SEARCH

SEARCH BY CITATION

Keywords:

  • Decidua basalis;
  • decidua parietalis;
  • extravillous trophoblast;
  • pre-eclampsia;
  • Treg cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References

Regulatory T cells (Tregs) have been identified as immunomodulatory cells, which induce tolerance. Evidence from numerous recent studies implicates these cells as critical for maternal tolerance to the fetal-derived placenta, proper functioning of which is required for normal gestation and growth and development of the fetus in utero. This review focuses on the recent studies of Tregs at the maternal–fetal interface in pregnancy complications, as well as their identification in specific inflammatory lesions in the placenta.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References

One of the most intriguing questions in the realm of immunology is the mystery of maternal tolerance to the semi-allogeneic fetus. In the recent past, one key discovery related to this area has been identification of regulatory T cells (Tregs), defined as a subset of T-helper cells, which co-express surface CD4 and CD25, and the transcription factor FOXP3.[1] Tregs modulate the functions of other T cells (both CD4+ and CD8+), primarily through secretion of cytokines, including IL-10 and TGFβ.[1, 2] Evidence for the role of Treg cells in establishing fetal tolerance during pregnancy comes from multiple animal studies, demonstrating a significantly increased rate of fetal resorption in allogeneic gestations of Treg-deficient mice.[3-5] Interestingly, syngeneically-mated Treg-deficient mice had a normal fetal outcome, indicating that Tregs suppress the maternal immune response against fetal alloantigens, rather than responses against paternal minor histocompatibility antigens.[3]

Most of what we know about the role of Tregs in human pregnancy comes from quantification of this cell population in peripheral blood, which may or may not be representative of events at the maternal–fetal interface. Recent studies have shown a differential distribution of Tregs in decidua versus peripheral blood and provided evidence for the selective migration of fetus-specific Tregs from peripheral blood to decidua, further emphasizing the importance of studying Tregs at the maternal–fetal interface.[6, 7] In this review, we will focus on studies of Tregs in the human decidua and placenta, in both normal and abnormal pregnancies.

Tregs in normal gestation

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References

Following ovulation, during the luteal phase of the menstrual cycle, the corpus luteum secretes progesterone, which leads to changes in the endometrial glands and stroma, preparing it for implantation. After fertilization, the continued secretion of progesterone leads to hypersecretory changes in the endometrial glands and ‘decidualization’ of the stroma. This altered endometrial stroma, or ‘decidua’, is the maternal portion of the maternal–fetal interface and in humans is subdivided into decidua basalis and decidua parietalis. The former is adjacent to the basal plate of the placental disk and is normally invaded by the interstitial extravillous trophoblast (EVT) (Fig. 1A); the latter forms the maternal portion of the chorioamniotic membranes, adjacent to the EVT of the chorion (Fig. 1B). The decidua parietalis is easily examined in late gestation, as part of the fetal membrane ‘roll’ generated during routine placental examination (Fig. 1B), while the amount of decidua basalis available for review within the delivered placenta is generally more variable, depending on the separation point of the placental disk. Optimal examination of the decidua basalis generally requires obtaining placental site biopsies, following delivery.

image

Figure 1. The two types of decidua and their relationship to extravillous trophoblast (EVT). (A) Decidua basalis, located at the ‘base’ of the placental disk, showing a mononuclear inflammatory infiltrate (likely to be composed mostly of monocytes and T cells) and multiple invasive interstitial EVT (arrowheads), both normal components of this tissue (400× magnification). (B) Decidua parietalis (labeled ‘d’), located in fetal membranes, adjacent to the EVT in the chorion (labeled ‘c’). Amnion is designated ‘a’. Decidual (maternal) vessels are designated with arrowheads. Ample decidua parietalis can be evaluated as part of the fetal membrane ‘roll’ during placental examination. (100× magnification).

Download figure to PowerPoint

Treg cells were first identified in the human decidua in 2004, by two independent groups.[8, 9] Heikkinen et al.[8] analyzed decidual mononuclear cells by flow cytometry and found that 14% of decidual CD4+ cells expressed surface CD25; further, phenotypic characterization of these cells confirmed the presence of Treg cells in the human decidua. Sasaki et al.[9] showed that these cells are able to inhibit the proliferation of autologous CD4+CD25 T cells in a dose-dependent fashion, thus confirming their immunosuppressive capability.

Recent studies by Tilburgs et al.[6] have found Treg cells to be much more abundant (as a proportion of total CD4+ cells) in decidual samples compared to peripheral blood samples in pregnant patients; furthermore, they have shown that the decidua parietalis has a significantly higher percentage of Treg cells compared to the decidua basalis. These decidual Tregs are functional, with the ability to downregulate both fetus-specific and non-specific immune responses.[7] Finally, this group has also reported that the number of decidual Tregs correlates with the degree of HLA mismatch between mother and fetus in healthy term pregnancies, giving further credence to the notion that Tregs act locally to facilitate maternal acceptance of an allogeneic fetus.[10]

Our own study found the median proportion of decidual Tregs to peak in the second trimester and decrease thereafter.[11] These results differ from Tilburgs et al.[6] which found no difference in the proportion of Tregs in the decidua basalis across gestation, but did find an increase in the decidua parietalis from second to third trimester. The difference in these two studies is likely due to a combination of different methods of Treg cell identification (immunohistochemistry for FoxP3 in our study versus FACS for CD4/CD25 in Tilburgs et al.) and different gestational ages of the second trimester group (<16 weeks in our study, versus >16 weeks in the Tilburgs study).[6, 11]

Treg cells in pre-eclampsia

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References

Pre-eclampsia (PE) is characterized by shallow invasion of EVT at the decidua basalis, leading to placental insufficiency and oxidative stress.[12] Particularly when severe with an early onset (<34 weeks gestation), PE is commonly associated with fetal growth restriction and often requires preterm delivery to release the fetus from the harmful intrauterine environment.[13] On gross examination, PE placentas are characterized by small size (disk weight of <10th percentile for gestational age) and multiple central infarcts. Microscopically, the chorionic villi are hypermature, showing an increased number of syncytial knots for gestational age.[14] In addition, spiral arterioles in the maternal decidua show evidence of vasculopathy, characterized by perivascular inflammation, fibrinoid necrosis, and/or atherosis (Fig. 2). The latter involves infiltration of the vessel wall with lipid-laden macrophages. These gross and microscopic changes correlate with severity of the symptoms of PE and are most common in early-onset severe PE.[11, 15]

image

Figure 2. Decidual vasculopathy. (A) Multiple thickened decidual vessels in decidua parietalis in a membrane roll of a patient with severe pre-eclampsia (100× magnification). (B) Closer examination shows perivascular inflammatory infiltrate as well as fibrinoid necrosis of the vessel walls. (200× magnification). Arrowheads in both panels point to decidual vessels.

Download figure to PowerPoint

The regulation of EVT invasion is incompletely understood; however, work over the past two decades has established a role for uterine immune cells in this process, including uterine NK (uNK) and, more recently, Treg cells.[16, 17] Specifically, it is thought that crosstalk between EVT, uNK, and Treg cells is required to achieve a balance between EVT invasion and apoptosis.[17, 18] In fact, fetal resorption following allogeneic mating of Treg-deficient mice is accompanied by defective remodeling of spiral arteries.[5]

The majority of studies of Treg cells in PE have focused on measuring this cell population in peripheral blood.[19-24] The results have been mixed, although most point to a reduction in Treg cells in PE.[22-24] The major reason for the mixed results is the heterogeneity of the cases of PE, with most studies not separating mild/severe or term/preterm PE. One study included mostly severe and preterm (early onset) PE cases and evaluated both peripheral blood and decidual Treg cells in these patients; the results confirmed the proportion of Treg cells to be decreased in both locations in these patients, compared to normal term controls.[25] Our own study expanded on this, evaluating cytotoxic T (CD8+) and Treg cells in the decidua parietalis, in both early- and late-onset severe PE, in comparison with normal term membranes.[11] We showed that, while decidual cytotoxic T cells are increased overall in both early- and late-onset severe PE, the median proportion of decidual Tregs was significantly lower only in pregnancies complicated by early-onset severe PE (proportion of Tregs as a function of total CD3+ cells: 0.46% in early-onset severe PE, compared to 5.21% in uncomplicated term pregnancies, and 3.34% in late-onset severe pre-eclampsia; P < 0.001 and P = 0.004, respectively).[11] This decrease in decidual Tregs, specifically in early-onset severe PE, was accompanied by increased apoptosis of extravillous trophoblast (EVT) in the chorion, adjacent to the decidua parietalis.[11] We believe the altered balance of T cells at the maternal–fetal interface may help tip the balance toward increased EVT apoptosis and away from EVT invasion. However, the EVT population we studied here was the chorionic trophoblast in the fetal membranes and not the invasive (interstitial and endovascular) EVT. To best study this population and determine a direct correlation between Treg numbers and EVT invasion, a study of the decidua basalis, requiring placental bed biopsies, would be most optimal.

Tregs in preterm labor/premature rupture of membranes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References

Similar to PE, immunologic mechanisms have also been shown to play a pivotal role during labor and rupture of membranes. For example, it has been suggested that fetal macrophages, along with the maternal immune system, play a role in labor initiation.[26] During the early stages of labor, fetal macrophages produce and secrete IL-6, a known inhibitor of Treg activity.[27] There are few studies of decidual Tregs in the onset of labor or rupture of membranes: most focus on circulating Tregs and show a decrease in either the proportion or suppressive activity of Tregs in labor or with premature rupture of membranes (PROM), compared to women without labor or PROM.[28-31] A single study has been carried out on Tregs in the decidua basalis and shows that their proportion is decreased in women with term labor, in comparison with those at term without labor.[32]

We have characterized the proportion of Tregs in the decidua parietalis of placentas from normal pregnancies and those complicated by either preterm premature rupture of membranes (PPROM) or preterm labor (PTL) and found the median proportion of decidual Treg cells to be significantly lower in PPROM (1.19%; P = 0.03) and PTL (0.25%; P = 0.019), compared to uncomplicated term pregnancies (5.21%) (Quinn et al., unpublished data).

Tregs in inflammatory lesions of the placenta

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References

Inflammation in the placenta occurs either in the membranes (‘chorioamnionitis’) or in the chorionic villi (‘villitis’) and can be acute (predominantly neutrophils) or chronic (predominantly lymphocytes and macrophages).[33] Acute chorioamnionitis is the most common lesion, is clinically associated with amniotic fluid infection, and is a primary cause of preterm births.[33] Chronic chorioamnionitis, characterized by a predominantly mononuclear infiltrate in the fetal membranes (Fig. 3A–B), is both a more rare and less recognized lesion, and is perhaps most significant for its association with non-infectious chronic villitis (‘villitis of unknown etiology’ or VUE) (Fig. 3C–D).[33, 34] While the majority of the cells in chronic chorioamnionitis are T cells,[34] the mononuclear infiltrate in VUE is a mixture of placental (fetal) CD14+ macrophages (Hofbauer cells) and maternal lymphocytes, predominantly T cells.[33-35] Both chronic chorioamnionitis and VUE are thought to represent immunological lesions similar to transplant rejection or graft-versus-host disease.[34] In fact, similar to the latter lesions, the inflammation in chronic chorioamnionitis tends to extend to decidual vessels and can even be associated with frank vasculopathy, with fibrinoid necrosis in the vessel wall (Fig. 3B). Most recently, a study by Katzman et al.[36] has shown that Tregs are present in VUE, although their presence in chronic chorioamnionitis remains unknown. Furthermore, the mechanism of recruitment and function of Tregs in these lesions has yet to be explored. It is possible that the recruitment of Tregs to these lesions may dampen the immune response or limit its spread. Further studies are needed to determine the relationship between the extent of Treg infiltration in these inflammatory placental lesions and perinatal outcome.

image

Figure 3. Inflammatory lesions in the placenta associated with immune dysregulation. (A) Chronic chorioamnionitis is characterized by a predominantly mononuclear infiltrate in the fetal membranes, extending up from the decidua into the chorion and toward the amnion. (200× magnification). (B) The chronic inflammatory infiltrate in chronic chorioamnionitis commonly involves the decidual vessels, which in this case show a mild amount of fibrinoid necrosis. (200× magnification). (C) Chronic villitis of unknown etiology (VUE) is characterized by involvement of chorionic villi with lymphocytes (predominantly maternal T cells) and macrophages (predominantly fetal/placental CD14+ Hofbauer cells) and typically extends up from the basal plate (maternal surface of the placental disk). Chronic chorioamnionitis is commonly present with VUE in the same placenta (100× magnification). (D) Close up of chorionic villi with VUE shows infiltration of the villous stroma with a chronic inflammatory infiltrate, loss of fetal vessels, and stromal fibrosis. (200× magnification).

Download figure to PowerPoint

Summary

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References

Much has been learned about the development and function of Treg cells; however, their role in normal implantation, initiation of labor, and rupture of membranes, as well as pregnancy complications, still requires further study. While the evaluation of these cells at the maternal fetal interface is at times more difficult, we believe that such studies are needed to determine, not only the true nature of the decidual T reg cells, but also their interaction with the fetal-derived trophoblast, as well as their contribution and responses to the local microenvironment during pregnancy.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Tregs in normal gestation
  5. Treg cells in pre-eclampsia
  6. Tregs in preterm labor/premature rupture of membranes
  7. Tregs in inflammatory lesions of the placenta
  8. Summary
  9. Acknowledgements
  10. References
  • 1
    Saito S, Nakashima A, Shima T, Ito M: Th1/Th2/Th17 and Regulatory T-cell paradigm in pregnancy. Am J Reprod Immunol 2010; 63:601610.
  • 2
    Sakaguchi S, Yamaguchi T, Nomura T, Ono M: Regulatory T cells and immune tolerance. Cell 2008; 133:775787.
  • 3
    Aluvihare V, Kallikourdis M, Betz A: Regulatory T cells mediate maternal tolerance to the fetus. Nat Immunol 2004; 5:266271.
  • 4
    Saito S, Shiozaki A, Sasaki Y, Nakashima A, Shima T, Ito M: Regulatory T cells and regulatory natural killer (NK) cells play important roles in feto-maternal tolerance. Semin Immunopathol 2007; 29:115122.
  • 5
    Samstein R, Josefowicz S, Arvey A, Treuting P, Rudensky A: Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict. Cell 2012; 150:2938.
  • 6
    Tilburgs T, Roelen DL, Van der Mast BJ, Van Scip JJ, Kleijburg C, de Groot-Swings GM, Kanhai HH, Claas FH, Scherjon SA. Differential distrubution of CD4+CD25bright and CD8+CD28- T-cells in decidua and maternal blood during human pregnancy. Placenta 2006; 27 Suppl. A: S47S53.
  • 7
    Tilburgs T, Roelen D, van der Mast B, de Groot-Swings GM, Kleijburg C, Scherjon S, Claas F: Evidence for a selective migration of fetus-specific CD4+CD25bright regulatory T cells from the peripheral blood to the decidua in human pregnancy. J Immunol 2008; 180:57375745.
  • 8
    Heikkinen J, Mottonen M, Alanen A, Lasila O: Pheontypic characterization of regulatory T cells in the human decidua. Clin Exp Immunol 2004; 136:373378.
  • 9
    Sasaki Y, Sakai M, Miyazaki S, Higuma S, Shiozaki A, Saito S: Decidual and peripheral blood CD4+ CD25+ regulatory T cells in early pregnancy subjects and spontaneous abortion cases. Mol Hum Reprod 2004; 10:347353.
  • 10
    Tilburgs T, Scherjon SA, van der Mast BJ, Haasnoot GW, Versteeg VD, Voort-Maarschalk M, Roelen DL, van Rood JJ, Claas FHJ: Fetal-maternal HLA-C mismatch is associated with decidual T cell activation and induction of functional T regulatory cells. J Reprod Immunol 2009; 82:148157.
  • 11
    Quinn KH, Lacoursiere DY, Cui L, Bui J, Parast MM: The unique pathophysiology of early-onset severe preeclampsia: role of decidual T regulatory cells. J Reprod Immunol 2011; 91:7682.
  • 12
    Burton GJ, Woods AW, Jauniaux E, Kingdom JC: Rheological and physiological consequences of conversion of the maternal spiral arteries for uteroplacental blood flow during human pregnancy. Placenta 2009; 30:473482.
  • 13
    Gilstrap L, Ramin S: ACOG Practice Bulletin Number 33. 2002:19.
  • 14
    Loukeris K, Sela R, Baergen RN: Syncytial knots as a reflection of placental maturity: reference values for 20 to 40 weeks' gestational age. Pediatr Dev Pathol 2010; 13:305309.
  • 15
    Taché V, LaCoursiere DY, Saleemuddin A, Parast MM: Placental expression of vascular endothelial growth factor receptor-1/soluble vascular endothelial growth factor receptor-1 correlates with severity of clinical preeclampsia and villous hypermaturity. Hum Pathol 2011; 42:12831288.
  • 16
    Lash GE, Robson SC, Bulmer JN: Review: functional role of uterine natural killer (uNK) cells in human early pregnancy decidua. Placenta 2010; 31(Suppl):S87S92.
  • 17
    Munoz-Suano A, Hamilton AB, Betz AG: Gimme shelter: the immune system during pregnancy. Immunol Rev 2011; 241:2038.
  • 18
    von Rango U: Fetal tolerance in human pregnancy–a crucial balance between acceptance and limitation of trophoblast invasion. Immunol Lett 2008; 115:2132.
  • 19
    Paeschke S, Chen F, Horn N, Fotopoulou C, Zambon-Bertoja A, Sollwedel A, Zenclussen M, Casalis P, Dudenhausen J, Volk H, Zenclussen A: Pre-eclampsia is not associated with changes in the levels of regulatory T cells in peripheral blood. Am J Reprod Immunol 2005; 54:384389.
  • 20
    Darmochwal-Kolarz D, Saito S, Rolinski J, Tabarkiewicz J, Kolarz B, Leszczynska-Gorzelak B, Oleszczuk J: Activated T lymphocytes in pre-eclampsia. Am J Reprod Immunol 2007; 58:3945.
  • 21
    Hu D, Chen Y, Zhang W, Wang H, Dong M: Alteration of peripheral CD4+ CD25+ regulatory T lymphocytes in pregnancy and pre-eclampsia. Acta Obstet Gynecol Scand 2008; 87:190194.
  • 22
    Toldi G, Svec P, Vasarhelyi B, Rigo J, Tulassay T, Treszl A: Decreased number of FoxP3+ regulatory T cells in preeclampsia. Acta Obstet Gynecol Scand 2008; 87:12291233.
  • 23
    Santner-Nanan B, Peek M, Khanam R, Richarts L, Zhu E: Fazekas de St Groth B, Nanan R. Systemic increase in the ratio between Foxp3+ and IL-17 producing CD4+ T cells in healthy pregnancy but not in preeclampsia. J Immunol 2009; 183:70237030.
  • 24
    Prins J, Boelens H, Heimweg J, Van der Heide S, Dubois A, Van Oosterhout A, Erwich J: Preeclampsia is associated with lower percentages of regulatory T cells in maternal blood. Hypertens Pregnancy 2009; 1:112.
  • 25
    Sasaki Y, Darmochwal-Kolarz D, Suzuki D, Sakai M, Ito M, Shima T, Shiozaki A, Rolinski J, Saito S: Proportion of peripheral blood and decidual CD4+ CD25bright regulatory T cells in pre-eclampsia. Clin Exp Immunol 2007; 149:139145.
  • 26
    Steinborn A, Sohn C, Sayehli C, Saudendistel A, Huwelmeier D, Solbach C, Schmitt E, Kaufmann M: Spontaneous labor at term is associated with fetal monocyte activation. Clin Exp Immunol 1999; 117:147152.
    Direct Link:
  • 27
    Lehner T: Special regulatory T cell review: the resurgence of the concept of contrasuppression in immunoregulation. Immunology 2008; 123:4044.
  • 28
    Kisielewicz A, Schaier M, Schmitt E, Hug F, Haensch G, Mauer S, Zeier M, Sohn C, Steinborn A. A distinct subset of HLA-DR+ regulatory T cells is involved in the induction of preterm labor during pregnancy and in the induction of organ rejection after transplantation. Clin Immunol 2010; 137: 209220.
  • 29
    Xiong H, Zhou C, Qi G: Proportional changes of CD4+CD25+FoxP3+ regulatory T cells in maternal peripheral blood during pregnancy and labor at term and preterm. Clin Invest Med 2010; 33:E422.
  • 30
    Schober L, Radnai D, Schmitt E, Mahnke K, Sohn C, Steinborn A: Term and preterm labor: decreased suppressive activity and changes in composition of the regulatory T-cell pool. Immunol Cell Biol 2012; 90:935944.
  • 31
    Seol H, Oh M, Lim J, Jung N, Yoon S, Kim H: The role of CD4+ CD25bright regulatory T cells in the maintenance of pregnancy, premature rupture of membranes, and labor. Yonsei Med J 2008; 49:366371.
  • 32
    Galazka K, Lukasz W, Pitynski K, Kijowski J, Zajac K, Bednarek W, Dutsch-Wicherek M, Rytlewski K, Kalinka J, Basta A, Majka M: Changes in the subpopulation of CD25+ CD4+ and FoxP3+ regulatory T cells in decidua with respect to the progression of labor at term and the lack of analogical changes in the subpopulation of suppressive B7–H4+ macrophages- a preliminary report. Am J of Reprod Immunol 2009; 61:136146.
  • 33
    Kraus FT, Redline RW, Gersell DJ, Nelson DM, Dicke JM: Placental Pathology. Washington, DC, Armed Forces Institute of Pathology, 2004.
  • 34
    Kim CJ, Romero R, Kusanovic JP, Yoo W, Dong Z, Topping V, Gotsch F, Yoon BH, Chi JG, Kim JS: The frequency, clinical significance, and pathological features of chronic chorioamnionitis: a lesion associated with spontaneous preterm birth. Mod Pathol 2010; 23:10001011.
  • 35
    Kim MJ, Romero R, Kim CJ, Tarca AL, Chhauy S, LaJeunesse C, Lee DC, Draghici S, Gotsch F, Kusanovic JP, Hassan SS, Kim JS: Villitis of unknown etiology is associated with a distinct pattern of chemokine up-regulation in the feto-maternal and placental compartments: implications for conjoint maternal allograft rejection and maternal anti-fetal graft-versus-host disease. J Immunol 2009; 182:39193927.
  • 36
    Katzman PJ, Murphy SP, Oble DA: Immunohistochemical analysis reveals an influx of regulatory T cells and focal trophoblastic STAT-1 phosphorylation in chronic villitis of unknown etiology. Pediatr Dev Pathol 2011; 14:284293.