end mixing for 3 h. A sample of 50 µl was plated out in duplicate using the pour-plate technique into blood agar and bacterial colonies were counted after an overnight incubation at 37°C. Assays were valid if the bacterial population increased >32 times the inoculum in the presence of control IgG, PMN and complement (6,8,9,15,16).
PMN kinetic opsonization assay
Bacteria were grown overnight in THB, washed twice in HBSS without Ca2+ and Mg2+ (HBBS-CM), and resuspended at an OD600 of 0.8 in HBBS-CM. PMN were isolated as above with the exception that cells were suspended in HBBS-CM at 2x107 PMN/ml. To inactivate complement, sera, including whole immune sera (IS) and whole control sera (CS), and complement source sera were heated at 56°C for 30 min. The classical complement pathway was inhibited by incubation of whole sera samples or purified affinity-purified p145-specific IgG and control IgG in complement source sera, with 10 mM EGTA for 20 min at room temperature (17). Bacteria (50 µl) were added to 50 µl of untreated, heat-inactivated or EGTA-treated human IS, CS, and to p145-specific IgG and control IgG with 50 µl of untreated, heat-inactivated or EGTA-treated complement source. Samples were incubated at room temperature for 20 min prior to addition of 50 µl of PMN in a final volume of 500 µl HBBS-CM and then incubated with rotation at 37°C for 180 min. At times indicated in the figures, 10 µl aliquots were removed, diluted 1:100 in saline and colony counts were determined by the pour-plate method using 2.5% THB agar. Results of experiments are given as total number of viable bacteria.
Complement binding assay
This assay was performed essentially as described (18). The assay mixture included antibody dilutions in a final volume of 25 µl, 25 µl of antigen (M5 GAS, OD650 = 1) and 25 µl of guinea pig complement, added to a V-bottomed microtiter plate. The assay mixture was then mixed and incubated at 37°C for 30 min. Following incubation, 25 µl of sensitized sheep red blood cells (sSRBC; coated with anti-SRBC antibodies) (18) was added to each well, mixed and then incubated for a further 30 min at 37°C. Plates were then centrifuged for 5 min at 200 g, and supernatants were transferred to a flat-bottomed microtiter plate and read in an ELISA plate reader at 405 nm. Results were expressed as the concentration of antibody required to give 50% lysis of sSRBC.
Statistical analysis
Geometric means and SD were calculated using standard formulas. Comparison between groups of patients was performed using the two-tailed Student's t-test for unpaired means. Statistical significance was taken as P < 0.05. Relationships between p145-specific sIgA and sera IgG levels were analyzed by linear and non-linear least-squares methods.
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Results
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Prevalence of sIgA to the conserved epitope in an age-stratified population
Saliva samples obtained from 128 Australian Aboriginals living in areas of high GAS endemicity were examined for the presence of p145-specific sIgA by ELISA. Patients were grouped into three age categories: 19 children (aged 110 years, mean age 4.6), 29 teenagers (1119 years, mean age 15.2) and 80 adults (>20 years, mean age 31.8). All subjects had detectable p145-specific sIgA (10120, 103600 and 103480 ng/ml saliva respectively) (Fig. 1
). Both teenage and adult Aboriginals had a significantly higher mean concentration of sIgA to p145 than children (P = 0.02 and P = 0.04 respectively), but there was no significant difference in the mean anti-p145 sIgA concentration between teenagers and adults (P > 0.05) (Fig. 1
). No significant difference in mean anti-p145 sIgA concentration was found between any of the patients grouped according to disease categories (RHD, ARF, OHD or controls) (P > 0.05).

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Fig. 1. Salivary IgA response to p145 in Aboriginal subjects grouped by age. Horizontal bars represent mean anti-p145 IgA concentration (ng/ml saliva). Each data point may represent more than one set of measurements. Mean anti-p145 specific IgA concentration in teenagers and adults grouped by disease: RHD, 385.71 and 450.94; controls, 90 and 284.44; ARF, 602.86 and 200 ng/ml saliva respectively.
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Sera samples, obtained at the time of saliva collection, were examined for the presence of anti-p145-specific IgG. Significant levels of IgG were detected in all patients as previously described (8). Serum p145-specific IgG and sIgA levels were compared in children aged 110 years and in individuals aged 11+. Although no correlation was observed between the p145-specific IgG and sIgA concentration in children (R2 = 0.041), a significant inverse trend was seen in individuals aged 11 years and over (R2 = 0.1; P = 0.001). Patients within the age group 11 years and over were then grouped according to disease category (RHD/ARF or controls). While control individuals had no significant correlation between p145-specific serum IgG and sIgA level, (R2 = 0.0001) (Fig. 2A
), the disease category, RHD/ARF, revealed a significant inverse relationship (R2 = 0.489; P < 0.00001) (Fig. 2B
).

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Fig. 2. Relationship between Aboriginal p145-specific serum IgG and sIgA levels grouped by disease categories. (A) Controls. (B) RHD/ARF. Model: IgG = a + b/IgA, where a and b are contants to be estimated by the least-squares method.
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Opsonization of M5 GAS by human sIgA and IgG to p145
To assess a functional role for p145-specific human sIgA, we tested whether these antibodies could opsonize M5 GAS. sIgA was purified from pooled saliva of four RHD patients (with specific titer to p145 exceeding 1600) by passage over an anti-human IgAagarose column followed by affinity purification to p145 (see Methods). Control sIgA was also purified from the pooled saliva of two Caucasians with no salivary opsonizing activity for M5 GAS and no detectable anti-p145 sIgA, and no antibodies to a 20 amino acid M5 N-terminal peptide (p160), known to be a target of type-specific opsonic antibodies (6). Control IgG and p145-specific IgG were also purified from pooled human sera and used in an opsonization assay as decribed above.
Peptide 145-specific sIgA decreased colony counts by 86% (Fig. 3A
). Similarly, p145-specific IgG reduced mean c.f.u. by 81% compared to control IgG (Fig. 3B
). Free p145, but not an irrelevant peptide (pNS) with only 12% identity to p145 (13), was able to completely block opsonization, confirming the specificity of the antibodies.

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Fig. 3. Bactericidal effect against M5 GAS by mucosal and systemic antibodies, and blocking by p145 and pNS. (A) Opsonization of M5 GAS in the presence of p145-specific sIgA. IgA titer to p145 by p145-specific IgA = 640 and control IgA 10. Inoculum size = 6; donor blood and bacteria control = 200 c.f.u. (B) Opsonization of M5 GAS by p145-specific serum IgG. IgG titer to p145 by p145-specific IgG = 6400; control IgG < 20. Inoculum size = 98; donor blood and bacteria control = 12,000 c.f.u. Data is given as the mean colony count from duplicate experiments. For all experiments, growth of the bacteria in donor blood alone was >32 times the inoculum.
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Opsonization by sIgA and IgG is dependent on the presence of PMN and complement
To determine the specific requirements for PMN and complement in opsonization, a PMN bactericidal assay was used (see Methods). In the presence of PMN and complement, p145-specific IgA specifically reduced the mean c.f.u. of bacteria by 58% in comparison to control sIgA and total immune saliva reduced mean c.f.u. of GAS by 87% compared to control saliva (Fig. 4A
). Affinity-purified p145-specific serum IgG antibodies and total immune sera could kill M5 GAS by >90% compared to control antibodies (Fig. 4B
). Serial dilution of immune saliva and sera completely abrogates the bactericidal activity of these samples but has no effect on mean colony count for control saliva and sera (data not shown). In contrast, in the presence of complement alone or PMN alone, neither p145-specific sIgA, IgG, immune saliva nor sera could inhibit bacterial growth by >32% (average = 7.9%) (Fig. 4A and B
).
The role of complement in PMN-mediated killing of GAS was further assessed by depletion studies. Control IgG or p145-specific IgG were mixed with non-opsonic serum as a complement source that had either been left untreated, heat-inactivated or pre-treated with EGTA to inhibit activation of the classical complement pathway (17). sIgA was not examined as sufficient quantities were unavialable. In the presence of p145-specific IgG, PMN and a complement source, the total number of viable bacteria was significantly reduced by 180 min (Fig. 5A
). Bactericidal activity was completely abrogated by heat inactivation of the complement source or EGTA treatment (Fig. 5A
). No opsonization of M5 GAS was seen in the presence of control IgG nor was opsonization seen in the absence of PMN (Fig. 5A
) or antibody (data not shown). Similar results to these with affinity-purified p145-specific antibodies were seen using immune sera (Fig. 5B
).

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Fig. 5. PMN-mediated killing of M5 GAS. (A) Affinity-purified p145-specific IgG and control IgG. (B) Total IS and CS. Sample definitions include: untreated (neat), heat inactivated (hi), serum pre-treated with EGTA (EGTA) or no PMN.
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The complement fixation test can be used to demonstrate whether antibody can bind antigen (GAS) in the presence of complement. Sensitized SRBC (sSRBC coated with anti-SRBC antibodies) are added to complement and antibodyantigen complex. In the presence of free complement, sSRBC are lysed (see Methods). Using this assay, both affinity-purified p145-specific sIgA and IgG were capable of fixing complement when bound to M5 GAS as free complement was not available to lyse indicator sSRBC. In contrast, control sIgA and IgG, which do not opsonize M5 GAS, were unable to fix complement in the presence of M5 GAS resulting in 100% lysis of sSRBC (Table 1
).
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Discussion
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This study describes the ability of human p145-specific sIgA and IgG to opsonize M5 GAS in vitro, and demonstrates roles of these antibodies and complement in immunity to GAS. We have shown that human sIgA, specific to a conserved region peptide, p145, is highly prevalent within an Aboriginal population living in an area endemic for GAS and that acquisition of p145-specific sIgA increases with age, consistent with a role of such antibodies in immunity to GAS. Our data show a significant inverse correlation between p145-specific serum IgG and sIgA levels in individuals aged >11 years, and more specifically with RHD/ARF. This relationship may influence immunity to GAS infections and therefore development of post-infectious sequelae in adults. One interpretation of these data is that individuals who are able to mount only a salivary or systemic response (or no response at all) are most at risk of infection and subsequent sequelae. Interestingly, even though the prevalence of RF/RHD in Australian Aboriginals is amongst the highest worldwide and the current dogma suggests that RF/RHD is associated with throat infection, the throat carriage of GAS in these populations is recorded to be very low (<13%), compared to pyoderma (up to 70%), and symptomatic GAS pharyngitis is uncommon (19). It is still very possible that pharyngitis is a pre-requisite in this population for RF and that throat organisms are transmitted from skin lesions initially. Further studies investigating throat and skin carriage of GAS in Aboriginal communities and its association with anti-streptococcal sIgA and IgG are required to fully understand immunity to GAS in Aboriginal communities.
While the conserved region has previously been shown to be important in immunity against GAS (29), the current study demonstrates that sIgA specific for the conserved region peptide p145 can opsonize M5 GAS in vitro. Other studies have suggested the importance of sIgA in preventing streptococcal infection. Human M6-specific sIgA was shown to passively protect mice against systemic infection following intra-nasal challenge with M6 GAS, although these antibodies were unable to opsonize bacteria in an in vitro bactericidal assay (20). Others have also shown that mice intra-nasally immunized with a conserved region peptide conjugated to CTB showed decreased colonization of GAS following challenge even though the salivary IgA response to the peptide was low (2,3); however, the mechanism for protection was not delineated. Although the role of sIgA as an opsonin is disputed (21), there is increasing evidence that this may be one of its primary roles in immunity to bacterial infection (2224). The ability of sIgA to activate the complement system has previously been described (2526). In addition, PMN and monocytes express Fc receptors specific for IgA, and numerous reports suggest these phagocytes are capable of ingesting IgA-coated targets (10,2730). We have now shown that affinity-purified sIgA to p145 is capable of fixing complement in the presence of M5 GAS, and that this sIgA can opsonize GAS in the presence of complement and PMN. Thus, opsonization of GAS mediated by IgA antibodies to the conserved region of the M protein may contribute to protection against GAS. Although our data show that specific sIgA can promote opsonization of GAS in vitro this may not occur in vivo. Even though complement components have been detected in saliva from patients with peridontal disease and healthy subjects (31,32), it is not known whether sufficient complement is present in the mucosa to be involved in opsonization. Our results show that in the absence of added complement no salivary-mediated opsonization occurs. However, this scenario may change during inflammation, indeed, in crevicular fluid from patients with inflammatory peridontal disease hemolytically functional complement was recognized (31,33).
Opsonization does not occur in the presence of specific antibody when the activation of the classical complement pathway is blocked or when the complement source is heat-inactivated. Other studies have suggested that antibodies to the conserved region do not opsonize GAS although they are capable of binding GAS and complement (34). However, these conserved region antibodies do not target p145. It has also been suggested that the binding of Factor H to the M protein, which inhibits or reverses the formation of C3b, may obstruct opsonization mediated by conserved region antibodies (35). The binding site for Factor H overlaps the p145 sequence by the first 7 amino acids of the p145 sequence. However, two B cell epitopes within the p145 sequence have been definedamino acid residues 214 and 719 (8,36). Opsonization may thus be mediated by antibodies binding to a region outside the Factor H binding site, therefore allowing these conserved region antibodies to bind to GAS, fix complement and opsonize the bacteria.
The results reported here have important implications in terms of development of a vaccine to prevent GAS infection. The advantage of oral vaccines is that they target the organism at the portal of entry and that they are easier to administer, especially to large populations, a feature that would be of particular benefit for immunizing children in GAS-endemic locations. In addition, intra-nasal immunization of mice with p145 admixed with the mucosal adjuvant cholera toxin B can induce both mucosal IgA and systemic IgG to p145 (37). However, one consideration for a vaccine is immunological cross-reactivity between the M protein and host tissue (36,38). We have previously identified minimal opsonic sequences within p145 that do not evoke immunological host cross-reactivity and may also be useful as mucosal vaccine candidates (36,38). An oral vaccine based on such a conserved region epitope may provide immunity against a broad range of GAS strains both at the point of infection at the mucosa and systemically.
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Acknowledgments
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We would like to thank all the blood and saliva donors for making this study possible, Dr Gary Waine for donation of the non-specific peptide and Yvonne Wood for technical assistance. This work was supported by NHMRC (Australia), National Heart Foundation of Australia, The Prince Charles Hospital Foundation, the Cooperative Research Centre for Vaccine Technology, and The Australian Centre for International and Tropical Health and Nutrition.
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Abbreviations
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ARF | acute rheumatic fever |
CS | control sera |
GAS | group A streptococci |
HBBS-CM | HBSS without Ca2+ and Mg2+ |
IS | immune sera |
OHD | other non-rheumatic heart disease |
RND | rheumatic heart disease |
sIgA | salivary IgA |
PMN | polymorphonuclear leukocytes |
RF | rheumatic fever |
sSRBC | sensitized sheep red blood cells |
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Notes
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Transmitting editor: S. H. Kaufmann
Received 24 August 1998,
accepted 17 December 1998.
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