Induction Of Immunological Tolerance Research Articles

Methylene blue treatment for cytokine release syndrome-associated vasoplegia following a renal transplant with rATG infusion: A case report and literature review.

Rabbit anti-thymocyte globulin (rATG) is an infusion of polyclonal rabbit-derived antibodies against human thymocyte markers, which can be used to prevent and treat acute rejection following organ transplantation. However, the product monograph issued by the manufacturer (Sanofi Canada) reports that serious immune-mediated reactions have been observed following the use of rATG, consisting of anaphylaxis or severe cytokine release syndrome (CRS), which is a form of vasoplegic syndrome (VS), in which distributive shock occurs refractory to norepinephrine (NE) and vasopressin (VP). Severe infusion-associated reactions are consistent with CRS and can cause serious cardiac or respiratory problems, or in certain cases, mortality. CRS is a form of systemic inflammatory response syndrome (SIRS). In SIRS, the substantial activation of endothelial inducible nitric oxide synthase (iNOS) and smooth muscle guanylate cyclase (GC) is observed, which can produce severe hypotension that is unresponsive to conventional vasopressors. Methylene blue (MB) is a direct inhibitor of iNOS and GC and has been used as an effective treatment for VS following cardiothoracic surgery. In the present study, the successful use of MB as a rescue therapy for CRS in a patient receiving rATG following a renal transplant was reported. Following an uneventful cadaveric kidney transplant involving the intravenous (IV) administration of rATG for the induction of immunological tolerance, the patient became markedly hypotensive and tachycardic. The patient required high doses of VP and NE infusions. Following the protocol described for treating refractory VS in post-cardiac surgery patients, the decision was made to initiate the patient on an IV MB infusion. This treatment protocol was shown to improve the hemodynamic status of the patient, which enabled the withdrawal of vasopressors and suggests an important role for methylene blue in the management of refractory VS.

The Research of Treg Cells: Progress and Challenge

T regulatory cells (Tregs) play an important role in the induction and maintenance of immunological tolerance to self and alloantigens. Recent findings in experimental transplant models have demonstrated that Treg could control and delay allograft rejection. Induction of immune tolerance decreases the risk of acute and chronic graft rejection after solid organ transplantation and can improve transplanted organ survival. Tregs are being tested in trials as a potential therapy in cell and solid organ transplantation. However, as we know, regulatory T cells (Tregs) are crucial for peripheral tolerance and are intimately involved in cancer. The influence of Tregs on cancer progression has been demonstrated in a large number of preclinical models and confirmed in several types of malignancies. Neoplastic processes trigger an increase of Treg numbers in draining lymph nodes, spleen, blood, and tumors, leading to the suppression of anti-tumor responses. In this review, we summarize some of the critical aspects of the immunoregulatory function of Treg cells in cancer and transplantation and discuss their potential research progress and challenge.

The roles of IL-2 and IL-10 enhance anti-CD45RBmAb immune inhibition in allograft skin.

As a new type of immune tolerance inducer, anti-CD45RB monoclonal antibodies (anti-CD45RBmAb) can prolong the graft survival time of animal organs or cell transplantation as well as induce stable immune tolerance. Both interleukin (IL)-2 and IL-10 have important roles in the induction and maintenance of immunological tolerance. However, whether these cytokines combined with anti-CD45RBmAb can promote immune tolerance is poorly understood. Therefore, we investigated the effect of IL-2 and IL-10 in vitro and in vivo on the tolerance induction by anti-CD45RBmAb. The changes of Treg and Th17 cells and Th1/Th2 cytokines in anti-CD45RBmAb induced prolongation of skin allograft survival in mice. The finding of a role for IL-2 is novel, of interest, IL-2 promoted anti-CD45RBmAb-induced CD4(+) T cell differentiation into Treg and Th2 cells and suppressed Th17 and Th1 cells. IL-2 enhanced the induction of immune tolerance by anti-CD45RBmAb and significantly prolonged skin graft survival time in vivo. In contrast, this effect should be demonstrated experimentally by neutralizing IL-2 and inhibition of the effect of anti-CD45RBmAb, and neutralizing IL-10 showed no effect for anti-CD45RBmAb-induced tolerance. These data reveal that IL-2 significantly enhances anti-CD45RBmAb-induced immune tolerance via up-regulated T regulatory (Treg) cells and the balance of Th1/Th2 shifts. Conversely, IL-10 showed no effect on anti-CD45RBmAb-induced tolerance.

Polymeric synthetic nanoparticles for the induction of antigen-specific immunological tolerance

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

In vitro Studies of the Antibody Response: Antibodies of Different Specificity are Made in Different Populations of Cells.

In vitro Studies of the Antibody Response: Antibodies of Different Specificity are Made in Different Populations of Cells.

An increase of CD83+ dendritic cells ex vivo correlates with increased regulatory T cells in patients with active eosinophilic granulomatosis and polyangiitis.

BackgroundEosinophilic granulomatosis with polyangiitis (EGPA) is a rare disease characterized by the presence of allergic granulomatosis and necrotizing vasculitis with eosinophilic infiltration. The etiology of EGPA is unknown. Dendritic cells (DCs) are not only critical for the induction of primary immune responses; they may also be important for the induction of immunological tolerance and the regulation of the type of T-cell-mediated immune response. To investigate whether DC maturation is associated with EGPA disease status, we examined the relationship between the maturation of DCs and the differentiation of regulatory T (Treg) cells in EGPA patients. We exposed the CD14+ blood monocytes of 19 patients with EGPA in remission or relapse to stimulation with GM-CSF and IL-4 for 6 d and lipopolysaccharide for 24 h to obtain mature CD83+ DCs and immature CD206+ DCs. Using immunohistochemistry, we examined four patients for the presence of CD83+ and CD206+ DCs in the lung at the onset of EGPA.ResultsThe percentage of CD83+ cells among DCs differentiated from CD14+ monocytes was lower for EGPA patients in relapse than in remission. The percentage of CD83+ DCs was inversely correlated with the percentage of CD206+ DCs and was significantly correlated with the numbers of naturally occurring CD4+ regulatory Treg (nTreg; FOXP3+CD4+) cells and inducible Treg (iTreg; CD4+CD25+ T cells producing IL-10 or TGF-β) cells but not the number of eosinophils. The percentage of CD206+ DCs was significantly inversely correlated with the percentages of nTreg and iTreg cells but not the number of eosinophils. Immunohistochemistry revealed both CD206+ DCs and CD83+ DCs in alveoli and interstitial spaces at the onset of EGPA.ConclusionThe maturation of DCs from monocytes was related to disease activity in patients with EGPA. Increased CD83+ DCs in EGPA patients may induce the differentiation of iTreg and nTreg cells, thereby suppressing inflammation and disease activity.

Attenuation of early atherosclerotic lesions by immunotolerance with β2 glycoprotein I and the immunomodulatory effectors interleukin 2 and 10 in a murine model

This study assessed the effect of cellular and humoral autoimmune response inhibition after immunization with β2-glycoprotein I (β2-GPI) and the effect of immunomodulation with interleukin (IL)-2 and IL-10 in the development of early atherosclerotic vascular lesion in a murine model. Atherosclerosis is increasingly considered a chronic inflammatory disease with pathogenic autoimmune processes. Regulatory T cells, and their cytokines, have been implicated in the inhibition of the development of atherosclerotic lesions and involved in the immunologic tolerance induction. Eight-week-old male C57BL6 LDL-receptor deficient (LDLR(-/-)) mice were fed a cholesterol-rich (2.8%), high-saturated-fat (82%) diet for a week and divided in five groups. The groups received the following intravenous immunizations: group I (control group): one dose of 5 μg β2-GPI; group II: 5 μg β2-GPI I and 1 μg IL-2; group III: 5 μg β2-GPI and 0.75 μg of IL-10; and group IV: 5 μg β2-GPI, 1 μg IL-2, and 0.75 μg IL-10. The aortas of the mice were assessed 8 weeks after inoculation to determine the aortic lesion size and composition in all groups. β2-GPI immunization attenuated the early atherosclerotic lesions development compared with the control group (P = .001). Macroscopic and histologic aortic atherosclerotic lesions were significantly decreased in the IL-2 and IL-10-treated groups in β2-GPI-tolerant mice compared with the β2-GPI-tolerant group without cytokine injection (P = .001). The association of both cytokines did not provoke a major inhibition in the atherosclerosis development when compared with groups injected with the two cytokines separately. The immunotolerance induction against β2-GPI attenuates the development of atherosclerosis lesions in an animal model, enhanced by downregulation of the cellular and humoral autoimmune response provoked by IL-2 and IL-10.

Induction of Immunological Tolerance With In Vitro Generated Suppressive Donor Blood Monocytes.

Our previous studies showed that incubation of dendritic cells (DCs) with the chemotherapeutic drug mitomycin C (MMC) renders them immunosuppressive. Donor-derived MMC-DCs injected into the recipient before transplantation prolonged heart allograft survival in rats. Whereas generation of DCs is labour-intensive, peripheral blood mononuclear cells (PBMCs) can be easily harvested. Here we analyse under which conditions DCs can be replaced by PBMCs and study their mode of action. When injected into rats, MMC-incubated donor PBMCs (MICs) strongly prolonged heart allograft survival. Removal of monocytes from PBMCs abrogated their suppressive effect, showing that monocytes are the active cell population. Suppression of rejection was donor-specific. Recipients treated with MICs showed an increased number of CD4+CD25+FoxP3+ Tregs in their peripheral blood and FoxP3-cell infiltration of heart allograft. Most important, tolerance could be transferred to syngeneic recipients with blood cells, whereas depletion of Tregs annihilated the effect. This argues for mediation of suppression by CD4+CD25+FoxP3+ Tregs. Cell culture studies showed that MMC-treated monocytes give rise to myeloid cells, which do neither resemble monocytes, nor DCs but show morphological and phenotypic similarities with monocytic myeloid-derived suppressor cells (MDSCs) occurring in some forms of cancer. Donor-derived MICs also prolonged kidney allograft survival in pigs. MICs were applied for the first time in a patient with therapy-resistant rejection of a haploidentical stem cell transplant, showing that they can be easily generated and used in humans. In conclusion, we describe here a simple method for in vitro generation of monocytic suppressor cells for potential use in clinical organ transplantation.

Effect of Tolerogenic Dendritic Cells Repetitive Injection in Skin Allograft Survival.

Objective. Dendritic cells(DC) gained considerable attention as antigen presenting cells that are also capable of immunologic tolerance induction. The study of DC injection frequency and amount for tolerance induction have not been perforemd. This study assesses the effect of tolerogenic dendritic cell multiple injection in a mouse skin allograft model. Materials and Methods.Peripheral blood mononuclear cells (PBMC) were harvested from ICR mouse and cultured in the presence of recombinant mouse IL-10 for tolergenic DC induction. IL-10-imDCs were co-cultured with donor mononuclear cells for 24 h and injected subcutaneously. From the donor BALB/C mouse, 1.5cm x 1.5cm sized dorsal skin was grafted to recipient ICR mouse. Recipient animals were divided into five groups according to the treatment, each comprising five rats (n=5):Group I(Untreated control), Group II( IL-10-imDC alone, 2 x 104/mouse, SC injection), Group III (FK-506 only group, 2mg/kg, IM injection), Group IV (FK-506 +IL-10-imDC 1 time injection), Group V(FK-506+IL-10-imDCinjectionevery 2days during 2weeks). Results. The group IV(FK-506+IL-10-imDC 1 time injection) showed longest graft survival rate(20.4days, p<0.05) than other groups; Group I(5.8days), Group II(13.6 days), Group III(16.8days), Group V(17.2). Conclusion. We demonstrated that multiple subcutaneous injection of IL-10 treated immature DC failed to show prolonged skin allograft survival.Figure: No Caption available.

Large Animal Models for Vascularized Composite Allotransplantation

Large animal models have served as the foundation for clinical trials in all solid organ transplantation. Likewise, pre-clinical studies in vascularized composite allotransplantation (VCA) have provided a foundation for initial human applications in VCA. The growth of clinical VCA correspondingly benefits from large animal studies investigating the unique immunobiology, improved immunosuppressive regimens, and the potential induction of immunologic tolerance. VCA studies in large animal models of nonhuman primates, swine, and canines have and continue to build knowledge towards improved clinical outcomes.

Phagocytosis of apoptotic or necrotic cells differentially regulates the transcriptional expression of IL-12 family members in dendritic cells

Uptake of apoptotic cells by DCs is considered to contribute to induction and maintenance of immunological tolerance. TolDCs are sought after as cellular therapy in transplantation and autoimmunity and can be generated in vitro using GCs. In this study, we investigated how uptake of dead cells affects the production and expression of different members of the IL-12 family by immature DCs or TolDCs. We show that compared to regular immature DCs, TolDCs display elevated levels of PS-recognizing bridge molecule receptors αvβ5 and CD36, and have enhanced phagocytic abilities with accelerated uptake of apoptotic cells. We confirm that apoptotic cell uptake results in diminished production of IL-12p40 and IL-12p70 by DCs. We now show that this also results in increased expression of IL-12p35 and Ebi3. TolDCs completely lack expression of IL-12p40 yet have enhanced levels of Ebi3 and IL-12p35. Uptake by TolDCs of apoptotic or necrotic cells does not affect the expression of Ebi3/IL-12p35 and also does not increase IL-12p40. This is distinct from the culture of immature DCs with necrotic cells, which is sufficient to induce IL-12p40 secretion. Conversely, ingestion of apoptotic cells by DCs leads to increased expression of IL-12p35 and Ebi3 without affecting IL-12p40. In conclusion, we have shown that uptake of apoptotic versus necrotic cells by DCs differentially regulates members of the IL-12 family. Apoptotic cells favor expression of Ebi3 and IL-12p35, and we propose that differential regulation of the IL-12 family is an additional mechanism in determining the immune response to dying cells.

Tobacco Smoke–Induced Immunologic Changes May Contribute to Oral Carcinogenesis

ObjectiveThe objective of this study was to determine if tobacco smoke (TS), a risk factor for cancers of the aerodigestive tract, may contribute to oral carcinogenesis, in part, by suppressing...

Vascularized Composite Allograft Tolerance Across MHC Barriers in a Large Animal Model

Vascularized composite allograft (VCA) transplantation can restore form and function following severe craniofacial injuries, extremity amputations or massive tissue loss. The induction of transplant tolerance would eliminate the need for long-term immunosuppression, realigning the risk-benefit ratio for these life-enhancing procedures. Skin, a critical component of VCA, has consistently presented the most stringent challenge to transplant tolerance. Here, we demonstrate, in a clinically relevant miniature swine model, induction of immunologic tolerance of VCAs across MHC barriers by induction of stable hematopoietic mixed chimerism. Recipient conditioning consisted of T cell depletion with CD3-immunotoxin, and 100 cGy total body irradiation prior to hematopoietic cell transplantation (HCT) and a 45-day course of cyclosporine A. VCA transplantation was performed either simultaneously to induction of mixed chimerism or into established mixed chimeras 85-150 days later. Following withdrawal of immunosuppression both VCAs transplanted into stable chimeras (n=4), and those transplanted at the time of HCT (n=2) accepted all components, including skin, without evidence of rejection to the experimental end point 115-504 days posttransplant. These data demonstrate that tolerance across MHC mismatches can be induced in a clinically relevant VCA model, providing proof of concept for long-term immunosuppression-free survival.

Association of ESR and FOXP3 gene polymorphisms with outcome of ovarian stimulation in infertile females undergoing IVF

Background Estrogen receptor (ER) gene plays a major role in folliculogenesis, maturation of oocytes and fertilization of embryo. FOXP3 gene is a crucial regulatory factor for the development and function of Treg cells; it takes part as a central role in the induction and maintenance of fetal-maternal immunologic tolerance. Therefore, the present study was aimed to investigate the association of ESR and FOXP3 gene polymorphisms in infertile women who are undergoing in vitro fertilization (IVF) with poor ovarian reserve upon controlled ovarian hyperstimulation (COH) induced by follicle-stimulating hormone (FSH).

Regulatory Dendritic Cells for Immunotherapy in Immunologic Diseases

We recognize well the abilities of dendritic cells to activate effector T cell (Teff cell) responses to an array of antigens and think of these cells in this context as pre-eminent antigen-presenting cells, but dendritic cells are also critical to the induction of immunologic tolerance. Herein, we review our knowledge on the different kinds of tolerogenic or regulatory dendritic cells that are present or can be induced in experimental settings and humans, how they operate, and the diseases in which they are effective, from allergic to autoimmune diseases and transplant tolerance. The primary conclusions that arise from these cumulative studies clearly indicate that the agent(s) used to induce the tolerogenic phenotype and the status of the dendritic cell at the time of induction influence not only the phenotype of the dendritic cell, but also that of the regulatory T cell responses that they in turn mobilize. For example, while many, if not most, types of induced regulatory dendritic cells lead CD4+ naïve or Teff cells to adopt a CD25+Foxp3+ Treg phenotype, exposure of Langerhans cells or dermal dendritic cells to vitamin D leads in one case to the downstream induction of CD25+Foxp3+ regulatory T cell responses, while in the other to Foxp3− type 1 regulatory T cells (Tr1) responses. Similarly, exposure of human immature versus semi-mature dendritic cells to IL-10 leads to distinct regulatory T cell outcomes. Thus, it should be possible to shape our dendritic cell immunotherapy approaches for selective induction of different types of T cell tolerance or to simultaneously induce multiple types of regulatory T cell responses. This may prove to be an important option as we target diseases in different anatomic compartments or with divergent pathologies in the clinic. Finally, we provide an overview of the use and potential use of these cells clinically, highlighting their potential as tools in an array of settings.

Long-term prevention of chronic allograft rejection by regulatory T-cell immunotherapy involves host Foxp3-expressing T cells

AbstractDespite the use of immunosuppressive drugs, chronic allograft rejection remains a major hurdle in transplantation medicine. Induction of specific immunologic tolerance to antigens expressed by the graft would avoid its eventual functional loss and the severe side effects of paralyzing the immune system. We previously showed that donor-specific regulatory T-lymphocytes prevent rejection of fully allogeneic bone marrow (BM) grafts in mice. Thus generated hematopoietic chimeras then accepted skin and heart allografts of the same donor. We noticed that injected regulatory T-cells (Tregs) disappeared with time and investigated the mechanisms involved in the nevertheless long-term persistence of allograft tolerance. Using Tregs that can be depleted in vivo with diphtheria toxin, we show that injected cells are required for induction but not for maintenance of tolerance to BM allografts. We observed progressive deletion of donor-specific T-lymphocytes, accounting at least in part for maintenance of tolerance. Toxin-induced depletion of administered as well as host Tregs did not affect hematopoietic chimerism but it led to rapid loss of skin allografts. Therefore, our data show that newly generated host Tregs can prevent chronic allograft rejection. Long-lasting tolerance to allografts is thus achieved.

Induction of immunological tolerance by pulmonary macrophages during chronic exposure to house dust mite antigen (P6212)

Abstract Allergic asthma is a leading cause of morbidity in industrialized nations. While the emphasis of most studies pertaining to the attenuation of asthma after chronic antigen exposure has focused on the local generation of Tregs, little attention has been paid to the potentially crucial role of pulmonary macrophages (MΦ) in disease resolution. To determine whether these MΦ are involved in tolerance generation, C57BL/6 mice were challenged intranasally with house dust mite (HDM) extract both acutely (2 and 5 weeks) and chronically (11 weeks) to induce an asthma-like phenotype (n=3-6 per group). Compared to acutely-challenged mice, MΦ frequency increased over two-fold in the airways of chronic HDM-exposed mice (P&amp;lt;0.001). This finding correlated with increases in local Foxp3+ Tregs as well as resolution of pulmonary eosinophilia (P&amp;lt;0.01) and airway hyper-reactivity (P&amp;lt;0.01). Flow cytometric analysis of naïve IL-10 GFP mice confirmed that pulmonary MΦ produce high amounts of IL-10. Following chronic HDM exposure, these MΦ may expand in the lungs and influence tolerance development directly through IL-10 or indirectly through effects on Tregs. We also observed 3- to 7-fold increases in serum MCP-1, MIP-1α, and MIP-1β following chronic but not acute HDM exposure, which suggests that these MΦ are derived from monocytes recruited to the site of inflammation. Collectively, these data suggest a novel role for pulmonary MΦ in the generation of tolerance in an experimental model of asthma.

Bronchiolitis Obliterans Syndrome-Free Survival after Two Years in Patients with Higher Frequencies of Regulatory T Cells Early after Lung Transplantation

Purpose Regulatory T cells (Treg) play important roles in the induction and maintenance of immunological tolerance to self and non-self antigens. They have the potential to regulate alloantigens and thus may counteract the development of acute and chronic rejection (i.e. BOS) in lung transplant recipients. BOS affects over 60% of lung transplant recipients within 5 years after transplantation. Here, we present a cohort of 89 patients, in which we analyzed Tregs in peripheral blood prospectively during the first 2 years after lung transplantation and correlate with the onset of BOS. Methods and Materials In consecutive routine lung transplant recipients, the number of circulating Treg was detected by flow cytometry before transplantation and 3 weeks, 3 6,12, 18 and 24 months after transplantation. Treg were defined as CD4+CD25high T cells and were further analyzed for relevant surface as well as intracellular markers such as, amongst others, CTLA4, CD127, FoxP3 and IL-2. Additionally, lung function results at that time points were analyzed. We divided the cohort into two groups based on the development of BOS stage 0-p versus BOS stage 1 or higher. Results A total of 89 consecutive patients were included into the study. 25 patients developed a BOS stage 1 or higher 2 years after transplant. 64 patients showed a good clinical course with stable lung function. A statistically significant positive correlation could be detected between the frequencies of CD4+CD25highCD127low T cells, CD4+CD25highCTLA-4+ T cells, CD4+CD25highFoxP3+ T cells and CD4+CD25highIL-2 + T cells and the absence of BOS within the first 2 years after lung transplantation (p 0.05). Conclusions Higher frequencies of CD4+CD25highCD127low T cells, CD4+CD25highCTLA-4+ T cells, CD4+CD25highFoxP3+ T cells and CD4+CD25highIL-2 + T cells early after transplantation are associated with protection against development of BOS during the initial 2 years after lung transplantation.

Induction of Tolerance of Vascularized Composite Allografts

Vascularized composite allotransplantation has become established as a clinical specialty since the first successful hand transplant was performed in 1998. Data now available indicate that hand and face transplants offer patients good functional outcomes and significant improvements in quality of life. Despite the debilitating nature of the injuries treated by such transplants, the defects are generally not life threatening, making it difficult for physicians to recommend life-long immunosuppression that can itself have grave consequences. One potential solution to this dilemma is the induction of immunologic tolerance of the tissue transplants because tolerance would eliminate the need for such immunosuppression. Transplant tolerance may also prevent chronic rejection, a significant source of late graft loss after organ transplantation.Induction of mixed hematopoietic chimerism is a robust approach to establishing such transplant tolerance, which recently led to the first clinical application of a tolerance induction protocol for kidney transplantation. In this manuscript, we review the current status of VCA and of research directed toward bringing a tolerance approach to the VCA field. We also discuss the potential clinical significance of these studies and outline the remaining obstacles to introduction of a tolerance induction protocol to clinical practice in hand or face transplantation.

Recipients with higher percent predicted FEV1 early after lung transplantation have more circulating regulatory T cells

Purpose: Regulatory T cells (Treg) play important roles in the induction and maintenance of immunological tolerance to self and non-self antigens. They have the potential to regulate alloantigens and thus may counteract the development of acute and chronic rejection in lung transplant recipients. Clinically, the most valuable parameter of lung allograft function is the % predicted FEV1 measured in spirometry for diagnosis of both acute and chronic rejection. Stable high values may thus represent an immunologically favourable course. In this analysis, we present a cohort of 89 patients, in which we analyzed Tregs after lung transplantation as well as lung function results.