Antibody-Dependent NK Cell Activation Is Associated with Late Kidney Allograft Dysfunction and the Complement-Independent Alloreactive Potential of Donor-Specific Antibodies.

Although kidney transplantation remains the best treatment for end-stage renal failure, it is limited by chronic humoral aggression of the graft vasculature by donor-specific antibodies (DSAs). The complement-independent mechanisms that lead to the antibody-mediated rejection (ABMR) of kidney allografts remain poorly understood. Increasing lines of evidence have revealed the relevance of natural killer (NK) cells as innate immune effectors of antibody-dependent cellular cytotoxicity (ADCC), but few studies have investigated their alloreactive potential in the context of solid organ transplantation. Our study aimed to investigate the potential contribution of the antibody-dependent alloreactive function of NK cells to kidney graft dysfunction. We first conducted an observational study to investigate whether the cytotoxic function of NK cells is associated with chronic allograft dysfunction. The NK-Cellular Humoral Activation Test (NK-CHAT) was designed to evaluate the recipient and antibody-dependent reactivity of NK cells against allogeneic target cells. The release of CD107a/Lamp1(+) cytotoxic granules, resulting from the recognition of rituximab-coated B cells by NK cells, was analyzed in 148 kidney transplant recipients (KTRs, mean graft duration: 6.2 years). Enhanced ADCC responsiveness was associated with reduced graft function and identified as an independent risk factor predicting a decline in the estimated glomerular filtration rate over a 1-year period (hazard ratio: 2.83). In a second approach, we used the NK-CHAT to reveal the cytotoxic potential of circulating alloantibodies in vitro. The level of CD16 engagement resulting from the in vitro recognition of serum-coated allogeneic B cells or splenic cells was further identified as a specific marker of DSA-induced ADCC. The NK-CHAT scoring of sera obtained from 40 patients at the time of transplant biopsy was associated with ABMR diagnosis. Our findings indicate that despite the administration of immunosuppressive treatments, robust ADCC responsiveness can be maintained in some KTRs. Because it evaluates both the Fab recognition of alloantigens and Fc-driven NK cell activation, the NK-CHAT represents a potentially valuable tool for the non-invasive and individualized evaluation of humoral risk during transplantation.

Plasma Xanthine Oxidase Activity Is Predictive of Cardiovascular Disease in Patients with Chronic Kidney Disease, Independently of Uric Acid Levels.

BACKGROUND: Chronic kidney disease (CKD) is associated with increased cardiovascular morbidity and mortality. Oxidative stress seems to play a pivotal role in this process, and purine metabolism may be involved in CKD-related oxidative stress. Xanthine oxidase (XO) is an enzyme involved in purine metabolism and is also responsible for the production of reactive oxygen species. METHODS: This prospective study aimed to analyze the relation between plasma dosages of molecules involved in redox balance, purine metabolism and cardiovascular events in patients with non-diabetic CKD stages 3-5 or on chronic hemodialysis (HD). CKD (n = 51) and HD (n = 50) patients were compared to matched healthy controls (n = 38) and followed-up for 3 years. RESULTS: Both CKD and HD patients had decreased plasma levels of antioxidants (selenium, zinc, vitamin C). HD patients had decreased levels of the antioxidant enzyme superoxide dismutase and increased levels of oxidation products (ischemia-modified albumin, malondialdehyde [MDA]). The following substrates and enzymes involved in purine metabolism were increased in the HD cohort: adenosine, adenosine deaminase and the pro-oxidant XO. XO activity was negatively correlated with super oxide dismutase and positively with MDA. Interestingly, XO activity was an independent predictor of cardiovascular events in CKD and HD patients, regardless of uric acid levels. Uric acid was not predictive of events. CONCLUSION: This highlights a possible role of XO itself in CKD-related cardiovascular disease (CVD) and raises the hypothesis that beneficial effects observed with XO inhibitors on CVD in CKD may also be due to the reduction of oxidative stress.

[Antiphospholipids antibodies and hemodialysis: a frequent association linked to arteriovenous fistula thrombosis]. / Anticorps antiphospholipides et hémodialyse: une association fréquente corrélée à la thrombose de l'abord vasculaire.

Antiphospholipid antibodies (APL) are a heterogeneous family of auto-antibodies that recognize phospholipoproteins bound antigenic epitopes. APL prevalence in patients on chronic hemodialysis ranges from 11 to 37% in the literature. The association of APL with hemodialysis vascular access (VA) thrombosis has already been reported in small studies. In this single center and retrospective study, we defined the APL prevalence and APL risk factors in a large cohort of 192 hemodialysis patients. The association between history of VA thrombosis and APL presence was also analyzed. At least one type of APL was found in 38 patients (19.8%) of which 74% (n=28) had only lupus anticoagulant. Median age of APL positive patients was 68.1years vs. 71.3years in APL negative patients (P=0.02). Smoking history was associated with APL presence: 35.5% of APL positive patients had a smoking history vs only 18.3% of APL negative patients (P=0.04). The multivariate analysis showed an association between the history of VA thrombosis and patient age (HR [IC 95%]=1.04 [1.02-1.06]; P=0.001) or APL presence (HR [IC 95%]=3.03 [1.69-4.42]; P<10(-3)). In conclusion, the prevalence of APL in hemodialysis patients remains high despite hemodialysis techniques improvement: hemodiafiltration, biocompatibility improvements, ultrapure dialysis water. We report that a younger age and past history of smoking are associated with an increased risk of APL presence. The presence of APL, especially lupus anticoagulant, is associated to VA thrombosis in hemodialysis patients.

Acute tubulointerstitial nephritis complicating Legionnaires' disease: a case report.

INTRODUCTION: Legionnaires' disease is recognized as a multi-systemic illness. Afflicted patients may have pulmonary, renal, gastrointestinal tract and central nervous system complications. However, renal insufficiency is uncommon. The spectrum of renal involvement may range from a mild and transient elevation of serum creatinine levels to anuric renal failure requiring dialysis and may be linked to several causes. In our present case report, we would like to draw attention to the importance of the pathological documentation of acute renal failure by reporting a case of a patient with acute tubulointerstitial nephritis complicating Legionnaires' disease. CASE PRESENTATION: A 55-year-old Caucasian man was admitted to our hospital for community-acquired pneumonia complicated by acute renal failure. Legionella pneumophila serogroup type 1 was diagnosed. Although the patient's respiratory illness responded to intravenous erythromycin and ofloxacin therapy, his renal failure worsened, he became anuric, and hemodialysis was started. A renal biopsy was performed, which revealed severe tubulointerstitial nephritis. After initiation of steroid therapy, his renal function improved dramatically. CONCLUSIONS: This case highlights the importance of kidney biopsies in cases where acute renal failure is a complicating factor in Legionnaires' disease. If the presence of acute tubulointerstitial nephritis can be confirmed, it will likely respond favorably to steroidal treatment and thus irreversible renal damage and chronic renal failure will be avoided.

Comparative safety and efficiency of five percutaneous kidney biopsy approaches of native kidneys: a multicenter study.

BACKGROUND: Renal biopsy (RB) is necessary for the diagnosis, prognosis, and therapy guidance of native kidney diseases. Few studies have compared outcomes of RB procedures. We retrospectively compared the safety and efficiency of five biopsy procedures. METHODS: The number of glomeruli on light microscopy (LM) and on immunofluorescence (IF) and serious adverse events following RB performed in five nephrology units (C1-C5) were collected. C1 performed ultrasound (US) assessment before RB and used a 14-gauge core-cutting needle biopsy gun, C2 US guidance and a 14-gauge needle, C3 tomodensitometry guidance and a 14-gauge needle, C4 US guidance and a 16-gauge needle, and C5 tomodensitometry guidance and a 16-gauge needle. RESULTS: RB was performed in 943 adults between January 2006 and July 2010. Serious adverse events occurred in 1.5% of biopsies. The complication rate was not different between nephrology units. The mean number of glomeruli on biopsy was 14.2 ± 8.6 with LM and 4.4 ± 3.3 on IF. It was different according to the nephrology unit for LM (p = 0.01) and for IF (p < 0.001). The number of failed biopsies was influenced by the nephrology unit and radiological guidance technique, favoring real-time US guidance. Failed biopsies using US or tomodensitometry assessment before RB was certainly due to kidney imprecise localization since it was often non-renal tissue sampling. At least 10 glomeruli were found in 69% of biopsies on LM. This rate varied according to the nephrology unit (p = 0.004) and was higher when 14-gauge needles were used in comparison with 16-gauge needles. CONCLUSION: RB is safe regardless of the technical procedure, but radiological guidance and needle size influence the efficiency of biopsies.

A randomized trial with steroids and antithymocyte globulins comparing cyclosporine/azathioprine versus tacrolimus/mycophenolate mofetil (CATM2) in renal transplantation.

BACKGROUND: The best immunosuppressive regimen in benefit-risk ratio in renal transplantation is debated. Nowadays, tacrolimus (Tac) and mycophenolate mofetil (MMF) are considered more efficient than cyclosporine A (CsA) and MMF, but recent studies have challenged this assumption. METHODS: We conducted a monocentric, prospective, open-labeled, randomized, and controlled trial comparing CsA/azathioprine (Aza) versus Tac/MMF in 289 kidney transplant recipients treated with antithymocyte globulins and prednisone. Primary outcome was the number of patients with clinically suspected acute rejection at 1 year. Secondary outcomes were the number of patients with biopsy-proven acute rejection (BPAR), estimated glomerular filtration rate (eGFR), patient and graft survivals, and adverse events at 1 and 3 years. RESULTS: During the first year, 21 patients had clinically suspected acute rejection with CsA/Aza (14.4%) vs. 11 (7.7%) with Tac/MMF (P=0.07). BPAR, including borderline, was more frequent in the CsA/Aza group (14.4%) than in the Tac/MMF group (5.6%; P=0.013). At 1 year, patient and graft survivals were not different, and eGFR was 48±1 in the CsA/Aza group and 53±1 mL/min/1.73 m in the Tac/MMF group (P=0.007). There was no significant difference in diabetes after transplantation (16.8% and 18.8%, respectively). CONCLUSIONS: With antithymocyte globulins and steroids, clinically suspected acute rejections did not differ between CsA/Aza and Tac/MMF arms. Analysis of secondary endpoints showed a lower rate of BPAR, including border line, and a higher eGFR in the Tac/MMF group. CsA/Aza allowed a low acute rejection rate, but Tac/MMF seemed as a better regimen regarding severe secondary outcomes.

Membranoproliferative glomerulonephritis and mixed cryoglobulinemia after hepatitis C virus infection secondary to glomerular NS3 viral antigen deposits.

BACKGROUND: We report on 3 cases of membranoproliferative glomerulonephritis associated with mixed cryoglobulin in patients with hepatitis C virus (HCV) antibodies but a negative blood viral load. These cases explore the pathogenesis of the renal disease. METHODS: We searched for occult HCV infection in peripheral blood mononuclear cells, cryoprecipitate, bone marrow cells, and glomeruli using ultrasensitive PCR assays and immunohistochemistry. We also looked for infraclinical B cell lymphoma by computed tomodensitometry, bone marrow aspiration and biopsy, and lymphocyte typing. RESULTS: By PCR assays, we did not evidence occult hepatitis C infection in peripheral blood mononuclear cells, bone marrow cells, or cryoprecipitates. In the only patient with available kidney specimen, we evidenced HCV-NS3 antigen in glomeruli. HCV-associated lymphoma was excluded, but mild polyclonal B lymphocytosis was present in the 3 patients. Remission occurred spontaneously in 1 patient, and in another patient it occurred after rituximab treatment. The third patient was lost to follow-up. CONCLUSIONS: In patients with hepatitis C-negative viral load, membranoproliferative glomerulonephritis could be induced by the persistence of HCV antigen in the kidney but not in hematopoietic cells. Nonlymphomatous B cell proliferation may also be induced by chronic viral stimulation.

Evolution of Anelloviridae strains distribution in serial blood and biopsy samples from a renal transplant patient.

A few studies investigated the natural history of viruses belonging to the family Anelloviridae in transplant patient. The case of a 64-year-old kidney transplant recipient is described. Molecular analysis of serial blood samples collected before and after transplant was performed during a period of 510 days. Two kidney biopsies were also analyzed. All blood samples tested positive for Anelloviridae DNA, with the identification of sequences belonging to the three taxonomic genera identified in humans. Sequences distribution during the follow-up was multimodal. A sequence nearly identical to one present in the blood before transplant was further characterized in one biopsy sample.

Fractalkine expression induces endothelial progenitor cell lysis by natural killer cells.

BACKGROUND: Circulating CD34(+) cells, a population that includes endothelial progenitors, participate in the maintenance of endothelial integrity. Better understanding of the mechanisms that regulate their survival is crucial to improve their regenerative activity in cardiovascular and renal diseases. Chemokine-receptor cross talk is critical in regulating cell homeostasis. We hypothesized that cell surface expression of the chemokine fractalkine (FKN) could target progenitor cell injury by Natural Killer (NK) cells, thereby limiting their availability for vascular repair. METHODOLOGY/PRINCIPAL FINDINGS: We show that CD34(+)-derived Endothelial Colony Forming Cells (ECFC) can express FKN in response to TNF-α and IFN-γ inflammatory cytokines and that FKN expression by ECFC stimulates NK cell adhesion, NK cell-mediated ECFC lysis and microparticles release in vitro. The specific involvement of membrane FKN in these processes was demonstrated using FKN-transfected ECFC and anti-FKN blocking antibody. FKN expression was also evidenced on circulating CD34(+) progenitor cells and was detected at higher frequency in kidney transplant recipients, when compared to healthy controls. The proportion of CD34(+) cells expressing FKN was identified as an independent variable inversely correlated to CD34(+) progenitor cell count. We further showed that treatment of CD34(+) circulating cells isolated from adult blood donors with transplant serum or TNF-α/IFN-γ can induce FKN expression. CONCLUSIONS: Our data highlights a novel mechanism by which FKN expression on CD34(+) progenitor cells may target their NK cell mediated killing and participate to their immune depletion in transplant recipients. Considering the numerous diseased contexts shown to promote FKN expression, our data identify FKN as a hallmark of altered progenitor cell homeostasis with potential implications in better evaluation of vascular repair in patients.

High Resolution Melt analysis for mutation screening in PKD1 and PKD2.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disorder. It is characterized by focal development and progressive enlargement of renal cysts leading to end-stage renal disease. PKD1 and PKD2 have been implicated in ADPKD pathogenesis but genetic features and the size of PKD1 make genetic diagnosis tedious. METHODS: We aim to prove that high resolution melt analysis (HRM), a recent technique in molecular biology, can facilitate molecular diagnosis of ADPKD. We screened for mutations in PKD1 and PKD2 with HRM in 37 unrelated patients with ADPKD. RESULTS: We identified 440 sequence variants in the 37 patients. One hundred and thirty eight were different. We found 28 pathogenic mutations (25 in PKD1 and 3 in PKD2 ) within 28 different patients, which is a diagnosis rate of 75% consistent with literature mean direct sequencing diagnosis rate. We describe 52 new sequence variants in PKD1 and two in PKD2. CONCLUSION: HRM analysis is a sensitive and specific method for molecular diagnosis of ADPKD. HRM analysis is also costless and time sparing. Thus, this method is efficient and might be used for mutation pre-screening in ADPKD genes.

[Experiment of self blood pressure measurement at home in haemodialysis patients in a hospital unit]. / Expérience de l'automesure tensionnelle à domicile chez des patients hémodialysés dans une unité hospitalière.

We analyzed, by home blood pressure (BP) self-measurement and conventional predialytic measurement of BP, a cohort of haemodialysis patients in two hospital units between 2008 and 2010. All patients who already own a BP self-measurement device were included in this study. BP was recorded by the two methods for one week. The number of patients with a validated self-measurement device was 69 of 350 (21%) and 60 patients were included in analyses. These patients were divided into 23 (38%) permanent uncontrolled hypertensive (elevated BP at home and in hospital), 13 (22%) masked hypertensive (normal BP in hospital and elevated at home), eight (13%) white coat hypertensive (elevated BP in hospital and normal at home), and 16 (27%) permanent controlled normotensive (normal BP in hospital and at home). Patient compliance with all the self BP measurements was 95%. We did not find in this cohort the factors associated with masked hypertension in the general population such as being male, smoking and high body mass index. These results obtained in an in-hospital dialysis unit should be extrapolated with caution to all haemodialysis patients. However it is, to our knowledge, the first study on home BP self-measurement published in patients undergoing haemodialysis in France. A significant proportion of patients have masked hypertension. This should alert clinicians because of the poor cardiovascular prognosis associated with masked hypertension.

Osseous metaplasia in a kidney allograft.

Osseous metaplasia is defined by the presence of heterotopic normal bone tissue in a soft tissue. The bone matrix is associated with osteoblasts, osteoclasts, adipocytes and haematopoietic stem cells. Osseous metaplasia pathophysiology is not well known, but many factors have been incriminated including chronic inflammation and chronic ischaemia. We describe the second case of osseous metaplasia in a kidney allograft. Numerous factors might favour its development including factors linked to transplantation failure environment.

Mycophenolate mofetil monotherapy in membranous nephropathy: a 1-year randomized controlled trial.

BACKGROUND: Treatment of patients with membranous glomerulonephritis (MGN) is controversial because of the lack of clear benefit of the immunosuppressive regimens on patient or renal survival. The objective of this study is to evaluate the efficacy and safety of mycophenolate mofetil (MMF) for patients with MGN. STUDY DESIGN: 1-year prospective, randomized, and controlled clinical trial. SETTING & PARTICIPANTS: 36 patients with biopsy-proven idiopathic MGN and nephrotic syndrome. INTERVENTION: 19 patients received MMF (2 g/d) for 12 months and 17 patients were in the control group. All patients had the same conservative treatment based on renin-angiotensin blockers, statins, low-salt and low-protein diet, and diuretics in case of edema. OUTCOMES & MEASUREMENTS: End points were the mean proteinuria over creatinuria ratio in mg/g throughout the study and numbers of complete and partial remissions at 1 year (month 12). Data were analyzed on an intention-to-treat analysis. RESULTS: Mean proteinuria over creatinuria ratio was stable in both groups throughout the study (P = 0.1). Mean proteinuria over creatinuria ratio was 4,690 +/- 2,212 mg/g in the MMF group and 6,548 +/- 4,601 mg/g in the control group (95% confidence interval of the difference, -619 to +4,247; P = 0.1). Remission was complete in 3 patients (1 in the MMF group, 2 in the control group; P = 0.5) and partial in 11 patients (6 in the MMF group, 5 in the control group; P = 0.9). The probability of complete or partial remission did not differ between the 2 groups after 12 months (relative risk, 0.92; 95% confidence interval, 0.48 to 1.75; P = 0.7). Kidney function was stable in the 2 groups according to estimated glomerular filtration rate and serum creatinine level. LIMITATIONS: The small number of patients and short follow-up prevent generalizations. CONCLUSIONS: A 12-month regimen of MMF did not decrease mean proteinuria over creatinuria ratio or increase partial and complete remissions. Serious adverse effects were observed in 4 patients (20%) receiving MMF.

Nanobacteria are mineralo fetuin complexes.

"Nanobacteria" are nanometer-scale spherical and ovoid particles which have spurred one of the biggest controversies in modern microbiology. Their biological nature has been severely challenged by both geologists and microbiologists, with opinions ranging from considering them crystal structures to new life forms. Although the nature of these autonomously replicating particles is still under debate, their role in several calcification-related diseases has been reported. In order to gain better insights on this calciferous agent, we performed a large-scale project, including the analysis of "nanobacteria" susceptibility to physical and chemical compounds as well as the comprehensive nucleotide, biochemical, proteomic, and antigenic analysis of these particles. Our results definitively ruled out the existence of "nanobacteria" as living organisms and pointed out the paradoxical role of fetuin (an anti-mineralization protein) in the formation of these self-propagating mineral complexes which we propose to call "nanons." The presence of fetuin within renal calculi was also evidenced, suggesting its role as a hydroxyapatite nucleating factor.

[Hemodialysis procedure in a patient treated with radioactive iodine]. / Procédure d'hémodialyse chez un patient traité par iode 131.

The treatment of a patient with 131I at activity over 740 mega Becquerel (MBq) must be performed in a nuclear medicine department. Isolation is stopped if the patient radiation level is less than 20 muSv/hour at one meter. As regards patients with chronic renal failure treated with hemodialysis (HD), the first HD session will eliminate the major part of the radioactivity. French regulations do not give definite recommendations for this session. However, it imposes to collect liquid and solid wastes contaminated by radioactivity. Thus, it seems necessary to collect dialysate and solid wastes and to stock them in a room dedicated to radiation decay. The risk for dialysis staff is to be contaminated by an accidental ingestion of a biologic fluid from the patient. The usual protection barriers used during the HD session are sufficient: mask, gloves, overgarments, cap. There is no risk linked to external exposure to radiations. The maximal theoretical dose received by the staff during the session is 65 muSv, while annual maximal dose for public exposed to radiations is 1000 muSv. Although the dosimetric follow-up of dialysis staff is not mandatory, the nuclear medicine department of Marseille University Hospital has decided to do it in an information perspective. The session is performed in the presence of a radiation safety technician who gives film badges and active dosimeters to the dialysis staff. He reports the dialysis staff to the nuclear safety agency (Autorité de sûreté nucléaire).

Natural killer cell alterations correlate with loss of renal function and dialysis duration in uraemic patients.

BACKGROUND: Natural killer (NK) cells provide a first line of immune defence towards infections and tumours, and participate in atherosclerosis and pregnancy diseases, of which there is a higher incidence in uraemic patients. Still, their relative contribution to the immunodeficient state associated with renal failure is poorly documented. METHODS: A multivariate and comparative analysis of lymphocyte subsets in haemodialysed (HD) and undialysed (UD) uraemic patients in comparison to healthy donors (HC) is provided in this article. NK-mediated cytotoxicity, degranulation and interferon secretion were compared in HD and HC. RESULTS: Evaluation of NK cells in 210 HD patients concluded with a decrease in NK cell counts in comparison to HC. Multivariate analysis associated lowered NK cell counts in UD patients with decreased renal clearance and higher NK counts HD with male gender and age. The 32% NK cell count decrease observed in sex- and age-matched groups (n = 88) was associated with B- and CD8(+)T-lymphocyte defects. NK cell functions were similar in subgroups of HD and HC matched for NK cell counts. Longer dialysis duration was associated with improved NK cytototoxic activity. While the expression of receptors modulating NK cytotoxicity were not modified, expression of the activation markers CD69 and NKp44, CD94 and chemokine receptors CX3CR1 and CXCR4 was altered in HD. CONCLUSIONS: This study is the first to associate decrease in renal function with selective fading of NK cell number and identify haemodialysis duration as a factor influencing NK cell function. It further shows that lower cell counts rather than intrinsic NK cell dysfunction per se characterize immune disorders in HD.

Influence of haemodialysis and left ventricular failure on peripheral A(2A) adenosine receptor expression.

BACKGROUND: Haemodialysis (HD) sometimes accelerates left ventricular failure (LVF). As adenosine (ADO) is strongly implicated in cardiovascular functions, particularly via A(2A) receptor activation and as changes of peripheral A(2A) receptors mirror changes occurring in the cardiovascular system, we examined the influence of HD and LVF on both ADO plasma concentration and the expression of A(2A) receptors (i.e. Bmax, K(D) and mRNA amount) of peripheral blood mononuclear cells. METHODS: This cross-sectional study included 61 chronic renal failure (CRF) patients: 41 without LVF (24 haemodialysed and 17 undialysed) and 20 with LVF (9 haemodialysed and 11 undialysed). Ten LVF patients without CRF and 10 healthy subjects were also examined. RESULTS: (i) Bmax values of CRF patients without LVF were significantly decreased in undialysed patients compared with haemodialysed patients, and compared with controls (69 +/- 25 vs 98 +/- 33 vs 180 +/- 60 fmol/mg of protein, P < 0.05). Bmax values of CRF patients with LVF were lower in undialysed patients than in haemodialysed patients (60 +/- 27 vs 101 +/- 27 fmol/mg of protein, P < 0.05). Bmax values of LVF patients without CRF were lower than in controls (51 +/- 19 vs 180 +/- 60 fmol/mg of protein). (ii) A(2A) mRNA expression was increased in haemodialysed patients compared with controls (20.2 +/- 0.75 vs 17.6 +/- 1.3, P < 0.05). (iii) ADO plasma levels were high in haemodialysed patients and further increased during the HD sessions. CONCLUSION: The number of A(2A) receptors was decreased by CRF with or without LVF. However, this decrease was less important in haemodialysed patients. The changes in peripheral A(2A) receptor expression suggest a significant inflammatory response to HD and heart or kidney failure. Whether these changes do reflect alterations in cardiomyocytes needs further investigation.