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Nephrology Dialysis Transplantation
 

Nephrology Dialysis Transplantation

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    Nephrology Dialysis Transplantation Nephrology Dialysis Transplantation Document Transcript

    • Nephrol Dial Transplant (2000) 15: 28–33 Nephrology Dialysis Original Article Transplantation Humoral immunity against the proline-rich peptide epitope of the IgA1 hinge region in IgA nephropathy Tohru Kokubo, Kenjiro Hashizume, Hitoo Iwase1, Kenji Arai2, Atsushi Tanaka2, Kazunori Toma2, Kyoko Hotta1 and Yutaka Kobayashi Department of Medicine and 1Department of Biochemistry, School of Medicine, Kitasato University, Sagamihara-City and 2Analytical Research Center, Asahi Chemical Industry Co., Ltd, Fuji-City, Japan Abstract Key words: IgA nephropathy; humoral immunity; IgA1 Background. The human IgA1 hinge region is a unique hinge region; proline-rich peptide; O-glycosylation mucin-like O-linked proline-rich glycopeptide, and its core peptide was found to be exposed aberrantly by the underglycosylation in IgA nephropathy (IgAN ). Introduction We describe here the presence of humoral immunity against the IgA1 hinge peptide epitope in IgAN and evaluate the relationship between the underglycosyl- IgA nephropathy (IgAN ) is the most common glomer- ation of the IgA1 hinge region and humoral immunity. ular disease which is characterized immunohistolog- Method. The serum anti-IgA1 hinge peptide antibody ically by the predominant deposition of the IgA1 (anti-a1HP ab) titre was measured and compared subclass in the mesangial area [1,2]. The structural between the IgAN (n=37) and control groups (n=34) characteristics of the IgA1 molecule recently were associated with the pathogenesis of IgAN, because the by enzyme-linked immunosorbent assay ( ELISA) IgA1 molecule is obviously different from the other using a synthetic peptide corresponding to the human immunoglobulins in its hinge region which is a unique IgA1 hinge region, PVPSTPPTPSPSTPPTPSPS, as an mucin-like glycopeptide. The IgA1 hinge region has a antigen. Next, to evaluate the relationship between the proline-, serine- and threonine-rich amino acid underglycosylation of the IgA1 hinge region and the sequence in which the serine and threonine residues humoral immunity, the reactivity of the serum IgG are able to carry O-linked oligosaccharides consisting from the patients with IgAN against monoclonal IgA1 of sialic acid (NeuNAc), galactose (Gal ) and N- which had been digested enzymatically to remove the acetylgalactosamine (GalNAc) with microhetero- carbohydrates from the IgA1 hinge region was meas- geneity [3,4]. Some investigators, including us, revealed ured by ELISA. the presence of defective O-glycans in the hinge region Results. The anti-a1HP ab titre was significantly higher of the IgA1 molecules derived from sera of patients in the IgAN group than in the control group (OD with IgAN [5–9]. Additionally, we showed that the value: IgG class, 0.564±0.344 vs 0.331±0.154, P= core peptide of the IgA1 hinge region was exposed by 0.0014; IgM class, 0.272±0.148 vs 0.141±0.072, its underglycosylation in IgAN using a rabbit poly- P<0.0001) and it was positive in ~40% of the patients clonal antibody against a synthetic peptide correspond- with IgAN. In addition, the reactivity of the serum ing to the human IgA1 hinge region [10]. IgG from the IgAN patients against the monoclonal Regarding the biological significance of O-glycans, IgA1 was found to be increased as the carbohydrates very interesting findings have been reported in studies were enzymatically removed from the IgA1 hinge of mucin. In patients with several different kinds of region (when native=100; asialo, 122±9.5; agalacto, cancer, it was shown that the core peptide of mucin 167±11.5; naked, 188±3.9). expressed on the malignant cells was exposed by the Conclusion. These results suggested that the peptide underglycosylation, resulting in humoral and cellular epitope of the IgA1 hinge region which was aberrantly immune responses to its peptide epitope [11–13]. Since exposed by underglycosylation could induce the hum- the IgA1 hinge region is very similar to mucin in oral immune response in IgAN. proline residue abundance and the presence of O- glycans [14], the IgA1 hinge peptide core exposed by underglycosylation may induce some immune response Correspondence and offprint requests to: Tohru Kokubo MD, in patients with IgAN. Department of Medicine, School of Medicine, Kitasato University, In this study, we examined the antibody production 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. against the peptide epitope of the IgA1 hinge region © 2000 European Renal Association–European Dialysis and Transplant Association
    • Anti-IgA1 hinge peptide antibody in IgA nephropathy 29 in patients with IgAN by enzyme-linked immunosorb- Mann–Whitney U-test. A P-value of <0.05 was considered ent assay ( ELISA) using a synthetic peptide corres- to be a significant difference. ponding to the human IgA1 hinge region as an antigen, Preliminarily, we examined the specific reactivity of the and evaluated the relationship between the underglyco- IgG from patients with IgAN against the sa1HP. The dose-dependent reactivity of the IgG (serum dilution, sylation of the IgA1 hinge region and the humoral 1/1600–1/100) against the plate-coated sa1HP was immune response. tested using the sera from patients with IgAN (n=6) and controls (n=6) with the above-mentioned method. Each mean±SD of the six cases was expressed, and the statistical difference between IgAN and the control was Materials and methods analysed by ANOVA. In addition, the inhibition assays were performed using a free sa1HP and an unrelated 21mer Sera were collected from 37 patients (17 males, 20 females, synthetic peptide, human b-endorphin (1–5)+(16–31), mean age 37 years, range 16–56 years) with biopsy-proven YGGFMTLFKNAIIKNAYKKGE (American Peptide IgAN and 34 control patients (22 males, 12 females, mean Company, Sunnyvale, CA). Fifty ml of the sera (1/50 dilution) age 43 years, range 17–74 years) with other biopsy-proven from the patients with IgAN (n=6) were added with 50 ml renal diseases without glomerular IgA deposition (membran- of the free synthetic peptides (0–20 mg/ml ) to the sa1HP- ous nephropathy, n=6; non-IgA mesangial proliferative coated wells. After incubation for 3 h and washing, the IgG glomerulonephritis (GN ), n=4, membranoproliferative GN, binding to the plate-fixed sa1HP was detected with peroxid- n=4; tubulointerstitial nephritis, n=4; focal glomerulo- ase-conjugated anti-human IgG antibody in the same way. sclerosis n=3; lupus nephritis, n=3; lipoid nephrosis, n=2; The reactivity without inhibitors was regarded as 100%, and nephrosclerosis, n=2; rapidly progressive GN, n=2; amilo- each mean±SD of the percentage reactivity was expressed. idosis, n=2; acute GN, n=1; diabetic nephropathy, n=1). In order to evaluate the relationship between the undergly- A 21mer peptide corresponding to the amino acid cosylation of the IgA1 hinge region and the humoral immune sequence of the human IgA1 hinge region, response, the reactivity of the IgG from patients with IgAN PVPSTPPTPSPSTPPTPSPSC, was synthesized by and pur- against a monoclonal IgA1 which was digested enzymatically chased from Bio-Synthesis, Inc. (Lewisville, TX ). The purity to remove the carbohydrates from the IgA1 hinge region and molecular weight were confirmed by HPLC and MALDI- was measured by ELISA. Neuraminidase (NA) from TOFMS. The synthetic IgA1 hinge peptide (sa1HP) was Streptococcus 6646K (1 U/100 ml; Seikagaku Co. Tokyo, conjugated with bovine serum albumin (BSA, Sigma Japan), b-galactosidase (GA) from bovine testis (1.0 U/ml; Chemical Co., St Louis, MO) for plate coating. The conjuga- Sigma Chemical Co.) and a-N-acetylgalactosaminidase tion of BSA with the peptide was performed by the following (GNA) from Acremonium sp. (13 U/ml; Seikagaku Co.) were procedure. A 1.5 ml aliquot of BSA (13.4 mg/ml ) was used in this examination according to the following procedure mixed with 100 ml of N-e-maleimidocaproyloxy succinimide based on our previous study [15]. The monoclonal IgA1 (100 mg/ml, Wako Junyaku Co., Tokyo) and incubated for derived from a patient with IgA1-type multiple myeloma 1 h at room temperature. After desalting, 250 ml of the (Funakoshi Co. Tokyo, Japan) was divided into four samples peptide (20 mg/ml ) was added and incubated for 36 h at (1 mg each). The first sample was dissolved in 100 ml of 4°C. After desalting, the BSA-conjugated peptide was used 0.2 M sodium acetate buffer, pH 5.0 (SAB) without adding as the antigen for ELISA. any enzymes and was used as a control. The second sample The 96-well microtitration plates (Linbro/Titertek, Flow was dissolved in 95 ml of SAB and 5 ml of NA was added to General Company, McLean, IL) were coated with 100 ml of remove NeuNAc. The third sample was dissolved in 75 ml of the BSA-conjugated sa1HP diluted to a concentration of SAB and 5 ml of NA and 20 ml of GA were added to remove 10 mg/ml with 0.015 M carbonate buffer, pH 9.6. After NeuNAc and Gal. The fourth sample was dissolved in 65 ml blocking of the unreacted sites with 0.01 M phosphate buffer, of SAB and 5 ml of NA, 20 ml of GA and 10 ml of GNA were pH 7.5, 0.15 M NaCl (PBS ) containing 1% BSA, 100 ml of added to remove NeuNAc, Gal and GalNAc. All the IgA1 sera (1/100 dilution in PBS ) from patients with IgAN (n= samples containing the enzymes were incubated overnight at 37) and controls (n=34) were added to the sa1HP-coated 37°C and then dialysed against PBS. The non-treated, and non-coated wells. After incubation for 3 h at room NA-treated, NA+GA-treated and NA+GA+GNA-treated temperature and washing with PBS containing 0.1% BSA IgA1 were named ‘native’, ‘asialo’, ‘agalacto’ and ‘naked’ and 0.05% Tween-80 (PBS/BSA/Tween), 100 ml of peroxi- IgA1, respectively. The efficacy of the enzymatic treatments dase-conjugated goat anti-human IgG (1/500, Fc portion- was confirmed by binding assays with peanut agglutinin and specific, Organon Teknika Corp., West Chester, PA), anti- Vicia vilosa lectins according to the previously described human IgA (1/500, a-chain-specific, Organon Teknika Corp.) method [16 ]. The microtitration plates were coated with and anti-human IgM (1/500, m-chain-specific, Organon 100 ml of the IgA1 (10 mg/ml ). After blocking with 1% BSA, Teknika Corp.) antibodies were added to the wells. After 100 ml of the sera (1/100 dilution) from the patients with incubation for 1 h at room temperature and washing with IgAN (n=6) was reacted against the coated IgA1. Because PBS/BSA/Tween, the plates were exposed to an enzyme of the non-specificity of NA+GA to O-glycans, we also substrate consisting of 0.4 mg/ml of o-phenylenediamine performed the same assays using a monoclonal IgA2 dihydrochloride (Sigma Chemical Co.) and 0.03% hydrogen (Funakoshi Co., Tokyo, Japan). The IgG reactivity was peroxide in a solution containing 0.1 M disodium hydrogen detected with peroxidase-conjugated anti-human IgG anti- phosphate and 0.05 M citric acid monohydrate. The body using the above-mentioned ELISA method. The OD developed colour was read at 490 nm with a microplate value in the native IgA1 was regarded as 100% and each reader (Bio-Rad Laboratories, model 450, Richmond, CA). mean±SD of the percentage reactivity in the deglycosylated Each absorbance level was reduced by that of the non- IgA1 was expressed. sa1HP-coated well. All of the assays were performed in To examine the exposure of the IgA1 hinge peptide core triplicate. Each median was expressed. The statistical differ- in IgAN, the reactivity of a rabbit polyclonal anti-synthetic ence between the two groups was analysed using the IgA1 hinge peptide antibody (anti-sa1HP ab), which was
    • 30 T. Kokubo et al. reported in our previous study [10], against the serum IgA was measured by ELISA and compared in the IgAN and control groups. The microtitration plates were coated with 100 ml of goat anti-human IgA antibody (10 mg/ml, Fab fraction, Organon Teknika Corp). After blocking with BSA, 100 ml of the sera (1/100 dilution) from the IgAN group (n= 24) and control group (n=24) were added to the wells. After incubation for 3 h at room temperature and washing, 100 ml of the rabbit anti-sa1HP ab (1/100 dilution) was added to the wells. After incubation for 3 h at room temperature and washing, 100 ml of the peroxidase-conjugated goat anti-rabbit IgG antibody (1/500 in PBS, Organon Teknika Corp.) was placed in the wells. After incubation for 1 h at room temper- ature and washing, the absorbance levels were measured using the above-mentioned method. These assays were per- formed in triplicate. Each median was expressed. The statist- ical difference between the two groups was analysed using the Mann–Whitney U test. Fig. 2. Reactivity of serum IgG from patients with IgAN (n=6) against plate-fixed sa1HP was inhibited by the free sa1HP but not Results by the control peptide. Specific reactivity of serum IgG from IgAN patients against sa1HP As shown in Figure 1, the serum IgG from the patients with IgAN reacted against the plate-fixed sa1HP in a dose-dependent manner. The reactivity was signific- antly higher in the patients with IgAN (n=6) than in the controls (n=6) (ANOVA, P=0.017). In addition, the binding of the IgG from the patients with IgAN (n=6) to the plate-fixed sa1HP was clearly inhibited by the free sa1HP but not by a control peptide (Figure 2). Serum anti-a1HP ab titres in IgAN As shown in Figure 3, the serum anti-a1HP ab titres of the IgG and IgM classes were significantly higher in the IgAN group (n=37) than in the control group (n=34) (IgG class, 0.564±0.344 vs 0.331±0.154, P= 0.0014; IgM class, 0.272±0.148 vs 0.141±0.072, P<0.0001). However, the antibody titre of the IgA Fig. 3. Serum anti-a1HP ab titres of IgG and IgM classes were significantly higher in the IgAN group (n=37) than in the controls (n=34). The titres were positive in ~40% of patients with IgAN (positive level>mean+2 SD of controls). class was not detectable in all cases of both groups. When more than the mean+2 SDs of the control group was regarded as a positive level of the anti- a1HP ab titres ( IgG class, >0.639; IgM class, >0.285), the positive rate was 40.5% (15/37) in the IgG class and 43.2% (16/37) in the IgM class in the IgAN group, and 0% (0/34) in both antibody classes in the control Fig. 1. Serum IgG from patients with IgAN (n=6) reacted against group. There is a significant correlation between the sa1HP in a dose-dependent manner. The reactivity was significantly anti-a1HP ab titres of the IgG and IgM classes (R2= higher in IgAN patients than in controls (n=6). 0.366, P<0.0001). However, no significant correlation
    • Anti-IgA1 hinge peptide antibody in IgA nephropathy 31 was found between the anti-a1HP ab titres and the serum concentration of IgG or IgM. Reactivity of serum IgG from IgAN patients against deglycosylated IgA1 (monoclonal) The serum IgG from patients with IgAN (n=6) reacted strongly against the deglycosylated IgA1 compared with native IgA1. When the reactivity against native IgA1 was regarded as 100%, the values were 122±9.5% (mean±SD) against the asialo IgA1, 167±11.5% against the agalacto IgA1 and 188±3.9% against the naked IgA1. The reactivity was increased as the carbo- hydrates were removed from the IgA1 hinge region, and the reactivity against naked IgA1 was ~2-fold higher than that against native IgA1 (Figure 4). In addition, there was no significant difference in the reactivity of the patient’s IgG against a monoclonal IgA2 between native and deglycosylated IgA2. Fig. 5. Reactivity of rabbit polyclonal anti-sa1HP ab against serum Reactivity of rabbit polyclonal anti-sa1HP ab against IgA was significantly higher in the IgAN group (n=24) than in the controls (n=24). serum IgA in IgAN As shown in Figure 5, the reactivity of the rabbit and it was regarded as positive in ~40% of the patients polyclonal anti-sa1HP antibody against serum IgA with IgAN. It was also observed that this titre was was significantly higher in the IgAN group (n=24) increased in both the IgG and IgM classes but not than in the control group (n=24) (0.321±0.167 vs detectable in the IgA class, indicating that antibody 0.189±0.137; P=0.0042). There was no significant production against the peptide epitope of the IgA1 correlation between this reactivity and the serum IgA hinge region occurred in the IgG and IgM classes. level. These observations suggested that these humoral immune responses could result in the formation of IgA1–IgG and IgA1–IgM immune complexes in the Discussion circulation of patients with IgAN. Czerkinsky et al. previously reported that IgA-containing circulating The results of the ELISA using a synthetic peptide immune complexes were found in patients with IgAN corresponding to the human IgA1 hinge region, [17]. The IgA1–IgG and IgA1–IgM interactions medi- PVPSTPPTPSPSTPPTPSPS, as an antigen showed ated by the IgA1 hinge peptide observed in the present that the serum anti-a1HP ab titre was significantly study may be one of the mechanisms involved in the higher in the IgAN group than in the control group, formation of IgA-containing immune complexes in IgAN. Next, we examined the reactivity of serum IgG from the patients with IgAN against native and deglycosyl- ated monoclonal IgA1. This reactivity was found to be greatest in the naked, followed by the agalacto, asialo and native IgA1, in that order; i.e. the more carbohydrates that were removed from the IgA1 hinge region, the greater the reactivity. This result suggests that the accessibility of the anti-a1HP ab to the peptide epitope of the IgA1 hinge region was increased by the enzymatic removal of the carbohydrates from the IgA1 hinge region, and that the carbohydrate moieties including NeuNAc, Gal and GalNAc attached to the hinge region function to protect the peptide epitope. Therefore, if there is some defect in the O-glycans of the IgA1 hinge region, the probability of the peptide epitope being recognized by the anti-a1HP ab expressed on B lymphocytes is thought to increase and, con- sequently, the B cells will be activated and produce the Fig. 4. Reactivity of serum IgG from patients with IgAN (n=6) anti-a1HP ab. against a monoclonal IgA1 was increased as carbohydrates were Furthermore, we also showed that the reactivity of removed from the IgA1 hinge region. a rabbit polyclonal anti-sa1HP ab against serum IgA
    • 32 T. Kokubo et al. was significantly higher in the IgAN group than in the mucin. Therefore, if the immune response to the pep- control group, suggesting that the peptide core of the tide epitope of the IgA1 hinge region observed in the IgA1 hinge region is aberrantly exposed by the defect- present study also functions to eliminate the undergly- ive O-glycosylation in IgAN. This result also supports cosylated IgA1 molecules from the circulation of the hypothesis that the underglycosylation of the IgA1 patients with IgAN, then synthetic peptides corres- hinge region causes the humoral immune response to ponding to the IgA1 hinge region may be useful for its peptide epitope in IgAN. Tomana et al. recently an ‘immunotherapy’ against IgAN. It was suggested reported that the Gal deficiency in IgA1 molecules was recently that IgA1 molecules which have defective O- related to the formation of IgA1–IgG complexes in glycans in their hinge region could cause direct glomer- IgAN [8]. Our data are consistent with theirs in terms ular deposition by non-immunological mechanisms of the relationship between the formation of the [5–9,16,29–34]. However, there is also the possibility IgA1–IgG immune complex and the aberrant glycosyl- that the immune response to the IgA1 hinge peptide ation of the IgA1 hinge region. plays a pathogenetic role in IgAN as a result of the Concerning the structure and function of the O- IgA1–IgG immune complex formation or other linked glycopeptides, the study of mucin, a typical O- immune mechanisms. Therefore, it is necessary to linked glycoprotein, has been well developed in the evaluate carefully the relationship between the anti- field of cancer. Kotera et al. showed the presence of a1HP antibody titre and the clinical course of many humoral immunity against a tandem repeat peptide patients with IgAN and to reveal the pathophysiolog- epitope of the human mucin MUC-1 in sera from ical implication of this antibody production in IgAN. breast, pancreatic and colon cancer patients [11]. In We previously indicated that the underglycosylation that study, it was shown that the anti-MUC-1 antibody of the IgA1 hinge region caused the non-immunological titre was positive in 8–17% of these cancer patients by IgA1 self-aggregation and adhesion to extracellular ELISA using a synthetic peptide corresponding to the matrix ( ECM ) proteins such as type IV collagen, MUC-1, PDTRPAPGSTAPPAHGVTSA. Also, anti- fibronectin and laminin, and proposed that these phen- MUC-1 antibody production was suggested to result omena might be one of the mechanisms of the glomer- from the exposure of the MUC-1 core peptide by ular deposition of IgA1 in IgAN [16 ]. We also inferred underglycosylation of the mucin expressed on the that the IgA1 self-aggregation and adhesion to ECM malignant cells [18,19]. Since the IgA1 hinge region is proteins could result from the exposure of the proline- very similar to mucin in terms of the abundance of rich peptide core of the IgA1 hinge region due to the proline residues and the presence of O-glycans, it is defect of O-linked sugars in IgAN [10,32], because, as likely that the IgA1 hinge region has a structure and Williamson described, the proline-rich peptide can function similar to mucin [14,20–22]. mediate various molecular interactions by non-specific In addition, Rughetti et al. showed that a synthetic physicochemical reactions such as hydrogen bonding peptide corresponding to MUC-1 could induce a B-cell and/or hydrophobic interaction [21]. However, in this immune response in an ovarian cancer patient [12]. study, we showed that the exposure of the proline-rich Jerome et al. reported that cytotoxic T lymphocytes IgA1 hinge peptide by underglycosylation could cause derived from patients with breast adenocarcinoma not only non-immunological, but also immunological recognized a peptide core epitope of mucin preferen- IgA1-containing protein–protein interactions in IgAN. tially expressed by the malignant cells [13]. These In conclusion, it was shown that the serum anti- findings indicated that the mucin peptide core epitope a1HP ab titre was significantly higher in the IgAN could induce cellular immunity as well as humoral group than in the control group and it was positive in immunity in humans. Therefore, the production of ~40% of the patients with IgAN. The reactivity of the antibody against the peptide core epitope of the IgA1 serum IgG from the IgAN patients against a mono- hinge region may provide a possible explanation not clonal IgA1 was increased as the carbohydrates were only for the formation of the IgA1-containing immune enzymatically removed from the IgA1 hinge region. complexes, but also for the abnormalities in the activity These results suggested that the peptide epitope of the of the cellular immune system observed in IgAN [23]. IgA1 hinge region which was aberrantly exposed by Recent studies in this field provided further interes- the underglycosylation could induce the humoral ting information. Agrawal et al. described that T cells immune response in IgAN. from multiparous women specifically proliferated in response to the MUC-1 peptide epitope, indicating Acknowledgements. The authors thank Ms M. Saitoh for her excellent that there is a natural immunization against it during technical assistance. Part of this study was supported by the New Energy and Industrial Technology Development Organization. pregnancy [24,25]. These observations provided an explanation for a negative correlation with the occur- rence of breast cancer in maltiparous women [26 ] and References led to the potential use of synthetic peptides corres- ponding to MUC-1 as ‘vaccines’ for therapy of breast 1. Berger J. IgA glomerular deposits in renal disease. Transplant cancer [27,28]. Thus, it is clear that the immune Proc 1969; 1: 939–944 2. Conley ME, Cooper MD, Michael AF. Selective deposition of responses to the MUC-1 peptide epitope serve as a immunoglobulin A1 in immunoglobulin A nephropathy, ana- preventive factor against breast cancer by eliminating phylactoid purpura nephritis and systemic lupus erythematosus. the cancer cells expressing the underglycosylated J Clin Invest 1980; 66: 1432–1436
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Kokubo T, Hiki Y, Iwase H et al. Protective role of IgA1 nephropathy. J Am Soc Nephrol 1997; 8: 915–919 glycans against IgA1 self-aggregation and adhesion to extracellu- 33. Mestecky J, Hashim OH, Tomana M. Alterations in the IgA lar matrix proteins. J Am Soc Nephrol 1998; 9: 2048–2054 carbohydrate chains influence the cellular distribution of IgA1. 17. Czerkinsky C, Koopman WJ, Jackson S et al. Circulating Contrib Nephrol 1995; 111: 66–72 immune complexes and immunoglobulin A rheumatoid factor 34. Coppo R, Amore A, Gianoglio B et al. Macromolecular IgA in patients with mesangial immunoglobulin A nephropathies. and abnormal IgA reactivity in sera from children with IgA J Clin Invest 1986; 77: 1931–1938 nephropathy. Clin Nephrol 1995; 43: 1–13 Received for publication: 24.2.99 Accepted in revised form: 26.8.99