Treatment of EBV-Related Post-Renal Transplant Lymphoproliferative Disease with a Tailored Regimen Including EBV-Specific T Cells

The treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) poses a considerable challenge. Efforts have been made to define regimens based on combination of the available therapeutic agents, chosen and tailored on a patient-by-patient basis, with the aim of augmenting event-free patient and graft survival. Recently, autologous EBV-specific cytotoxic T-lymphocytes (CTL) have proved effective in enhancing EBV-specific immune responses and reducing viral load in organ transplant recipients with active infection. We investigated the use of a tailored combined approach including autologous EBV-specific CTL for the treatment of EBV-related PTLD developing after pediatric kidney transplantation. Five patients with disseminated monoclonal (n = 3) or localized polyclonal (n = 2) PTLD unresponsive to reduction of immunosuppression were enrolled. The patients with disseminated PTLD received 4-5 courses of reduced-dosage polychemotherapy, accompanied by rituximab on the first day of each course, while localized disease was removed surgically. At treatment completion, autologous EBV-specific CTL were infused. All patients showed a complete response to treatment, without therapy-related toxicity or rejection, and persist in remission with good renal function at a median follow-up of 31 months. These preliminary results suggest that a combined chemoimmunotherapy regimen including virus-specific T-cells is well tolerated and potentially effective as first-line treatment of EBV-related PTLD.     

Successful Renal Retransplantation after Post-Transplant Lymphoproliferative Disease

Post-transplant lymphoproliferative disease (PTLD) is a rare but severe complication of renal transplantation. Reduction of immunosuppression is essential for controlling PTLD but may induce graft loss. Retransplantation after PTLD is considered dangerous, because immunosuppressive treatment resumption may trigger hematological relapse. We retrospectively report six patients (five adults, one child) who underwent a second renal transplantation after successfully treated PTLD. Epstein-Barr virus (EBV) serology was positive before the first transplantation in all patients except the child. Post-transplant lymphoproliferative disease was detected 6.6 months (range 4.5-9.4) after transplantation. Lymphoproliferation was always monomorphic, B-cell, and EBV-related. Post-transplant lymphoproliferative disease was confined to the renal allograft (n = 4), multilocular (n = 1) or cerebral (n = 1). Immunosuppression tapering (6/6) and transplantectomy (5/6) led to hematological remission in all patients. Retransplantation was performed 77 months (range 50-128) after PTLD diagnosis. Immunosuppression included steroids (n = 6), ATG (n = 2), anti-CD25 (n = 2), cyclosporine (n = 4), tacrolimus (n = 2), mycophenolate mofetil (n = 4) and azathioprine (n = 1). After a median follow up of 30 months (range 24-47) patient survival was 100%, with no recurrence of PTLD. In conclusion, renal retransplantation can be considered in patients with monomorphic PTLD history, without major risk of hematological recurrence.     

Chronic High Epstein-Barr Viral Load State and Risk for Late-Onset Posttransplant Lymphoproliferative Disease/Lymphoma in Children

Increased use of serial EBV-PCR monitoring after pediatric transplantation has led to the identification of asymptomatic patients who carry very high viral loads over prolonged periods. The significance of this high-load state is unknown. We speculated that this state may identify patients at high risk for development of late PTLD/lymphoma. We reviewed data on 71 pediatric heart recipients who had serial viral load monitoring since 1997. Chronic high-load state was defined as the presence of >16 000 genome copies/mL whole blood on ≥50% of samples over at least 6 months. Among 20 high-load carriers (eight following prior PTLD, seven with prior symptomatic EBV infection, five without previous EBV disease), 9 (45%) developed late-onset PTLD 2.5–8.4 years posttransplant (including with four Burkitt's lymphoma). Among 51 controls with low (n = 39) or absent (n = 12) loads, only 2 (4%; p < 0.001 absent/low vs. high load) developed late PTLD/lymphoma. By multivariable analysis, high-load carrier state (OR = 12.4, 95% CI 2.1–74.4) and prior history of PTLD (OR = 10.7, 95% CI 1.9–60.6) independently predicted late PTLD. A chronic high EBV-load state is not benign and is a predictor of de novo or recurrent PTLD.     

Post-transplant Lymphoproliferative Syndrome in the Pediatric Liver Transplant Population

Post-transplant lymphoproliferative disease remains a complication with a high morbidity and mortality. The present study examined 291 pediatric liver transplants performed in 263 children from October 1984 to December 1999. Post-transplant lymphoproliferative disease has an overall incidence of 12%. Tacrolimus and cyclosporine had a similar incidence of post-transplant lymphoproliferative disease. Fifty-six per cent of patients who developed post-transplant lymphoproliferative disease were Epstein-Barr virus negative at the time of transplantation. Mean time of conversion to Epstein-Barr virus positivity was 1.1 years after liver transplantation. Ten per cent of those who developed post-transplant lymphoproliferative disease never had Epstein-Barr virus detected. Mean time from Epstein-Barr virus positivity to detection of post-transplant lymphoproliferative disease was 2.68 years, and 3.13 years from liver transplantation (OLTx) to post-transplant lymphoproliferative disease. There was a 35% incidence of mortality. Deaths occurred a mean of 0.76 years after diagnosis of post-transplant lymphoproliferative disease. Most cases of post-transplant lymphoproliferative disease had extranodal location. There was one recurrence in 10% of patients, and two in 3%. All recurrent cases were seen in recipients who became Epstein-Barr virus positive after transplantation. There has been a decrease in the incidence of post-transplant lymphoproliferative disease from 15% to 9% to 4%. Post-transplant lymphoproliferative disease should be diagnosed promptly and treated aggressively. The best treatment, however, seems to be prevention, starting in the immediate postoperative period. Survivors should be monitored for both recurrence of post-transplant lymphoproliferative disease and acute cellular rejection.     

Post-Transplant Lymphoproliferative Disorders Following Liver Transplantation: Incidence, Risk Factors and Survival

This study investigates retrospectively the incidence, risk factors and mortality of post-transplant lymphoproliferative disorders (PTLD) in adult orthotopic liver transplant (OLT) recipients. Among 1206 OLT recipients at a single institution, 37 developed a PTLD. The incidence of PTLD was highest during the first 18 months and relatively constant thereafter with cumulative incidence of 1.1% at 18 months and 4.7% at 15 years. The risk of PTLD was approximately 10% to 15% of the risk of death without PTLD. During the first 4 years following OLT, PTLD were predominantly related to EBV, while afterward most PTLD were EBV negative. Significant risk factors for PTLD in OLT recipients were transplantation for acute fulminant hepatitis during the first 18 months following OLT (HR = 2.6, p = 0.007), and rejection therapy with high-dose steroids (HR = 4.5, p = 0.049) and OKT3 (HR = 3.9, p = 0.016) during the previous year. Therapy with high-dose steroids or OKT3 (HR = 3.6, p = 0.0071) were also significant risk factors for PTLD-associated mortality. OLT recipients remain at risk for PTLD years after transplantation. The strong association of PTLD with rejection therapy and the worse post-PTLD prognosis among recipients of rejection therapy indicate the need to balance the risk of immunosuppression against the risk of PTLD following rejection treatment.     

CMV-IVIG for Prevention of Epstein Barr Virus Disease and Posttransplant Lymphoproliferative Disease in Pediatric Liver Transplant Recipients

A randomized controlled trial of CMV-IVIG (cytomegalovirus-intravenous immunoglobulin) for prevention of Epstein Barr virus (EBV) posttransplant lymphoproliferative disease (PTLD) in pediatric liver transplantation (PLTx) recipients was begun in Pittsburgh and subsequently expanded to four additional sites. Protocol EB viral loads were obtained in a blinded fashion; additional loads could be obtained for clinical indications. Patients were followed for 2 years post-LTx. Eighty-two evaluable patients (39 CMV-IVIG, 43 placebo) developed 18 episodes of EBV disease (7 CMV-IVIG, 11 placebo) including nine cases of PTLD (three CMV-IVIG, six placebo). No significant differences were seen in the adjusted 2-year EBV disease-free rate (CMV-IVIG 79%, placebo 71%) and PTLD-free rate (CMV-IVIG 91%, placebo 84%) between treatment and placebo groups at 2 years (p > 0.20). The absence of significant effect of CMV-IVIG may be explained by a lack of efficacy of the drug or limitations of sample size.     

单倍体相合造血干细胞移植联合间充质干细胞输注治疗异染性脑白质营养不良

目的探讨单倍体相合造血干细胞移植联合间充质干细胞输注治疗异染性脑白质营养不良的疗效。方法 7岁患儿,采用单倍体相合造血干细胞移植联合间充质干细胞输注的方法,经氟达拉滨、马利兰、环磷酰胺和抗人胸腺细胞球蛋白方案进行预处理,供者应用G-CSF动员和联合免疫抑制剂(包括环胞菌素A、氨甲喋呤、霉酚酸酯、CD25单抗)预防移植物抗宿主病。造血重建后,每周输注脐带来源的间充质干细胞1次,连续4周。结果患儿造血重建迅速,11 d时中性粒细胞0.5×109/L、血小板20×109/L;14 d时血白细胞芳基硫酸酯酶A水平明显上升;28 d时达正常水平。患者无急性GVHD发生。移植后1个月经植入证据检测,证实为完全供者造血,神经系统症状逐渐恢复。结论单倍体相合造血干细胞移植,联合间充质干细胞输注,治疗异染性脑白质营养不良,安全、有效,血白细胞芳基硫酸酯酶A水平回升迅速。这种新型疗法可能是治疗缺乏HLA全相合供者的异染性脑白质营养不良疾病患者的可靠选择。     

Lymphoproliferative disorder post renal transplantation: Recent experience at a single centre

Summary: Post-transplant lymphoproliferative disorder was diagnosed in six renal transplant recipients at the Royal Melbourne Hospital over a 5 year period to December 1994. This constituted a detection rate of 2.2%. All patients were treated with cyclosporine, azathioprine, prednisolone and orthoclone (OKT3). the median time of onset was 2 months (range 0.3 months-3 years) after transplant. Two patients who ultimately died presented with the diffuse form of the disease where immunoblasts massively infiltrated all organs. These tumours were monoclonal B cell lymphomas and Epstein-Barr virus (EBV) reactivation could be demonstrated. One patient developed a polyclonal B cell tumour with primary EBV infection and the remaining 3 patients tresented with T cell polyclonal proliferations and were EBV negative. No cytomegalovirus (CMV) infections, primary or reactionary were observed. Therapy in all cases consisted of reducing or ceasing immunosuppressive treatment and in three patients ganciclovir was used. Four polyclonal tumours responded to the treatment measures.     

Dissociation of Depletional Induction and Posttransplant Lymphoproliferative Disease in Kidney Recipients Treated With Alemtuzumab

Transplant patients are at the risk for posttransplant lymphoproliferative disease (PTLD), a virally-driven malignancy. Induction with the depleting antibody preparations Thymoglobulin and OKT3 is associated with PTLD suggesting that the T-cell depletion increases PTLD risk. We therefore studied 59 560 kidney recipients from the Organ Procurement and Transplantation Network/United Network for Organ Sharing (OPTN/UNOS) database for a relationship between induction agent use and PTLD. Two agents with comparable T-cell depletional effects, alemtuzumab and Thymoglobulin, were compared to nondepletional induction agents or no induction. The overall incidence of PTLD was 0.46% and differed significantly by induction strategy (p < 0.01): without induction (0.43%), basiliximab (0.38%), daclizumab (0.33%), Thymoglobulin (0.67%) and alemtuzumab (0.37%). Thymoglobulin was associated with significantly increased PTLD risk (p = 0.0025), but alemtuzumab (p = 0.74), basiliximab (p = 0.33) and daclizumab, which trended toward a protective effect (p = 0.06), were not. Alemtuzumab and Thymoglobulin treated patients did not differ in any established parameter affecting PTLD risk although alemtuzumab is known to have a more pronounced B-cell depleting effect. Interestingly, maintenance therapy with an mTOR inhibitor was strongly associated with PTLD (0.71%, p < 0.0001). Thus, depletional induction is not an independent risk factor for PTLD. Rather, maintenance drug selection or perhaps the balance between B- and T-cell depletion may be more relevant determinants of PTLD risk.     

CXCL13 as a Novel Marker for Diagnosis and Disease Monitoring in Pediatric PTLD

Posttransplant lymphoproliferative disease (PTLD) is a severe complication of immunosuppressive treatment in organ‐grafted children. Early diagnosis of PTLD is hampered by both unspecific clinical symptoms and lack of easy accessible markers. The homeostatic chemokine CXCL13, which plays a crucial role in B‐cell homing and lymphoid organ development, is expressed in some lymphomatous diseases. This study aims to investigate whether serum CXCL13 (sCXCL13) levels correlate with occurrence and regression of PTLD in pediatric solid‐organ graft recipients. Serum samples from PTLD patients (n = 21), patients with Epstein–Barr virus (EBV) reactivation (n = 18), and healthy age‐matched controls (n = 19) were tested for CXCL13 using a commercially available ELISA kit. sCXCL13 levels were significantly higher in PTLD patients than in healthy children. PTLD patients had also higher sCXCL13 values than pediatric solid‐organ recipients with EBV reactivation. An increase in sCXCL13 levels was observed from EBV reactivation to PTLD diagnosis in most cases. Elevated sCXCL13 levels were detected up to 2 years prior to PTLD diagnosis and correlated well with response to cytoreductive treatment in individual patients. sCXCL13, thus, may be a readily available surrogate marker for the diagnosis of PTLD and for monitoring of response to treatment in patients with initially elevated sCXCL13 levels.     

肾移植术后淋巴增殖性疾病的诊治体会

目的探讨肾移植术后淋巴细胞增殖性疾病(PTLD)的发病原因、临床特点、诊治方法及预后。方法回顾性分析2000年1月至2019年1月海军军医大学长征医院2844例肾移植受者中术后并发PTLD的13例受者的临床资料,收集受者性别、年龄、血肌酐、药物浓度、糖尿病、肾移植术后有无移植肾功能延迟恢复、急性排斥反应和免疫抑制剂方案的使用等相关资料。13例PTLD受者中,男11例,女2例,确诊时年龄为55岁(31~78岁)。结果PTLD病变位置分布在肺部1例,胃肠道8例,区域淋巴结2例,皮肤1例,颅内1例,均病理诊断为弥漫性大B淋巴细胞淋巴瘤,距肾移植手术中位时间为86个月(12~204个月)。76.9%受者病理组织EB病毒检测呈阳性。确诊后给予免疫抑制剂减量为主的综合治疗,无法耐受利妥昔单抗+CHOP(R-CHOP)方案化学药物治疗的受者切换为利妥昔单抗+来那度胺(R2)方案,2例受者肺部感染死亡,完全缓解10例,部分缓解1例,病情进展1例,总有效率91.6%。结论肾移植术后淋巴细胞增殖性疾病进展迅速,病死率高,与EB病毒感染密切相关,减少免疫抑制剂用量是综合治疗的核心,根据受者耐受性选择合理的化学药物治疗方案,R2方案为无法耐受一线R-CHOP方案的受者更多选择。     

Stem Cell Transplantation Enhances Endogenous Brain Repair after Experimental Stroke

Stem cell transplantation for stroke treatment has been a promising therapy in small and large animal models, and many clinical trials are ongoing to establish this strategy in a clinical setting. However, the mechanism underlying functional recovery after stem cell transplantation has not been fully established and there is still a need to determine the ideal subset of stem cells for such therapy. We herein reviewed the recent evidences showing the underlying mechanism of functional recovery after cell transplantation, focusing on endogenous brain repair. First, angiogenesis/neovascularization is promoted by trophic factors including vascular endothelial growth factor secreted from stem cells, and stem cells migrated to the lesion along with the vessels. Second, axonal sprouting, dendritic branching, and synaptogenesis were enhanced altogether in the both ipsilateral and contralateral hemisphere remapping the pyramidal tract across the board. Finally, endogenous neurogenesis was also enhanced although little is known how much these neurogenesis contribute to the functional recovery. Taken together, it is clear that stem cell transplantation provides functional recovery via endogenous repair enhancement from multiple ways. This is important to maximize the effect of stem cell therapy after stroke, although it is still undetermined which repair mechanism is mostly contributed.     

Comparative analysis of post‐transplant lymphoproliferative disorder after kidney transplantation versus hematopoietic stem cell transplantation

Post‐transplant lymphoproliferative disorder (PTLD) is a major complication caused by immune‐suppression after transplantation. Survival outcome is known to be poor and the characteristics are not fully understood because of its rare incidence. This single center retrospective study enrolled 41 adult PTLD patients after kidney‐transplantation (KT, n = 28) and hematopoietic stem cell transplantation (HSCT, n = 13) from 1992 to 2012. We compared the characteristics and estimated the survival outcomes according to several factors [age‐adjusted‐IPI (aaIPI), pathologic subtype, viral status, extranodal manifestation] and added some significant parameters to aaIPI scoring system. Post‐HSCT‐PTLD patients were younger and showed earlier onset, and viral status was more frequently identified. Ten‐year OS of the entire group was 44% but the 10‐year OS was not significantly different between post‐KT‐PTLD and post‐HSCT‐PTLD (39% vs. 56%, P = 0.860). The time onset of PTLD and viral statuses were not meaningful, however, aaIPI, age > 50, extranodal manifestation and monomorphic subtype were predictive for OS. We used those factors for PTLD‐specific scoring which showed intermediate‐risk (HR = 7.1, P = 0.019) and high‐risk (HR = 16.5, P = 0.001) presented worse OS compared to low‐risk subgroup. Although the treatment strategies were heterogenous, this study showed comprehensive PTLD data between KT versus HSCT, and our PTLD‐specific scoring might be validated by another larger studies.     

Primary Cutaneous Posttransplant Lymphoproliferative Disorders in Solid Organ Transplant Recipients: A Multicenter European Case Series

Primary cutaneous posttransplant lymphoproliferative disorders (PTLD) are rare. This retrospective, multicenter study of 35 cases aimed to better describe this entity. Cases were (re)‐classified according to the WHO‐EORTC or the WHO 2008 classifications of lymphomas. Median interval between first transplantation and diagnosis was 85 months. Fifty‐seven percent of patients had a kidney transplant. Twenty‐four cases (68.6%) were classified as primary cutaneous T cell lymphoma (CTCL) and 11 (31.4%) as primary cutaneous B cell PTLD. Mycosis fungoides (MF) was the most common (50%) CTCL subtype. Ten (90.9%) cutaneous B cell PTLD cases were classified as EBV‐associated B cell lymphoproliferations (including one plasmablastic lymphoma and one lymphomatoid granulomatosis) and one as diffuse large B cell lymphoma, other, that was EBV‐negative. Sixteen (45.7%) patients died after a median follow‐up of 19.5 months (11 [68.8%] with CTCL [6 of whom had CD30⁺ lymphoproliferative disorders (LPD)] and 5 [31.2%] with cutaneous B cell PTLD. Median survival times for all patients, CTCL and cutaneous B cell PTLD subgroups were 93, 93, and 112 months, respectively. Survival rates for MF were higher than those for CD30⁺ LPD. The spectrum of primary CTCL in organ transplant recipients (OTR) is similar to that in the general population. The prognosis of posttransplant primary cutaneous CD30⁺ LPD is worse than posttransplant MF and than its counterpart in the immunocompetent population. EBV‐associated cutaneous B cell LPD predominates in OTR.     

EBV PCR in the Diagnosis and Monitoring of Posttransplant Lymphoproliferative Disorder: Results of a Two-Arm Prospective Trial

While EBV PCR is used in the management of PTLD, the optimal primer set, relative importance of intracellular versus free plasma EBV, and the baseline profile in an organ transplant population remains unclear. We performed a prospective 2-arm trial utilizing an EBV PCR panel measuring LMP-1, EBER-1 and EBNA-1 in both free plasma as well as intracellular whole blood. Control Arm A consisted of 31 lung transplant patients and Arm B consisted of 35 transplant patients being evaluated for possible PTLD. In Arm A, 1/31 (3%) patients developed a transient plasma EBV load. Thirteen of 31 (42%) had detectable intracellular EBV. In Arm B, 17 (49%) patients were diagnosed with PTLD. Thirteen (76%) had EBV-positive PTLD with 12/13 (92%) having detectable EBV by PCR. The EBV PCR panel had a high sensitivity (92%), specificity (72%), positive predictive value (PPV) (71%) and negative predictive value (NPV) (93%) for diagnosing EBV-positive PTLD and followed patients' clinical course well (p < 0.001). Comparing the individual PCR assays, plasma EBNA PCR was superior with high sensitivity (77%), specificity (100%), PPV (100%) and NPV (86%). We conclude that EBV PCR is a useful test for managing PTLD patients. While plasma EBNA PCR is the best single assay for diagnosing and monitoring PTLD, the complete PCR panel is superior for ruling out its presence.     

干细胞移植与心脏生物起搏

干细胞是一类具有高度自我复制和多向分化潜能的细胞,特定条件下干细胞可分化成具有自律性和传导性的细胞。心脏生物起搏即利用生物学及其相关技术,修复或替代受损的自律性节律点和/或特殊传导系统,使心脏的起搏和传导功能得以恢复。干细胞移植心脏生物起搏即诱导干细胞分化成具有起搏和传导功能的细胞,重建心脏的起搏和传导功能。研究干细胞移植实现心脏生物起搏,治疗缓慢型心律失常已成为研究热点。     

Management of Epstein–Barr Virus-induced Post-transplant Lymphoproliferative Disease in Recipients of Solid Organ Transplantation

The optimal management of Epstein-Barr virus (EBV)-induced post-transplant lymphoproliferative diseases (PTLD) remains controversial. While withdrawal or reduction of immunosuppression is widely accepted as the strategy for the treatment of EBV/PTLD, the role of additional therapeutic interventions remains less clear. Newer strategies, including anti-B-cell monoclonal antibodies and adoptive immunotherapy offer the promise of impaired efficacy and outcome against EBV disease, but lack data demonstrating how and when to use these approaches. The current review provides an overview of potential strategies and presents guidelines for the management of EBV/PTLD in solid-organ transplant recipients.     

Concentrated Ascites Reinfusion Therapy for Sinusoidal Obstructive Syndrome After Hematopoietic Stem Cell Transplantation

Sinusoidal obstruction syndrome (SOS) is one of the severe complications of hematopoietic stem cell transplantation (HSCT). Systemic management including respiratory and circulatory support is necessary. In addition, abdominal paracentesis is often needed for pain relief and to reduce the pressure of tense ascites. Concentrated ascites reinfusion therapy (CART) involves the filtration, concentration, and reinfusion of drained ascites, which contributes to reuse of autologous proteins. CART has been reported as supportive therapy for patients with liver cirrhosis and cancer. We retrospectively reviewed the efficacy and safety of CART in three patients (two with acute myelogenous leukemia and one with chronic myeloid leukemia) who developed SOS after allo‐HSCT. They all had symptomatic, tense, and diuretic‐refractory ascites with right costal pain and marked weight gain. Two patients showed immediate improvement after CART. However, one patient experienced four CARTs with slow recovery. All patients are now alive and are being monitored as outpatients over 2 years with remission. No severe adverse event was observed related to CART, and 25.2–98.0 (median 30.2) grams of albumin was collected and reinfused. CART after paracentesis reduces protein loss in ascites by reinfusion of autologous protein instead of exogenous albumin preparations. Although transient fever is reported as a frequent adverse event, no events like severe bleeding or infection were observed. While its safety has not been fully established in patients with hematological disease after HSCT, CART may be a considerable supportive therapy for SOS with tense ascites.     

Female‐Versus‐Male Alloreactivity as a Model for Minor Histocompatibility Antigens in Hematopoietic Stem Cell Transplantation

H‐Y encoded gene products were the first to be recognized as clinically relevant minor histocompatibility antigens. Compared to other gender combinations, female donor/male recipient (FDMR) transplants are associated with increased graft‐versus‐host disease (GvHD), increased transplant‐related mortality (TRM) and reduced risk of relapse. Still, their relative impact on transplant outcome remains controversial. We analyzed donor/recipient sex combination in 53 988 patients treated with allogeneic hematopoietic stem cell transplantation (HSCT) between 1980 and 2005. We found a strong increase in chronic GvHD and late TRM and decreased survival in FDMR transplants irrespective of underlying disease. Conversely, FDMR patients had lower relapse rates. The negative effect on survival decreased with advancing disease stage as relapse protection became more important. Effects of H‐Y alloreactivity were most pronounced in patients transplanted from HLA‐matched donors and in those receiving transplants from an adult donor. Adjustment for acute and chronic GvHD only partially corrected the effects of H‐Y alloreactivity. Analysis of the FDMR proportion over time indicated that the frequency of this gender combination has declined in unrelated transplants over the last 10 years. These data define the role of H‐Y mismatching in allogeneic HSCT and support the current practice of avoiding female donors for male patients, if possible.