Interferon-beta-1a treatment increases CD56 natural killer cells and CD4+CD25+ Foxp3 expression in subjects with multiple sclerosis

Disease modifying effects of interferon (IFN)-β therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56^bright subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-β-1a(IM) treatment of relapsing–remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56^bright NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-β-1a in MS include control of pernicious autoimmunity.     

Repeated bouts of aerobic exercise enhance regulatory T cell responses in a murine asthma model

We have reported previously that moderate intensity aerobic exercise training attenuates airway inflammation in a murine asthma model. Recent studies implicate regulatory T (Treg) cells in decreasing asthma-related airway inflammation; as such, the current study examined the effect of exercise on Treg cell function in a murine asthma model. Mice were sensitized with ovalbumin (OVA) prior to the start of exercise training at a moderate intensity 3×/week for 4 weeks; exercise was performed as treadmill running (13.5 m/min, 0% grade). Mice were OVA challenged repeatedly throughout the exercise protocol. At protocol completion, mice were analyzed for changes in the number and suppressive function of CD4⁺CD25⁺Foxp3⁺ cells isolated from lungs, mediastinal lymph nodes, and spleens. Results show that exercise increased significantly the number of Foxp3⁺ cells within the lungs and mediastinal lymph nodes, but not the spleens, of OVA-treated mice as compared with sedentary controls. Exercise also enhanced the suppression function of CD4⁺CD25⁺Foxp3⁺ Treg cells derived from OVA-treated mice as compared with sedentary controls. Specifically, Treg cells from exercised, OVA-treated mice more effectively suppressed CD4⁺CD25^? cell proliferation and Th2 cytokine production in vitro. Enhanced suppression was associated with increased protein levels of TGF-β and lesser amounts of IL-10 and IL-17; however, blocking TGF-β had no effect on suppressive functions. These data demonstrate that exercise-mediated increases in Treg cell function may play a role in the attenuation of airway inflammation. Further, these results indicate that moderate intensity aerobic exercise training may alter the Treg cell function within the asthmatic airway.     

CD4CD25 regulatory T cell in childhood ocular myasthenia gravis

Dysfunction of CD4⁺CD25⁺ regulatory T cell (Treg) has been demonstrated to play an important role in the development of autoimmune myasthenia gravis. This T cell subset, which has potent regulatory properties against immune response, has been reported to have a numerical or functional defect in patients with myasthenia gravis. We examined various T cell subsets, including CD4⁺CD25⁺Treg in peripheral blood mononuclear cells using flow cytometry in a pediatric patient suffering from ocular myasthenia gravis. Contrary to previous reports, the percentage of CD4⁺CD25⁺Treg in peripheral blood decreased significantly after successful treatment with prednisolone. This discrepancy could result from diversity within the immunopathogenesis of myasthenia gravis and may underpin a particular subgroup of myasthenia gravis seen in the East-Asian pediatric population.     

Effect of IFN-ß therapy on the frequency and function of CD4+CD25+ regulatory T cells and Foxp3 gene expression in relapsing–remitting multiple sclerosis (RRMS): A preliminary study

Interferon-ß (IFN-ß) is an immunomodulatory drug of choice to control relapsing–remitting multiple sclerosis (RR-MS), although its function is still unclear. A reduced suppressive function of CD4⁺CD25⁺ regulatory T cells (T_reg) has been shown in RR-MS patients. In this study, to understand the effect of IFN-ß on CD4⁺CD25⁺ regulatory T cells, we analyzed the frequency and function of these cells and Foxp3 gene expression before and after treatment.We evaluated the frequency and function of CD4⁺CD25⁺Foxp3⁺ regulatory T cells by flow cytometry and co-culture inhibition test respectively and gene expression of Foxp3 by real-time PCR in a longitudinal follow-up study in 18 relapsing–remitting MS patients. Our data revealed that IFN-ß significantly improved frequency and suppressive function of T_reg cells (P < 0.05) without any significant effect on gene expression of Foxp3 after 6 months. The results of the present study indicate that IFN-ß therapy in some of patients with RR-MS may restore function of regulatory T cells and control the unchecked immune cascade activity. Larger longitudinal studies on more MS patients are required to confirm our findings.     

Double filtration plasmapheresis benefits myasthenia gravis patients through an immunomodulatory action

Double filtration plasmapheresis (DFPP) is used to treat myasthenia gravis (MG). However, the definite mechanism is unclear. This study investigated whether DFPP improves MG through an immunomodulatory action. Thirty-five MG patients were randomly divided into two treatment groups: Group A (DFPP combined with oral methylprednisolone) and Group B (oral methylprednisolone alone). Their antibody levels, clinical scores, cytokine levels, and CD4⁺CD25^highFoxp3⁺ (regulatory T cell [Treg]) levels were then determined. Anti-titin antibody levels were significantly lower in Group A compared with Group B after treatment. The clinical remission rate in Group A was significantly higher than in Group B. The changes in cytokine levels (interleukin [IL]-2, IL-4, IL-10, and interferon-γ) in sera and the peripheral blood mononuclear cell culture supernatants did not significantly differ before and after the treatments in both groups (p < 0.05). The soluble intercellular adhesion molecule-1 (sICAM-1) levels were lower in Group A than in Group B (p < 0.05). MG patients exhibited a lower percentage of Treg cells than normal patients. DFPP combined with methylprednisolone treatment increased the Treg cell percentage more than treatment with methylprednisolone alone (p < 0.05). DFPP treatment more effectively lowers sICAM-1 and increases Treg cell expression, consequently benefiting MG patients.     

CD25 regulatory T cells determine secondary but not primary remission in EAE: impact on long-term disease progression

Multiple sclerosis (MS) is often characterized by several relapses and remissions during long-term disease, but neither the responsible cells nor the mechanisms are known to date. Using an animal model of multiple sclerosis, relapsing experimental autoimmune encephalomyelitis (R-EAE) CD4+CD25+ Treg cells expressing Foxp3 and CTLA-4 intracellularly and T lymphocytes expressing surface CTLA-4 were identified in the CNS. The first remission occurred even after depletion of Treg cells, but secondary remissions from EAE were ablated. Despite the unaltered first remission autoantigen rechallenge revealed already an amplified cytokine response during acute phase. These results indicate that the cellular composition during first attack of MS predicts long-term disease progression.     

EAE大鼠胸腺CD4~+ CD25~+ Foxp3~+ Treg细胞的动态变化及α-硫辛酸的干预作用

目的探讨实验性变态反应性脑脊髓炎(EAE)大鼠不同病程中胸腺CD4+CD25+Foxp3+Treg细胞变化情况及α-硫辛酸对EAE大鼠胸腺的干预作用。方法取不同时期对照组、自然病程EAE组及α-硫辛酸EAE组大鼠的胸腺组织做流式细胞学,动态检测CD4+CD25+Foxp3+Treg细胞的变化情况。结果 EAE组大鼠急性期、复发期CD4+CD25+Foxp3+Treg细胞较同时期对照组明显减少(P<0.05),缓解期有所上升;α-硫辛酸组与同期EAE组相比CD4+CD25+Foxp3+Treg细胞无明显变化;半年期三组大鼠胸腺CD4+CD25+Foxp3+Treg细胞都明显下降,各组间无统计学差异。结论 CD4+CD25+Foxp3+Treg细胞参与了EAE的发病,与病程的发展密切相关;α-硫辛酸对EAE大鼠的干预作用并非通过CD4+CD25+Foxp3+Treg细胞发挥其治疗作用;随着年龄的增长,胸腺不再是机体的主要免疫器官。     

The sympathetic nervous system modulates CD4+Foxp3+ regulatory T cells via noradrenaline-dependent apoptosis in a murine model of lymphoproliferative disease

The sympathetic nervous system (SNS) plays a crucial role in the course and development of autoimmune disease in Fas-deficient lpr/lpr mice. As regulatory T cells (Tregs) are considered important modulators of autoimmune processes, we analyzed the interaction between the SNS and Tregs in this murine model of lymphoproliferative disease. We found that the percentage of Tregs among CD4⁺ T cells is increased in the spleen, lymph nodes, and thymus of lpr/lpr mice as compared to age-matched C57Bl/6J (B6) mice. Furthermore, noradrenaline (NA), the main sympathetic neurotransmitter, induced apoptosis in B6- and lpr/lpr-derived Tregs. NA also reduced the frequency of Foxp3⁺ cells and Foxp3 mRNA expression via β₂-adrenoceptor (β₂-AR)-mediated mechanisms in a concentration and time-dependent manner. Destruction of peripheral sympathetic nerves by 6-hydroxydopamine significantly increased the percentage of Tregs in B6 control mice to an extent comparable to aged-matched lpr/lpr mice. The concentration of splenic NA negatively correlated with the frequency of CD4⁺Foxp3⁺ Tregs. Additionally, 60 days after sympathectomy, a partial recovery of NA concentrations led to Treg percentages comparable to those of intact, vehicle-treated controls. Immunohistochemical analysis of the spleen revealed localization of single Foxp3⁺ Tregs in proximity to NA-producing nerve fibers, providing an interface between Tregs and the SNS. Taken together, our data suggest a relation between the degree of splenic sympathetic innervation and the size of the Treg compartment. While there are few examples of endogenous substances capable of affecting Tregs, our results provide a possible explanation of how the magnitude of the Treg compartment in the spleen can be regulated by the SNS.     

Circulating regulatory B cell subsets in patients with neuromyelitis optica spectrum disorders

This study analyzed the populations of three different subsets of regulatory B cells (Bregs) in the peripheral blood mononuclear cells (PBMCs) of patients with neuromyelitis optica spectrum disorders (NMOSDs) and explored the relationship between the changes in these subsets of Bregs and the severity of NMOSD. A total of 22 patients with relapsed NMOSDs before treatment were recruited in our study, along with 20 age and gender-matched healthy controls, from May 2015 to March 2016. The percentages and numbers for three different subsets of Bregs including the CD19⁺CD24^hiCD38^hi, CD19⁺CD24^hiCD27⁺, and CD19⁺CD5⁺CD1d^hi populations were evaluated in parallel by flow cytometry. Afterwards, correlations between the change of three different subsets of Bregs and disease severity were analyzed. We found significantly lower percentages of CD19⁺CD24^hiCD38^hi and CD19⁺CD5⁺CD1d^hi Bregs in NMOSDs patients than in healthy individuals. In contrast, the CD19⁺CD24^hiCD27⁺ Bregs population was significantly higher in NMOSDs patients than in healthy individuals. However, the three different Bregs subsets showed no significant correlation with expanded disability status scale (EDSS) or annualized relapse rate (ARR). Our findings suggest that the subsets of Bregs may play complex roles in the pathogenesis of NMOSDs and are not correlated with clinical disease severity. Further insights into the potential role of subsets of Bregs could increase our basic knowledge of NMOSDs pathogenesis.     

Clinical significance of Tim3-positive T cell subsets in patients with multiple sclerosis

The present study evaluated associations between the percentages of T cell immunoglobulin and mucin domain 3 (Tim3)-positive T cells and related cytokines and multiple sclerosis (MS). We collected peripheral blood samples from 30 MS patients and 30 healthy controls. Flow cytometry was used to determine the proportions of CD3⁺Tim3⁺, CD4⁺Tim3⁺, and CD4⁺CD25⁺Tim3⁺ in peripheral blood mononuclear cells (PBMCs) and related cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ also were determined using enzyme-linked immunosorbent assays (ELISA). The percentages of Tim3-positive T cells in CD4⁺ and CD4⁺CD25⁺ T cell subsets were significantly lower among MS patients than among controls. This difference was particularly evident in the CD4⁺CD25(high) T cell subset. The proportions of CD4⁺Tim3⁺ and CD4⁺CD25⁺Tim3⁺ cells in PBMCs were significantly lower in the MS group than in the control group, whereas no significant differences were detected regarding the percentages of CD3⁺Tim3⁺ in PBMCs and T cell subsets. The serum concentrations of galectin-9, IL-17, and IFN-γ all were increased in MS patients compared with healthy controls. Our results support that Tim3 and related cytokines may be involved in the onset of MS.     

CD4+CD25+ T Cells are Essential for Behavioral Effects of Lactobacillus rhamnosus JB-1 in Male BALB/c mice

Over the past decade there has been increasing interest in the involvement of the microbiota-gut-brain axis in mental health. However, there are major gaps in our knowledge regarding the complex signaling systems through which gut microbes modulate the CNS. The immune system is a recognized mediator in the bidirectional communication continuously occurring between gut and brain. We previously demonstrated that Lactobacillus rhamnosus JB-1 (JB-1), a bacterial strain that has anxiolytic- and antidepressant-like effects in mice, modulates the immune system through induction of immunosuppressive T regulatory cells. Here we examined a potential causal relationship between JB-1 induced regulatory T cells and the observed effects on behaviour.We found that depletion of regulatory T cells, via treatment with monoclonal antibody against CD25, inhibited the antidepressant- and anxiolytic-like effects induced by 4-week oral administration of JB-1 in mice. Ly6C^hi monocytes were found to be decreased in JB-1 fed mice with intact regulatory T cells, but not in JB-1 fed mice following depletion. Furthermore, adoptive transfer of CD4⁺CD25⁺ cells, from JB-1 treated donor mice, but not from controls, induced antidepressant- and anxiolytic-like effects in recipient mice. Ly6C^hi monocytes were also significantly decreased in mice receiving CD4⁺CD25⁺ cells from JB1 fed donors.This study identifies cells within the CD4⁺CD25⁺ population, most likely regulatory T cells, as both necessary and sufficient in JB-1-induced antidepressant- and anxiolytic-like effects in mice, providing novel mechanistic insight into microbiota-gut-brain communication in addition to highlighting the potential for immunotherapy in psychiatric disorders.     

Gintonin mitigates experimental autoimmune encephalomyelitis by stabilization of Nrf2 signaling via stimulation of lysophosphatidic acid receptors

Gintonin (GT), a glycolipoprotein fraction isolated from ginseng, exerts neuroprotective effects in models of neurodegenerative diseases such as Alzheimer’s disease. However, the in vivo role of GT in multiple sclerosis (MS) has not been clearly resolved. We investigated the effect of GT in myelin oligodendrocyte glycoprotein (MOG_35-55)-induced experimental autoimmune encephalomyelitis (EAE), an animal model of MS. GT alleviated behavioral symptoms of EAE associated with reduced demyelination, diminished infiltration and activation of immune cells (microglia and macrophage), and decreased expression of inflammatory mediators in the spinal cord of the EAE group compared to that of the sham group. GT reduced the percentages of CD4⁺/IFN-γ⁺ (Th1) and CD4⁺/IL-17⁺ (Th17) cells but increased the population of CD4⁺/CD25⁺/Foxp3⁺ (Treg) cells in the spinal cord, in agreement with altered mRNA expression of IFN-γ, IL-17, and TGF-ß in the spinal cord in concordance with mitigated blood–brain barrier disruption. The underlying mechanism is related to inhibition of the ERK and p38 mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways and the stabilization of nuclear factor erythroid 2-related factor 2 (Nrf2) via increased expression of lysophosphatidic acid receptor (LPAR) 1–3. Impressively, these beneficial effects of GT were completely neutralized by inhibiting LPARs with Ki16425, a LPAR1/3 antagonist. Our results strongly suggest that GT may be able to alleviate EAE due to its anti-inflammatory and antioxidant activities through LPARs. Therefore, GT is a potential therapeutic option for treating autoimmune disorders including MS.     

Temporal kinetics of CD8+CD28+ and CD8+CD28− T lymphocytes in the injured rat spinal cord

This study aims to explore the temporal changes of cytotoxic CD8⁺CD28⁺ and regulatory CD8⁺ CD28⁻ T‐cell subsets in the lesion microenvironment after spinal cord injury (SCI) in rats, by combination of immunohistochemistry (IHC) and flow cytometry (FCM). In the sham‐opened spinal cord, few CD8⁺ T cells were found. After SCI, the CD8⁺ T cells were detected at one day post‐injury (dpi), then markedly increased and were significantly higher at 3, 7, and 14 dpi compared with one dpi (p < 0.01), the highest being seven dpi. In CD8⁺ T cells, more than 90% were CD28⁺, and there were only small part of CD28⁻ ( < 10%). After 14 days, the infiltrated CD8⁺ T cells were significantly decreased, and few could be found in good condition at 21 and 28 dpi. Annexin V and propidium iodide (PI) staining showed that the percentages of apoptotic/necrotic CD8⁺ cells at 14 dpi and 21 dpi were significantly higher than those of the other early time‐points (p < 0.01). These results indicate that CD8⁺ T cells could rapidly infiltrate into the injured spinal cords and survive two weeks, however, cytotoxic CD8⁺ T cells were dominant. Therefore, two weeks after injury might be the “time window” for treating SCI by prolonging survival times and increasing the fraction of CD8⁺ regulatory T‐cells. © 2016 Wiley Periodicals, Inc.     

Circulating dendritic cells subsets and regulatory T-cells at multiple sclerosis relapse: differential short-term changes on corticosteroids therapy

Glucocorticoids remain the treatment of choice for MS relapses. However, little is known on the effect of intravenous methylprednisolone (IVMP) on dendritic cells (DCs) and regulatory T-cells (TReg). Our main goal was to quantify circulating myeloid and plasmacytoid DCs (mDCs and pDCs), and TReg at MS relapse versus healthy controls; and to analyse the short-term changes after IVMP for MS relapse. MS patients at relapse compared to controls showed higher %CD4+CD25high+ TReg (p<0.01). After 5-days of IVMP, activated T-lymphocytes (p=0.001), pDCs (p<0.0001), and CD11c+ mDCs (p<0.0001) decreased. By contrast, CD4+CD25+ and CD4+CD25high+ TReg further increased (p<0.0001 both). Changes on these subsets may play a relevant role in the immunosuppressive activity of this drug.     

Regulatory T cells increased while IL-1β decreased during antidepressant therapy

Background

Regulatory T cells (Tregs, CD4⁺CD25^hi) are specialized in steering the immune response and cytokine release to maintain tolerance to self-antigens. As cytokines such as interleukin (IL)-1β, IL-6 and interferon (IFN)-α have been shown to be involved in the pathophysiology of depression and cytokine levels have been shown to change during successful antidepressant treatment, we tested the involvement CD4⁺CD25^hi Tregs in these immunological processes during antidepressant therapy.

Methods

16 patients suffering from a depressive episode were included into the study and treated with antidepressants according to their doctor’s choice. Blood samples were collected during the first week after admission and after 6 weeks of treatment. Therein, we determined plasma levels of IL-1β, and measured IL-1β, IL-6 and IFN-α levels in the stimulated blood by performing a whole blood assay. We distinguished lymphocytes and identified CD4⁺CD25^hi Tregs by multiparameter flow cytometry. The psychopathological status was assessed using the Hamilton Depression Rating Scale (HAMD-21).

Results

HAMD-21 score, IL-1β serum levels as well as LPS-stimulated IL-1β and IL-6 production had decreased significantly at the end of treatment. In contrast, the amount of CD4⁺CD25^hi cells increased significantly from 2.74% ± 0.88 (mean value ± standard deviation) to 3.54% ± 1.21; p = 0.007. No significant changes in virus-induced IFN-α production was observed.

Conclusions

The increase in CD4⁺CD25^hi Tregs during antidepressant therapy may be the reason for the decrease in cytokine production and the recovery from depression.     

IFN-β induces the proliferation of CD4+CD25+Foxp3+ regulatory T cells through upregulation of GITRL on dendritic cells in the treatment of multiple sclerosis

IFN-β is a major disease-modifying agent used for the treatment of multiple sclerosis (MS). Its mechanisms are complex and it has broad immunomodulatory effects on many types of immune cells. It was observed clinically that the quantity of CD4(+)CD25(+)Foxp3(+) regulatory T cells increases in some MS patients treated with IFN-β. In this study, we show that IFNAR engagement by IFN-β expands naturally occurring CD4(+)CD25(+)Foxp3(+) regulatory T cell population through the modulation of dendritic cells (DCs). IFN-β has no effect on the conversion of CD4(+)CD25(-) T cells to adaptive Treg cells. The IFN-β-induced upregulation of GITRL on DC and downregulation of CTLA-4 on Treg cell work together to facilitate the proliferation of anergic Treg cells. In MS patients treated with Avonex or Rebif (IFN-β), it was found that GITRL expression is markedly upregulated on peripheral CD14(+) cells. Our findings help the better understanding of the complex effects of IFN-β in the treatment of MS.     

Paralysis of CD4(+)CD25(+) regulatory T cell response in chronic autoimmune encephalomyelitis

Increasing evidence strongly suggest that CD4(+)CD25(+) regulatory T (Treg) cells play a pivotal role in suppressing the development of autoimmune diseases. However, it remains poorly understood how these cells are involved in the persistence of, or recovery from, the diseases. In the present study, we examined the role of CD4(+)CD25(+) Treg cells in chronic EAE and compared the results with those obtained in acute EAE. In EAE lesions, CD25(+) cells decreased rapidly at the beginning of chronic EAE, whereas these cells were maintained at high levels during the recovery from acute EAE. The number of Foxp3(+)CD4(+)CD25(+) Treg and levels of Foxp3 mRNA in the lymphoid organ were significantly lower in chronic EAE. Importantly, the regulatory function of individual CD4(+)CD25(+) Treg cells was maintained in animals with chronic EAE. Furthermore, adoptive transfer of activated CD4(+)CD25(+) Treg cells suppressed the development of chronic EAE. These findings suggest that impairment of the CD4(+)CD25(+) Treg response is critical for development of chronic autoimmune diseases, and can be adjustable by autologous Treg transplantation.     

Multiple sclerosis relapses: a multivariable analysis of residual disability determinants

Background – Recovery from multiple sclerosis (MS) relapses is variable. The factors influencing persistence of residual disability (RD) after a relapse are still to be thoroughly elucidated. Aims of study – To assess RD after MS relapses and to define the factors associated with persistence of RD. Methods – Data were retrospectively collected for all relapses in a population of relapsing–remitting MS patients during 3 years. Relapse severity and RD after 1 year were calculated on Expanded Disability Status Scale basis. A multivariable analysis for factors influencing RD and relapse severity was performed (variables: age, gender, disease duration, oligoclonal bands, relapse severity, monosymptomatic/polysymptomatic relapse, immunomodulating treatment, incomplete recovery at 1 month). Results – A total of 174 relapses were assessed. RD after 1 year was observed in 54.5% of the relapses. Higher risk of RD was associated with occurrence of a severe relapse (P = 0.024). Incomplete recovery at 1 month was highly predictive of RD at 1 year (P < 0.0001). Risk of a severe relapse was associated with age ≤ 30 years (P = 0.025) and inversely associated with the use of immunomodulating treatment (P = 0.006). Conclusions – Incomplete recovery at 1 month is a predictor of long‐term persistence of RD. Higher relapse severity is associated with higher risk of RD. Risk of severe relapses is lower in patients treated with immunomodulating drugs.     

Expression of FcR2/CD23 and p55 IL-2R/CD25 on peripheral blood macrophages/monocytes in multiple sclerosis

Macrophages have been found histologically to be activated in multiple sclerosis. We analyzed the expression of CD23 and CD25 on monocytes/macrophages in peripheral blood obtained from patients with multiple sclerosis (MS) to investigate their role in the demyelinating process. Peripheral blood mononuclear cells were obtained from 30 patients with MS including for Baló's diseases (24 with acute relapsing type disease, six with chronic progressive type disease) and 12 healthy controls. The percentage of CD14⁺ CD23⁺ monocytes/macrophages and CD14⁺ CD25⁺ monocytes/macrophages were determined by two-color flow cytometry. The percentage of CD14⁺ CD23⁺ monocytes was significantly higher in patients with MS in the active phase as compared with controls (P < 0.01). Six patients with acute relapsing MS, who had received no therapy, had higher CD14⁺ CD23⁺ cells than did controls (P < 0.0001). CD14⁺ CD25⁺ monocytes/macrophages were not detected in peripheral blood monocytes/macrophages of patients with MS except Baló's concentric sclerosis. The four patients with Baló's concentric sclerosis had markedly elevated levels of CD14⁺ CD25⁺ monocytes/macrophages. Our findings suggest that monocytes/macrophages are activated during an exacerbatiion of MS. and that they may play an important role in the process of demyelination.