Obtaining regulatory T cells from uraemic patients awaiting kidney transplantation for use in clinical trials.

Adoptive transfer of regulatory T cells (T(regs)) has been proposed for use as a cellular therapy to induce transplantation tolerance. Preclinical data are encouraging, and clinical trials with T(reg) therapy are anticipated. In this study, we investigate different strategies for the isolation and expansion of CD4(+) CD25(high) CD127(low) T(regs) from uraemic patients. We use allogeneic dendritic cells (DCs) as feeder cells for the expansion and compare T(reg) preparations isolated by either fluorescence activated cell sorting (FACS) or magnetic activated cell sorting (MACS) that have been expanded subsequently with either mature or tolerogenic DCs. Expanded T(reg) preparations have been characterized by their purity, cytokine production and in-vitro suppressive ability. The results show that T(reg) preparations can be isolated from uraemic patients by both FACS and MACS. Also, the type of feeder cells used in the expansion affects both the purity and the functional properties of the T(reg) preparations. In particular, FACS-sorted T(reg) preparations expanded with mature DCs secrete more interleukin (IL)-10 and granzyme B than FACS-sorted T(reg) preparations expanded with tolerogenic DCs. This is a direct comparison between different isolation techniques and expansion protocols with T(regs) from uraemic patients that may guide future efforts to produce clinical-grade T(regs) for use in kidney transplantation.

Efficacy and safety of continuous local infusion of ropivacaine after retroperitoneoscopic live donor nephrectomy.

Morphine-based analgesia is effective but can compromise donor safety. We investigated whether continuous infusion of local anesthetics (CILA) can provide sufficient pain control and reduce morbidity related to opiate analgesics after hand-assisted retroperitoneoscopic (HARS) live donor nephrectomy. Forty consecutive live kidney donors underwent HARS and were treated with the ON-Q system providing CILA with 0.5% ropivacaine through two SilvaGard catheters placed in the retroperitoneal cavity and the rectus sheath, respectively. The case control group consisted of 40 donors matched with regard to sex, age, BMI and surgical technique. All donors were maintained on standardized multimodal analgesia combining nurse-controlled oxycodone treatment and acetaminophen. CILA donors had lower median cumulative consumption of morphine equivalents (CCME) (7 mg [0-56] vs. 42 mg [15-127]; p < 0.0000001), lower incidence of nausea (18 [45%] vs. 35 [87.5%] donors; p < 0.001), shorter time in postoperative care unit (160 vs. 242.5 min; p < 0.001) and shorter hospital stay (4 [4-7] vs. 6 [4-11] days; p < 0.001). In 32.5% of CILA donors the CCME was 0 mg (0% in matched control group, p < 0.001). CILA with 0.5% ropivacaine provides effective postoperative pain relief, reduces the need for opioid treatment and promotes postoperative recovery.

Time trends in risk and risk determinants of non-Hodgkin lymphoma in solid organ transplant recipients.

Organ transplantation increases risk of non-Hodgkin lymphoma (NHL), but long-term risk and time trends have seldom been evaluated. Immunosuppressive drug load is an important risk determinant, but the details are unclear. We studied NHL risk in a nationwide Swedish cohort of 11 081 graft recipients transplanted 1970-2008. Relative risks (RRs) were estimated within the cohort and versus the general population by age, sex, follow-up time and calendar period. NHL risk was also assessed by cumulative and average doses of immunosuppressive treatments in a nested case-control design throughout 1997 using conditional logistic regression. We observed 153 NHL cases during 97 853 years of follow-up. Compared with the general population, NHL risk was eightfold increased (RR 7.9; 95% confidence interval [CI] 6.6-9.4), and increased risks persisted after ≥15 years of follow-up among kidney (6.1; 95% CI 3.5-10) and nonkidney recipients (44; 14-103). Among nonkidney recipients, NHL risk was lower in the 2000s compared with the 1990s (0.5; 95% CI 0.3-1.0; p = 0.04). A high average dose of antithymocyte immunoglobulin (ATG) conferred an eightfold increased risk of NHL (OR 8.5; 95% CI 1.9-38). To conclude, posttransplant NHL risk decreased during the last decade among nonkidney recipients, possibly because of a more careful use of ATG, the introduction of new drugs, or both.

Hyaluronic acid accumulation and redistribution in rejecting rat kidney graft. Relationship to the transplantation edema.

By using biotin-labeled proteoglycan core protein and an avidin-enzyme system, hyaluronic acid (HA) was visualized in rat kidney. In the normal kidney, HA was localized in the extracellular space of the inner medulla and increased markedly towards the papillary tip. No staining for HA was seen in the interstitial tissue of the cortex or the outer medulla. During the development of rejection of allogeneic renal grafts, a progressive increase in accumulated HA was seen in the interstitial tissue of the cortex and outer medulla. The extractable amounts of HA increased, on average, 40 times in the cortex and outer medulla; no increase was measured in the inner medulla and papilla. The relative water content of the cortex and outer medulla also increased progressively and correlated with the HA accumulation. The extractable amounts of HA in syngeneic grafts increased by day 2 and then leveled off, indicating that surgical trauma may induce some transient HA accumulation after transplantation. Interstitial accumulation of HA, a glycosaminoglycan with unique water-binding qualities, would presumably influence water transport and osmotic activity and should thereby be implicated in the normal papillary function, but also in the development of the interstitial edema of the cortex and outer medulla during rejection of renal grafts.

Hyaluronidase treatment of graft pancreatitis in rats: marked effects on the blood perfusion of the transplanted pancreas.

Hyaluronan is known to accumulate in tissues during inflammatory diseases associated with graft implantation and rejection of organ allografts. The aim was to evaluate whether hyaluronidase treatment affected hyaluronan content and blood perfusion in graft pancreatitis. Syngeneic rat pancreatic-duodenal transplantations were performed. Two days later blood flow measurements were made with a microsphere technique in both grafted and endogenous pancreas in animals treated with daily injections of vehicle or hyaluronidase (20.000 U/kg). Non-transplanted rats served as controls. Also, samples for analysis of hyaluronan and water content were taken. The hyaluronan content of the pancreatic graft was increased after transplantation. Hyaluronidase treatment markedly reduced total pancreatic and islet blood flow in both grafted and endogenous pancreas, whereas duodenum blood flow was unaffected. No blood flow effects were seen in non-transplanted control rats. Hyaluronan content was increased in the grafted pancreas, but hyaluronidase treatment decreased it to levels comparable to those of the endogenous gland. There were no differences in hyaluronan content in the endogenous pancreases of transplanted and non-transplanted rats. Graft pancreatitis after rat pancreas transplantation is associated with an increased hyaluronan content, which can be reduced by treatment with hyaluronidase. Hyaluronidase treatment of the graft recipients effected a 50% reduction in total pancreatic and islet blood flow in the graft, as well as in the endogenous pancreas. The functional importance of this is at present unknown.

Positron emission tomography in clinical islet transplantation.

The fate of islets in clinical transplantation is unclear. To elude on this positron emission tomography combined with computed tomography (PET/CT) was performed for 60 min during islet transplantation in five patients receiving six transplants. A fraction of the islets (23%) were labeled with 18F-fluorodeoxyglucose ([(18)F]FDG) and carefully mixed with unlabeled islets just prior to intraportal transplantation. The peak radioactivity concentration in the liver was found at 19 min after start of islet infusion and corresponded to only 75% of what was expected, indicating that islets are lost during the transplantation procedure. No accumulation of radioactivity was found in the lungs. A nonphysiological peak of C-peptide was found in plasma during and immediately after transplantation in all subjects. Distribution in the liver was heterogeneous with wide variations in location and concentration. Islets found in areas with concentrations of >400 IEQ/cc liver tissue varied between 1% and 32% of the graft in different subjects. No side effects attributed to the PET/CT procedure were found. Clinical outcome in all patients was comparable to that previously observed indicating that the [(18)F]FDG labeling procedure did not harm the islets. The technique has potential to be used to assess approaches to enhance islet survival and engraftment in clinical transplantation.

Geographical prevalence, risk factors and impact of hepatitis B and C after renal transplantation.

BACKGROUND: Hepatitis B (HBV) and hepatitis C (HCV) virus infections are major risk factors affecting long-term morbidity and mortality after renal transplantation. Hepatitis prevalence is subject to geographical variations. OBJECTIVE: To compare and analyze the geographical prevalence, risk factors and impact of HBV and HCV infection in multinational cohorts of renal transplant recipients. METHODS: From 1989 - 2002, data on 12,856 kidney transplant recipients in 37 countries were collected within the prospective MOST (Multinational Observational Study in Transplantation). Subgroup analyses of hepatitis-related prevalence, risk factors and impact were conducted on patients whose HBV and HCV status was available at time of transplantation. Countries were substratified according to population prevalence of > or = 5% HBV or > or = 10% HCV. RESULTS: The prevalence of HBV was 2.9%, of HCV 8.7% and of HBV together with HCV 0.4%. Risk factors for hepatitis infection in renal transplant recipients were long dialysis time, retransplantation and blood transfusions. At each study endpoint up to 5 years after transplantation, no significant differences in graft function were observed, although the 1-year acute rejection rate tended to be lower in HCV+ patients. At 5 years post-transplant, there were no differences between the subgroups and regions regarding infections, post-transplant diabetes mellitus or malignancies including PTLD. CONCLUSIONS: Overall, HCV infections are more prevalent than HBV. Despite large geographical differences in prevalence, HBV and HCV status did not appear to have a significant impact on renal graft function, infections, malignancies and post-transplant diabetes mellitus up to 5 years after renal transplantation throughout the MOST countries.

The ethics of non-heart-beating donation: how new technology can change the ethical landscape.

The global shortage of organs for transplantation and the development of new and better medical technologies for organ preservation have resulted in a renewed interest in non-heart-beating donation (NHBD). This article discusses ethical questions related to controlled and uncontrolled NHBD. It argues that certain preparative measures, such as giving anticoagulants, should be acceptable before patients are dead, but when they have passed a point where further curative treatment is futile, they are in the process of dying and they are unconscious. Furthermore, the article discusses consequences of technological developments based on improvement of a chest compression apparatus used today to make mechanical heart resuscitation. Such technological development can be used to transform cases of non-controlled NHBD to controlled NHBD. In our view, this is a step forward since the ethical difficulties related to controlled NHBD are easier to solve than those related to non-controlled NHBD. However, such technological developments also evoke other ethical questions.

Accumulation of hyaluronan (hyaluronic acid) in myocardial interstitial tissue parallels development of transplantation edema in heart allografts in rats.

By using biotin-labeled proteoglycan core protein, hyaluronan (hyaluronic acid; HA) was visualized in rat heart grafts at different times (2, 4, and 6 d) after transplantation. In normal, nontransplanted hearts HA was present in the adventitia of arteries and veins and in the myocardial interstitial tissue. An increased accumulation of HA was evident in the edematous interstitial tissue, infiltrated with lymphocytes, on day 4 after allogeneic transplantation, and was even more pronounced by day 6. No apparent increase in HA was seen in syngeneic grafts. Biochemical assay of HA in heart tissue demonstrated that the myocardial content of HA had increased 60% by day 2 after transplantation in allogeneic as well as syngeneic grafts, indicating that surgical trauma may induce some HA accumulation in heart grafts. The extractable amount of HA declined during the following days in the syngeneic grafts, but increased progressively during the development of rejection in the allogeneic grafts, and increased on average three times by day 6. The relative water content also increased progressively during rejection of allogeneic grafts and correlated with the HA accumulation. The interstitial accumulation of HA, a glycosaminoglycan with unique water-binding qualities, is presumably implicated in the development of interstitial edema during rejection of heart grafts.

Tolerance induction ameliorates allograft vasculopathy in rat aortic transplants. Influence of Fas-mediated apoptosis.

Based on successful induction of donor-specific unresponsiveness by alloantigenic stimulation in several animal models of acute rejection, we hypothesized that similar immune manipulations would also inhibit the evolution of chronic rejection and transplant vasculopathy. To induce immune tolerance, DA rats received a PVG heart allograft and were immunosuppressed with cyclosporine for 30 d. At day 100 the animals were challenged with a PVG aortic allograft after either 1 or 18 h of cold ischemia. 8 wk after the aortic transplantation, the grafts were investigated for morphological changes, infiltrating cells, apoptosis, and Fas-Fas ligand expression. Control allografts showed advanced transplant arteriosclerosis, whereas tolerance-induced aortic allografts displayed reduced neointimal formation, less medial atrophy, fewer apoptotic cells, and fewer Fas- and FasL-expressing cells. Prolonged ischemic storage time did not profoundly alter the morphological changes of the allografts. Fas expression was found in T cells, macrophages, vascular smooth muscle cells, and endothelial cells, whereas FasL was expressed mainly by T cells and macrophages. FasL mRNA expression was evident throughout the entire allograft wall. In conclusion, induction of allospecific tolerance can effectively prevent transplant arteriosclerosis. Cold ischemia damage does not abrogate the beneficial effect of tolerance, but creates a separate identity of mainly endothelial lesions. Furthermore, Fas-mediated apoptosis appears to be involved in the pathological lesions seen in chronic rejection.

Low 1-year cyclosporine microemulsion doses are associated with better 5-year renal graft function: an insight from MOST, a multinational observational study.

BACKGROUND: In earlier registry analyses, cyclosporine at doses of < 3 mg/kg/d at 1 year post-renal transplantation has been associated with significant graft loss or reduction in renal function. Improvements in cyclosporine formulation with increased bioavailability, plus the use of more efficient comedications, may now confer better outcomes. To determine the effect of the 1-year cyclosporine microemulsion (CsA-ME) dose on renal allograft function at 5 years, we analyzed data collected from 2889 patients with documented graft survival to year 5 in a prospective, multinational, observational study-Neoral MOST. RESULTS: Glomerular filtration rate (GFR) at year 1 was 63 +/- 20 mL/min and 59 +/- 22 mL/min at year 5. The multivariate analysis including year 1 CsA-ME dose as factor and GFR at 1 year as covariate revealed the most significant factors affecting GFR at year 5 were 1-year GFR, donor age > 60 years, and CsA-ME dose at 1 year. Risk factors associated with reduction in 5-year GFR (<65 mL/min) included donor or recipient age >60 years, delayed graft function, cadaveric donor, previous graft, and acute rejection. CsA-ME dose <3 mg/kg/d was found to protect GFR. Analysis of GFR at each year posttransplantation (Wilcoxon model) found 1-year CsA-ME (cutoff 3 mg/kg/d) had a significant effect at each time point. CONCLUSIONS: Compared to higher doses, CsA-ME <3 mg/kg/d at year 1 posttransplantation is associated with increased preservation of renal allograft function at year 5.

Natural cell-mediated immunity to lymphoma cells. I. Characteristics of effector cells in a cytostasis assay in vitro.

Spleen cells from normal, nonimmune, CBA or (CBA X AKR)F1 mice markedly and rapidly inhibited the incorporation of [3H]thymidine by two different T-cell lymphomas in an in vitro cytostasis assay. These were the I-529 lymphoma of spontaneous AKR origin and the Moloney murine leukemia virus-induced YAC lymphoma of A mouse origin. Spleen cells were the most efficient inhibitors for both types of target cells, whereas lymph node cells were much less active and thymus cells showed little or no activity. Granulocytes, as well as conventional T- and B-lymphocytes, were excluded as important contributors to the cytostatic cell population. Spleen cells were separated on nylon wool, Sephadex G-10 columns, or plastic petri dishes and tested for activity in the cytostasis assay or for cytotoxicity against 51Cr-labeled lymphoma target cells. Adherent cells carried almost all cytostatic activity against the AKR lymphoma but also showed significant cytotoxic activity against these target cells. In addition, the cytostatic activity against the YAC lymphoma was mainly due to adherent spleen cells, but nonadherent cells were relatively more active against this target than against I-529 cells. Such nonadherent spleen cells further showed increased cytotoxic activity, compared to the whole spleen cell population.

Current status of clinical islet transplantation.

Islet transplantation is currently being explored as a treatment for patients with type 1 diabetes. At present, the number of patients becoming insulin-independent is rapidly increasing world-wide applying the transplantation protocol originally described by the group in Edmonton. A hallmark in this procedure is repeated infusions of islets obtained from 2 to 4 donors until normoglycemia is achieved. In order to establish islet transplantation as a widely accepted treatment modality, and make tolerance induction regimes applicable, it is essential that the donor:recipient ratio is brought down to 1:1. A conceivable strategy to achieve this goal in clinical islet transplantation is discussed.

Delayed graft function: risk factors, consequences and parameters affecting outcome-results from MOST, A Multinational Observational Study.

BACKGROUND: Delayed graft function (DGF) is a common complication after renal transplantation, and may affect graft function. The aim of this analysis was to evaluate risk factors for DGF, as well as parameters and events influencing graft function after DGF. We analyzed data collected in an ongoing international, prospective; observational study, the Neoral-MOST (Multinational Observational Study in renal Transplantation), and included in the analysis all patients with cadaveric kidney transplants for whom renal function at 1 year posttransplantation was documented (N = 8950). Logistic regression was used to evaluate the risk factors for DGF occurrence, and multifactorial analysis of variance (ANCOVA) to assess the relevance of different factors for GFR at 1 year. RESULTS: Higher donor age, longer CIT, male recipients, Caucasian recipients, high recipients body mass index, and PRA were all associated with a higher risk for DGF. Renal function of former DGF kidneys at 1 year was lower in kidneys of elder donors, or which had experienced rejection or CMV infection. Variations of the maintenance regimen at 1 year posttransplantation were not associated with better graft function. Multifactorial analysis showed donor age and acute rejection as significant independent factors. CONCLUSIONS: Most factors increasing the risk for DGF or having a negative impact on renal function at 1 year in grafts with DGF are predetermined. Additional posttransplant damage by acute rejection was associated with further reductions in GFR. Preventing acute rejection is an important step in achieving optimal function of DGF grafts.

Impact of mycophenolate mofetil dose posttransplantation on 12-month renal function: analysis of the MOST database.

INTRODUCTION: Mycophenolate mofetil (MMF) has greatly reduced the risk of acute rejection episodes (ARE) after renal transplantation, but dose reductions/withdrawals could jeopardize long-term results. METHODS: The MOST database of "de novo" patients treated with MMF at month 1 and functioning grafts at month 12 were divided into 2 groups: groups 1, 2 g MMF at month 1 and month 12; and group 2, 2 g MMF at month 1 but MMF <2 g at month 12 to evaluate renal function glonerular filtration rate (GFR). RESULTS: In this study, 1136 patients were receiving 2 g MMF at month 1. On month 12, 645 were on 2 g (56.8%, group 1) and 431 were on <2 g (43.2%, group 2). Group 1 included younger recipients of younger donors with fewer patients with delayed graft function (DGF). Group 1 showed more ARE during month 1 and more patients who received induction. Mean Neoral daily doses at month 1/month 12 were 5.3/3.0 and 5.3/3.1 mg/kg in group 1 and group 2, respectively (P = .05 at month 12). GFR in group 1 and group 2 were 59.06 (CI 57.10-60.60) and 53.81 (CI 52-55.7) at month 1 (P < .001); 63.7 (CI 62.1-65.30) and 55.9 (CI 54.1-57.7) mL/min*1.73 m(2) at month 12 (P < .001). The mean increases in GFR between month 1 and month 12 were 4.64 and 1.94 mL/min*1.73 m(2), respectively (P < .05). A multivariate analysis also included 795 patients from the "maintenance" patient database with retrospective detailed information. The following parameters were highly predictive for good renal function at month 12: donor age younger than 60 years, recipient age younger than 60 years, immediate graft function, 12-month MMF dose = 2 g, absence of CMV infection, and 12-month Neoral dose <3 mg/kg/d. CONCLUSIONS: Maintenance of MMF dose at 2 g/d during the first year appears to facilitate the attainment of optimal renal function at 12-months after kidney transplantation.

Key factors for human islet isolation and clinical transplantation.

BACKGROUND: To further improve the outcome of clinical islet transplantation analysis of the impact of donor- and process-related factors could be of great importance. MATERIALS AND METHODS: Thirty-eight consecutive clinical islet transplantations were performed with consecutive islet isolations. Univariate analysis for donor- and isolation-related variables were correlated with recipient C-peptide levels at 2 and 4 weeks after transplantation. "Warm ischemia time" was defined as the time from start of University of Wisconsin solution perfusion in the donor until the pancreas was removed to the back table. RESULTS: Short "warm ischemia time" (WIT), low expression of tissue factor (TF) in pancreatic tissue, and high creatinine levels in the donor were variables related to high C-peptide values after islet transplantation. Furthermore, hospitalization length longer than 4 days was associated with low C-peptide levels. The number of islet equivalents (IEQ) did not correlate with the clinical outcome, possibly due to the fact that IEQ number was included in the release criteria for clinical islet transplantation CONCLUSIONS: Successful clinical islet transplantation is strongly correlated with donor and pancreas procurement factors rather than isolation process-related variables. "WIT" may induce TF expression in the pancreatic tissues. TF has been identified as the main trigger of the instant blood-mediated-inflammatory reaction in clinical islet transplantation. Therefore, assay of TF expression in pancreatic tissues could be applied as useful screening tool to identify "good" pancreata for clinical transplantation.

Monitoring of intragraft pressure of rejecting organs: increased tissue pressure can be reduced by hyaluronidase therapy.

BACKGROUND: The present study was undertaken in order to: (a) develop a new technique for measurement of interstitial pressure, (b) study the intragraft pressure of rejecting and non-rejecting organs, and (c) study the effect of treatment with the hyaluronan-degrading enzyme hyaluronidase on intragraft pressure. Treatment with hyaluronidase has previously been demonstrated to result not only in reduction of tissue hyaluronan but also in ameliorated interstitial edema, and we suggested that the diminished edema would lead to a reduced interstitial pressure as well. METHODS: At day 5 after syngeneic or allogeneic rat heterotopic heart transplantation, the interstitial pressure of the cardiac grafts was measured using a microtip pressure sensor. Subsequently, the allogeneically grafted animals received a continuous intravenous infusion of either hyaluronidase (total dose: 60,000 U/kg) or vehicle during 2 hr; meanwhile, the interstitial pressure was monitored. RESULTS: The intragraft pressure measurement technique was found to give reproducible results. The interstitial pressure of the rejecting (allogeneic) grafts was considerably higher than that of the non-rejecting (syngeneic), i.e., 12.3+/-1.6 mmHg vs. 1.1+/-0.6 mmHg (P<0.001). Hyaluronidase infusion effectively reduced the interstitial pressure as compared with vehicle treatment. By 20 min, the pressure had been reduced by 28% (P<0.01 compared with vehicle treatment); after 1 hr, by 49% (P<0.001); and after 2 hr, by 68% (P<0.01). CONCLUSIONS: By using modern technology for tissue pressure measurements, we found that the strongly increased interstitial pressure of rejecting organs can be instantly reduced by intravenous administration of the hyaluronan-degrading enzyme hyaluronidase.

Recovery of hyaluronan during perfusion of small bowel transplantation reflects rejection.

Rejection seen after small bowel transplantation (SBT) in the rat is associated with an accumulation of hyaluronan (HA) in the lamina propria of the graft. In this study the intestinal intraluminal content of this connective tissue component was measured in order to determine if HA could be used as an index of rejection following SBT. Syngeneically and semiallogeneically transplanted rats were investigated by perfusion of a 4-cm segment of the intestinal graft on days 2, 4, and 6 posttransplantation. The amounts of HA recovered during perfusion were analyzed using a radiometric assay. In rejecting grafts the recovered HA amounts increased 15 times from day 2 to day 6, although after syngeneic transplantation there was only a minor increase from day 2 to day 4 and no further increase on day 6. Thus, the recovery of HA during perfusion of transplanted small bowel grafts has the potential value to reflect rejection.

Recipient-reactive antibodies occur during development of acute graft-versus-host reaction after small bowel transplantation.

After small bowel transplantation, not only rejection but also graft-versus-host reaction (GVHR) may occur. Donor T lymphocytes, transferred together with the graft, are a prerequisite for the development of GVHR. So far, however, little is known about the effector mechanisms in acute GVHR. It can be assumed that not only T lymphocytes but also other cells, i.e., B lymphocytes, monocytes/macrophages, and NK cells, together with inflammatory cytokines, are responsible for the lesions of recipient tissue. In the present study, the occurrence of recipient-reactive antibodies after semisyngeneic small bowel transplantation was investigated to elucidate the role of B lymphocytes in GVHR development. No antibodies reactive with recipient cells were detectable in serum from untreated, nontransplanted rats. Five days after transplantation, recipient-reactive antibodies started to appear in recipient serum. At the same time, a deposition of IgM antibodies became visible in the liver and native intestine, which are target organs for GVHR. No antibodies directed against either the donor strain or a third-party strain were detectable in serum. We conclude that a synthesis of antibodies against recipient tissue occurs during the development of GVHR. Whether these antibodies contribute to the disease remains unclear.