The effect of human anticardiolipin antibodies on murine pregnancy

JOURNALOF REPItOI)IKq'IVE IMMUNOLOGY ELSEVIER

Journal of Reproductive Immunology 27 (1994) 123-134

The effect of human anticardiolipin antibodies on murine pregnancy L.W. Chamley *a'l, N.S. Pattison a, E.J. M c K a y b ~Department of Obstetrics and Gynaecology, University of Auckland, Auckland, New Zealand bDepartment of Virology and Immunology, Auckland Hospital, Auckland, New Zealand Accepted 20 July 1994

Abstract

Anticardiolipin antibodies (aCL) were affinity purified or isolated in the IgG fraction of serum from 6 patients with antiphospholipid antibody syndrome. Anticardiolipin antibodies from one patient consistently compromised murine pregnancy. However in 92% (45 of 49) of cases injection of human anticardiolipin antibodies had no adverse effect on murine pregnancy, regardless of whether affinity purified aCL or IgG fractions were used. It is concluded that in most cases human anticardiolipin antibodies alone do not induce murine fetal loss.

Keywords: Anticardiolipin antiphospholipid; Beta-2 glycoprotein 1; Stillbirth; Murine pregnancy; Miscarriage

1. Introduction Anticardiolipin antibodies (aCL) are one of a family of autoantibodies, antiphospholipid antibodies, that are strongly associated with thrombotic conditions and recurrent human fetal loss. Two recent studies have demonstrated that IgG fractions of human serum containing aCL cause a * Corresponding author. Current address: Department of Immunology, University of Liverpool, PO Box 147 Liverpool, United Kingdom. 0165-0378/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0165-0378(94)00893-C

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reduction in litter size and viability when injected into pregnant mice (Branch et al., 1990; Blank et al., 1991). These studies were limited in that IgG fractions were used rather than specific aCL. Thus fetal losses may have been induced by antibodies other than aCL. This study reports the variability of the effects of human aCL on murine pregnancies. 2. Materials and methods

2.1. Affinity purification of anticardiolipin antibodies Cardiolipin liposomes were prepared as described by Pengo et al. (1987) and incubated with patient serum for 60 min at room temperature. The liposomes were harvested by centrifugation at 27 000 x g for 10 min and resuspended in 3 M NaC1. Eluted proteins were separated from the liposomes by passage through a 0.22-t~m filter and dialysed against PBS. Anticardiolipin antibodies were further purified by binding to Protein A Affigel (Biorad Laboratories). Impurities were washed off with a 0.2% solution of n-octyl/3-D-glucopryanoside (Sigma) as described by Galli et al. (1990). Anticardiolipin antibodies, free of contaminants, were eluted from the Protein A column with 0.1 glycine (pH 2.5) and dialysed against PBS. 2.2. Isolation of immunoglobulin fractions The IgG fraction of patient and control serum was isolated by incubating serum with Protein A Affigel (Biorad Laboratories) for 3 h at room temperature. The Protein A beads were then placed into a column and washed with 0.5 M NaC1 pH 7.2 to remove contaminants. The pure IgG was eluted with 0.1 M glycine pH 2.5 and dialysed against PBS. 2.3. Anticardiolipin antibody ELISA Anticardiolipin antibodies were assayed using our previously published method (Chamley et al., 1991). This assay, which employs new born calf serum as the diluent and blocking agent, is calibrated against the Kingston Antiphospholipid Antibody Standards (KAPS). The KAPS standards use the units GPL and MPL where 1 unit is approximately equivalent to 1 #g of affinity purified antibody of IgG or IgM class, respectively. 2.4. Date mating and injection of mice with human anticardiolipin antibodies A colony of Swiss White mice was established at the Green Lane Hospital vivarium. Virgin female mice had their oestrous cycles synchronised by exposure to a castrated male. Three days later they were placed into a cage with an intact male. Mating was confirmed by the presence of a vaginal plug. A weight gain of 10% by day 8 post-coitum (p.c.) predicted pregnancy in 95% of the animals in this colony. The mean delivered litter size for the colony was 11.3 (S.D., 2.7) and the mean length of gestation was 20.5 days (n = 18).

L.W. Chamley et al./J. Reprod. Immunol. 27 (1994) 123-134

125

Mice with confirmed pregnancies were injected with human aCL or control human IgG via the tail vein using a 30-gauge hypodermic needle. Weight gain was monitored daily and animals were sacrificed on day 15 of gestation or earlier if obvious fetal loss occurred. Sacrificed animals were examined for litter size and weight and sites of fetal reabsorption. These procedures were approved by the institutional ethics committee.

2.5. Polyacrylamide gel electrophoresis and Western blot Vertical slab sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) was carried out according to the method of Laemmli (1970). Molecular weight markers were purchased from Biorad Laboratories Ltd. and prestained markers from Sigma. Following separation on SDS PAGE, proteins were transferred to Biotrace RP membrane using a Gelman Sciences electroblot unit. The membranes were blocked with 5% non-fat milk in PBS (pH 7.2) overnight at 4°C. Antisera were diluted in the blocking agent and incubated with the membrane for 2 h at 37°C. The membranes were washed with PBS, and chloronaphthol (0.6 mg/ml in PBS (pH 7.2) containing 0.06% H202) was used to develop the blot. Rabbit anti-human/32 glycoprotein 1 antiserum was purchased from Behring. Horseradish peroxidase (HRP) conjugated, affinity purified, anti-rabbit IgG and H R P conjugated, affinity purified, anti-human gamma chains antiserum were purchased from Tago. 2.6. Patient data Serum samples were obtained from patients with aCL and stored at -20°C until used. Approval was granted by the institutional ethics committee. Patient J is a 32-year-old woman with primary antiphospholipid antibody syndrome (aPLS) who had 2 mid-trimester fetal deaths and 1 live birth following treatment with corticosteroids and aspirin. She has both lupus anticoagulant and aCL > I00 MPL and 60 GPL. Patient B is a 33-year-old woman with aPLS secondary to SLE. She had 2 mid-trimester fetal deaths. She was treated with heparin during her last pregnancy due to a previous deep vein thrombosis (DVT). She suffered a second DVT following her last fetal loss. She has both lupus anticoagulant and aCL < 5 MPL, and 45 GPL. This sample was drawn following her last fetal loss. Patient I is a 26-year-old female with primary aPLS who had a previous live birth but had a third trimester fetal death in her last pregnancy. She was suspected of having a DVT following her last pregnancy. She has both lupus anticoagulant and aCL < 5 MPL and > 100 GPL. Patient K is a 27-year-old female with primary aPLS. She had 1 thirdtrimester fetal death, a previous DVT and pulmonary embolism. During her last pregnancy, which was complicated by proteinuria and retinal ischaemia, she was treated with heparin, aspirin and corticosteroids. This pregnancy

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resulted in a live birth at 36 weeks gestation. She has both lupus anticoagulant and aCL 12 MPL and 39 GPL. Patient O'H is a 34-year-old female with aPLS secondary to SLE. This sample was drawn during a pregnancy complicated by severe hypertension of pregnancy and intrauterine growth retardation. She has both lupus anticoagulant and aCL < 5 MPL, > 100 GPL. Patient S is a 57-year-old male with aPLS secondary to SLE. This sample was drawn following a deep vein thrombosis and contained > 100 GPL. 3. Results

3.1. Effect of affinity purified anticardiolipin antibodies on mur&e pregnancy Cardiolipin liposome affinity chromatography was used to isolate aCL from the serum of Patient J. Three separate preparations containing increasing concentrations of aCL were produced. These preparations, labelled 1, 2 and 3 contained 20, 40 and 70 GPL respectively. SDS PAGE demonstrated the preparations to contain only IgG (Fig. 1). Fourteen pregnant mice were injected on day 9 p.c. with increasing volumes (range, 200-500/~1) of these preparations. All of these animals continued to gain weight with increasing gestation. On day 14 p.c. they were sacrificed and found to have normal sized litters (Table 1). 3.2. Effect of IgG fractions on the outcome of murine pregnancies Total IgG was obtained from the serum of Patient J, 2 further women (I and K) and one man (S) with serum aCL. These preparations which contained up to 122 #g/ml aCL (Table 2) were demonstrated to contain only IgG by SDS PAGE (Fig. 1). A fourth preparation from a woman with aCL (OH) was shown to contain, in addition to IgG, proteins of apparent molecular weights 53 and 136 kDa (Fig. 1). Western blotting using an affinity purified antibody to human gamma chains demonstrated these additional proteins to be fragments of IgG (data not shown). A total of 19 pregnant animals were injected with these preparations on day 9 p.c. Three animals also had an additional injection on day 13 p.c. Eighty-two percent (18 of 22) of these pregnancies were unaffected by the administration of human aCL (Table 2). The only IgG fraction which produced consistent adverse effects on murine pregnancies was that of Patient O'H. Injection of the IgG fraction from this patient (O'H) reduced fetal viability in all studied pregnancies (Table 2). Two animals injected with aCL from this patient had non-viable litters. Upon dissection the pups from these litters were found to be small sized and macerated (Fig. 2). Seven of the thirteen pups from a third animal injected with aCL derived from Patient O'H were found to be small and nonviable whilst the remaining 6 pups appeared normal.

L.W. Chamley et al./J. Reprod. Immunol. 27 (1994) 123-134

1

2

3

4

5

6

127

7

97--

66

50--

Fig. 1. Coomassie blue (G250) stained SDS polyacrylamide gel (8%) analysis of aCL containing IgG fractions of patients' serum and cardiolipin liposome affinity purified aCL. Samples were run under non-reducing conditions. Lane 1, lgG fraction of Patient O'H; Lane 2, IgG fraction of Patient S; Lane 3, IgG fraction of Patient I; Lane 4, IgG fraction of Patient B; Lane 5, lgG fraction of Patient J; Lane 6, IgG fraction of normal serum; Lane 7, affinity isolated aCL patient J.

The litter from one animal injected with aCL from Patient S contained 5 non-viable and 10 normal pups. The pregnancies of three further animals injected with this preparation of aCL were unaffected (Table 2). Injection of a control IgG fraction obtained from pooled normal serum also had no effect on the studied pregnancies. 3.3. Survival o f human anticardiolipin antibodies in the murine circulation In order to determine how rapidly aCL was cleared from the murine circulation after injection, three animals were administered 500 ttl of IgG containing aCL (53 GPL) from Patient B. Blood samples, obtained prior to and up to 7 days post-injection, were assayed for aCL (Fig. 3). The level of circula-

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Table 1 The effect of affinity purified aCL from Patient J on murine pregnancies Preparation number

GPL/ml ~

Gestational age at injection

Volume of Number of injection (#1) animals

Effect on pregnancy outcome

1

20

2

40

3

70

9 9 9 9 9 9 9 9

500 250 500 250 500 300 250 200

no no no no no no no no

3 1 2 2 2 2 1 1

effect effect effect effect effect effect effect effect

aKingston Antiphospholipid Antibody Workshop Units: 1 GPL is approximately equal to 1 ~g of affinity purified Ig aCL.

Table 2 The effect of aCL containing IgG fractions on murine pregnancies lgG fraction from patient

GPL/ml a

Gestational age at injection

Volume of in- Number of jection (/~1) animals

Effect on pregnancy outcome

5 1 1 I 1 1 1 2 1 1 3

no effect no effect no effect no effect no effect no effect no effect no effect no effect no effect 2-non viable litter 1-7/13 nonviable 2 no effect 1 - 5 / 1 5 nonviable no effect no effect

J

71

9

I

53

K

34

9 9 9/13 9/13 9

O'H

35

9

500 400 200 500 300 500/500 500/300 500 350 250 500

122

9

500

3

9/13 9

350/250 500

1 7

S

Control

0

aKingston Antiphospholipid Antibody Workshop Units: 1 GPL is approximately equal to 1 #g of affinity purified IgG aCL.

L. IV. Chamley et al. /J. Reprod. Immunol. 27 (1994) 123-134

IIII Ill IIII

129

IIIlil

Fig. 2. Uteri containing small sized, macerated fetuses from animals injected with aCL from Patient O'H (top and middle) and normal sized fetuses from an animal injected with normal human IgG (bottom). Scale is in cm.

Human aCL Level in Mouse Injected with Antibody with Time 0.5 0.4 0.3 OD o.2

O.1 0

I

I

1 2

I

L

6

24

//(

I

49

hours post injection

Fig. 3. The survival of human aCL in the murine circulation was examined by injecting 3 nonpregnant mice with 500 #l of the IgG fraction from Patient B (53 GPL) via the tail vein. At various intervals blood samples were drawn from the tail vein. Serum from these samples was examined for human aCL by ELISA. Diagram shows a typical survival aCL curve. Samples were assayed in quadruplicate. Bars indicate standard errors.

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L.W. Chamley et al./J. Reprod. Immunol. 27 (1994) 123-134

ting aCL declined rapidly during the first 24 h post-injection and thereafter decreased slowly during the remainder of the study period with aCL remaining detectable, albeit at low levels for at least 6 days.

3.4. The effect of a modified injection protocol The injection protocol was modified to ensure high levels of aCL were maintained in the murine circulation throughout the study period. Aliquots of the IgG fraction of serum from 2 patients (J and B) were injected into 13 animals with vaginal plugs on days 4 or 5, 9 and 12. None of these pregnancies were affected (Table 3). A further 5 pregnant animals were injected at the same gestational ages with normal IgG. There was no effect on these control pregnancies (Table 3). 3.5. The ability of mur&e serum to act as cofactor to human anticardiolip& antibody preparations In order to determine whether murine f32 glycoprotein 1 (/32 GP1) could act as cofactor to the purified human aCL we performed a modified aCL ELISA. The level of aCL in 5 of the above aCL-containing IgG fractions was measured using either New Born Calf Serum (NBCS), pooled normal murine serum (MS) or Bovine Serum Albumin (BSA) as the assay's blocking agent and sample diluent. For all of the samples tested the level of aCL binding

Table 3 The effect of aCL on murine pregnancies when injected at an early gestational age IgG fraction from patient

GPL/ml a

Gestational age at injection

Volume of in- Number of jection (tzl) animals

Effect on pregnancy outcome

J

70

B

53

5 5/9 5/9 5/9/12 4 5 4/9 9 5 5/9 5/9 9

500 250/500 500/500 500/500 500 500 500/500 500 500 500/500 250/250 500

no no no no no no no no no no no no

Controls

0

1 2 2 1 1 3 2 1 1 1 1 2

effect effect effect effect effect effect effect effect effect effect effect effect

aKingston Antiphospholipid Antibody Workshop Units: 1 G P L is approximately equal to 1 ~tg of affinity purified IgG aCL.

L.W. Chamley et al./J. Reprod. lmmunol. 27 (1994) 123-134

131

2.5 • NBCS [] Murine serum [] BSA

2.0 ¢R t-

1.5

O

1.0

D.

O

0.5

0.0 I

B

J

O'H Patient

S

Control

Fig. 4. IgG fractions containing aCL were assayed in a conventional aCL ELISA employing 10% New Born Calf Serum as the assay diluent and blocking agent. The samples were simultaneously assayed by modified aCL ELISA which employed 10% pooled normal murine serum as both the blocking agent and assay diluent. Because the IgG fractions do not contain the aCL cofactor, B2 GP1, any aCL binding in this modified assay will be dependent upon the cofactor activity present in the murine serum. A third aCL assay employing 1% BSA as the assay blocking agent and diluent acts as a control to indicate the apparent level of aCL binding in the absence of cofactor.

was significantly higher in the ELISA using NBCS than in either of the ELISAs using MS or BSA (P < 0.05, t-test) (Fig. 4). There was no aCL binding in any of the ELISAs for control IgG (Fig. 4). 4. Discussion

Antiphospholipid antibodies are strongly associated with both recurrent human fetal loss and recurrent thrombotic disease (Pattison et al., 1993; Lockwood et al., 1989; Birdsall et al., 1992; Hamsten et al., 1986; Asherson et al., 1987). While much recent attention has focused on possible mechanisms of action of aPL, little attention has been directed to the question of whether these antibodies are in fact pathogenic. We have studied the effect of isolated human aCL on murine pregnancies. Antieardiolipin antibodies from only 1 of 6 patients consistently compromised murine pregnancies (4 of 61 mice studied). Thus, there was no statistically significant relationship between the injection of aCL and fetal demise (P = 0.4, Fisher's exact test). This result differs from two similar previous studies (Branch et al., 1990; Blank et al., 1991). There are several possible ex-

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L.W. Chamley et al./J. Reprod. lmmunol. 27 (1994) 123-134

planations for the differences in the results of this study and those of earlier workers. 4.1. Differences in the quantity and route of administration by which the antibody is given to the pregnant mice Branch et al. (1990) administered 15 mg/animal i.p. of total IgG whilst Blank et al. (1991) administered 10 t~g/animal i.v., but neither of these teams indicated the amount of specific aCL contained within these preparations. In contrast, we administered up to 61 GPL aCL/animal i.v. (these preparations contained approximately 5 mg/ml total IgG). Our clinical experience indicates that this level of aCL is frequently found in women with the antiphospholipid antibody syndrome and recurrent fetal loss. Further, fetal loss did occur as the result of injecting one of our aCL preparations, suggesting that this hypothesis does not explain the failure of aCL to induce fetal loss in this study. 4.2. The aCL may have been cleared from the murine circulation before disrupting the pregnancy There are two arguments against this hypothesis. First, repeatedly injecting mice in order to maintain a consistently high circulating level of aCL did not compromise the studied pregnancies. Second, a single injection of aCL from one patient (O'H) was sufficient to induce fetal loss. 4.3. Differences in the purity of aCL administered Previous workers used IgG fractions obtained by Ion Exchange and/or Protein A chromatography. Both of these procedures can yield IgG contaminated by other proteins, notably,/32 GPI. The immunoglobulins used in this study were free of contaminants. It is possible that IgG preparations used by previous workers contained, in addition to aCL, other factors necessary to induce fetal loss. Alternatively, these preparations may have contained antibodies unrelated to aCL which caused the fetal loss. 4.4. A different antiphospholipid antibody may be responsible for fetal loss Some aCL are known to bind to one or more phospholipids other than cardiolipin. It is possible that previous workers have used IgG fractions containing cross-reactive antibodies which react with phospholipids such as phosphotidyl ethanolamine or phosphotidyl serine in addition to cardiolipin. Similarly, our preparation which caused fetal death (O'H) may also have contained cross-reactive antiphospholipid antibodies. 4.5. Murine 132 GP1 does not promote human aCL activity /32 GP1 has been shown to be essential to promote the binding of pathogenic aCL to phospholipid surfaces in vitro (Matsuura et al., 1990; McNeil et al., 1990). Murine serum, at best, poorly promotes the binding of human aCL to phospholipid surfaces. This indicates that structural differ-

L.W. Chamley et al./J. Reprod Immunol. 27 (1994) 123-134

133

ences between human and murine/32GP1 may prevent the murine molecule acting as a cofactor to human aCL both in vitro and in vivo. This suggestion is supported by the finding that monoclonal antibodies which react with human /32 GP1 do not react with bovine /32 GP1 despite bovine t32 GP1 being 89% homologous to the human molecule (Gao et al., 1993; Wagenknecht and Mclntyre, 1993).

4. 6. Variations in the reactivity of aCL from different patients may affect their activity in the murine model of fetal loss Variability in the antigenic reactivity of polyclonal aCL is well described (Harris et al., 1985). It is possible that aCL derived from some patients will react with murine/32GP1 and bind to phospholipid surfaces in vivo (e.g. utero-placental blood vessels) to cause fetal loss whilst aCL from other patients do not. This would closely reflect the clinical situation where patients with apparently similar antibodies (aCL) can have markedly different clinical presentations. 5. Conclusion This study demonstrates that in the majority of cases aCL alone are not sufficient to induce fetal loss. This result extends the finding of previous studies using murine models of fetal loss and complements numerous other studies which have shown that aCL require a cofactor in vitro. (Branch et al., 1990; Galli et al., 1990; McNeil et al., 1990; Matsuura et al., 1990; Blank et al., 1991). The present study indicates that aCL also require a cofactor in vivo. Further studies are required to determine the nature of this in vivo cofactor.

Acknowledgments The authors are grateful to the staff of the Green Lane Hospital Surgical Laboratory for their assistance in the housing and handling of the animals used in this investigation. This research was supported by a grant from the Auckland Medical Research Foundation, New Zealand.

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