The treatment for primary immune thrombocytopenia with romiplostim in adult and paediatric patients: use experience at a Spanish university hospital.

WHAT IS KNOWN AND OBJECTIVE: Primary immune thrombocytopenia (ITP) is characterized by accelerated platelet destruction, as well as suboptimal platelet production. Thrombopoietin (TPO) receptor agonists bind to and activate human TPO receptor, and have been shown to increase platelet counts. In this study, we assessed the effectiveness and safety of long-term administration of TPO agonist romiplostim in adult and paediatric patients. METHODS: This is a retrospective observational study that included every ITP patient (adults and children) who received romiplostim since its inclusion in our institutional formulary. Data on patients' demographics, romiplostim doses, platelet counts, use of rescue medication and concurrent therapies were collected. Outcomes for effectiveness evaluation were proportion of patients who achieved a platelet response (platelet count >50 × 10(9) per litre and double the platelet count at baseline on any scheduled visit, excluding counts obtained within 8 weeks after receipt of rescue medications), proportion of patients who achieved a durable response (platelet responses during 6 or more weeks of the last 8 weeks of treatment), proportion of patients needing rescue medication, proportion of patients able to stop or reduce concurrent treatment and mean number of weekly platelet responses. Safety was assessed on the basis of the incidence of adverse events documented on the patients' medical records. RESULTS AND DISCUSSION: This study enrolled ten adults and four paediatric patients. None of the paediatric patients and one adult patient had been splenectomized (contraindicated in the other adults). In the adult population, eight achieved a response at least once during treatment, and 1 achieved a durable response. Four patients needed rescue medication (mostly intravenous immunoglobulins). Three patients were able to stop concurrent ITP therapies, and the mean number of weekly platelet responses was 6. All four paediatric patients achieved a response at least once during treatment, and three achieved durable responses. Three patients needed rescue medication. The only patient who was receiving concurrent ITP medication was able to stop it, and the mean number of weekly platelet responses was 25. No serious adverse events were registered during treatment in either population. WHAT IS NEW AND CONCLUSION: The effectiveness of romiplostim was variable with few adult patients achieving a durable response. Our paediatric patients responded better with most achieving a durable response. The treatment was safe for both groups of patients. Studies should be conducted to identify patients more likely to benefit from this treatment.

Survival and toxicity outcomes of hematopoietic stem cell transplantation for pediatric patients with Fanconi anemia: a unified multicentric national study from the Spanish Working Group for Bone Marrow Transplantation in Children.

Hematopoietic stem cell transplantation (HSCT) is currently the only curative option for hematological manifestations in patients with Fanconi anemia (FA). We report the outcome of 34 patients with FA inside a collaborative multicenter national study based on recommendations of Spanish Working Group for Bone Marrow Transplantation in Children (GETMON) between 2009 and 2016. Fludarabine-based conditioning regimen was carried out in all patients, with low dose total body irradiation in unrelated transplants. Disease status before HSCT was bone marrow failure (BMF) in 30 patients and myelodysplastic syndrome (MDS) in four. Donors were matched siblings donors (MSD) in 18, matched unrelated donors (MUD) in 15, and one haploidentical donor. All except one patient engrafted. Cumulative incidence of grades II-IV acute graft-versus-host disease (GVHD) was 29% and 11% for chronic GVHD. Median follow-up after HSCT was 6.5 years. Seven patients (21%) died due to transplant-related causes, two (6%) because of MDS relapse, and one (3%) after a squamous cell carcinoma. Overall survival (OS) was 73% at 5 years post-transplant, with no differences between MSD and MUD transplants. OS for patients with BMF was 80% while for MDS was 25%. Our data suggest HSCT can cure hematologic manifestations of most FA patients with BMF.

[Thrombocytosis in the oncology-haematology clinic: description, aetiological diagnosis and progression thrombocytosis]. / Trombocitosis en la consulta de oncohematología. Descripción, diagnóstico etiológico y evolución.

INTRODUCTION: Thrombocytosis is a common cause of patient referral to a paediatric haematologist specialist which requires a significant number of laboratory tests and visits to confirm the diagnosis. The aim of our study has been to analyse the characteristics of patients referred to our centre for specialised thrombocytosis assessment. Based on this assessment we established the criteria patients must fulfil to be recommend for further hospital study. PATIENTS AND METHODS: We categorised the 33 patients referred for thrombocytosis assessment according to sex, age, origin, personal and family history, platelet count at diagnosis and the reason why the red and white blood count at diagnosis (Haemoglobin, mean corpuscular volume, mean corpuscular haemoglobin, leukocyte, neutrophils, lymphocyte and monocyte count), maximum platelet count during follow-up and other complementary examinations were done. The final diagnosis itself and number of previous visits before were also considered. The classification used to grade thrombocytosis was: low (500-700 X 10(3)/microl), mild (700-900 x 10(3)/microl), severe (900-1.000 x 10(3)/microl) and extreme (> 1.000 x 10(3)/microl). RESULTS: There was no predominance of males or females. 45 % of patients were under 2 years old and 55 % of them came from their primary care centre. The mean platelet count at the first medical visit was 669,000 (mild thrombocytosis). During follow-up, 24 % of the patients reached extreme platelets levels. In 28 % the initial blood count was performed because of an infection. The most frequently requested laboratory test was iron metabolism (82 % of the cases). All cases correspond to secondary thrombocytosis (48 % were reactive to infections, 24 % secondary to iron deficiency, and 15 % were associated to both causes). The mean number of visits before hospital discharge was 5.12. CONCLUSIONS: The finding of thrombocytosis in the majority of the cases studied was casual or in the context of an infectious process. Most of the thrombocytosis were mild. Due to the extremely low incidence of primary thrombocytosis in childhood and the fact that diagnosis is made by exclusion of other possibilities, the initial study of these patients should be done in primary care centres. The first conditions to be ruled out are infectious, inflammatory or bleeding processes. Once these causes are excluded, the most useful complementary test is to measure iron level given the relation between iron deficiency and thrombocytosis. Once these causes are ruled out and thrombocytosis persists, it would then be indicated to refer the patient to a paediatric oncology-haematology department for a more exhaustive follow-up.

[Evaluation of systematic neonatal screening for sickle cell diseases in Madrid three years after its introduction]. / Evaluación en el tercer año de implantación del cribado neonatal universal de anemia falciforme en la Comunidad de Madrid.

BACKGROUND: Sickle cell anemia is a hereditary disease which, as a result of migration, constitutes one of the most frequent genetic disorders in northwest Europe. Complications secondary to this disease are common during the first 3 years of life and early diagnosis has been recommended to reduce their development. The autonomous community of Madrid began to perform universal neonatal screening for hemoglobinopathies in May 2003. This study presents the results of the first 32 months of this screening program. METHODS: A prospective, descriptive study was designed to include all the neonates born in centers in the autonomous community of Madrid from May 2003 to December 2005. A heel prick dried blood spot from the Guthrie card was analyzed by high-performance liquid chromatography to detect hemoglobin F, A, S, C, D and E. RESULTS: A total of 190,238 newborns were analyzed, and 1060 hemoglobin variants (5.57 for every 1000 births) were detected. Thirty-one were sickle cell diseases and appropriate antibiotics, vaccination and comprehensive care were initiated. Prenatal diagnosis of subsequent pregnancies was performed in three families after parental investigation. Carrier parents were from 44 countries of origin. CONCLUSIONS: Although sickle cell disease was considered anecdotic in Spain until recently, the diagnosis of this entity has markedly increased as a result of immigration. The universal screening program is expected to reduce morbidity and mortality in the first years of life.

[Sickle cell disease: registry of the Spanish Society of Pediatric Hematology]. / Enfermedad de células falciformes: registro de la Sociedad Española de Hematología Pediátrica.

INTRODUCTION: Sickle cell disease (SCD), a genetic anemia, is currently an emerging health problem in Spain. Since 2000, The Spanish Society of Pediatric Hematology has maintained a registry of these patients. The data corresponding to 2004 are presented herein. PATIENTS AND METHODS: Information was sent by different national hospitals. Pediatric patients with SCD followed-up during 2003 were registered in the first quarter of 2004. Data on epidemiology, diagnosis, treatment and outcome in each patient were gathered. RESULTS: A total of 138 patients in 24 national hospitals were registered. Of these, 99 were still under follow-up. There was no significant difference in sex. The mean age was 8.2 years. Seventy-eight percent of the patients were homozygous. Forty-four percent were born in Africa but 76% had abnormal genes originating in Africa. Neurophysiologic disorders were detected in 36% of the patients. Symptomatic treatment was given in 65%, hydroxyurea in 27%, hypertransfusional therapy in 3%, and chelation therapy, indicated for ferric overload, was provided in 4%. None of the patients underwent stem cell transplantation. Acute complications requiring hospitalization occurred in 21%, and chronic complications were observed in 27%. The most frequent chronic complications were delayed height and weight gain and liver and biliary tract disorders. Two patients died. CONCLUSIONS: This study confirms a highly significant increase in the prevalence of pediatric patients with SCD in the last 4 years, requiring greater resources to be devoted to the diagnosis and follow-up of this disease.

Detection of DNA adducts formed by aristolochic acid in renal tissue from patients with Chinese herbs nephropathy.

A unique type of rapidly progressive renal fibrosis, designated Chinese herbs nephropathy (CHN), has been described in young Belgian women who had followed a slimming regimen including recently introduced Chinese herbs (Stephania tetrandra and Magnolia officinalis). Aristolochic acid (AA), a known nephrotoxin and carcinogen, was suspected as its causal factor. To substantiate this hypothesis, renal tissue from five patients with CHN and six patients with other renal diseases was analyzed for the presence of AA-derived DNA adducts, a described biomarker of AA exposure associated with its carcinogenic and mutagenic activity. Using the 32P-postlabeling method, a major distinct DNA adduct spot was found in all five cases of CHN and identified by cochromatographic analyses with authentic markers as the deoxyadenosine adduct of AA-I [7-(deoxyadenosin-N6-yl)-aristolactam I], the major component of the plant extract AA. This DNA adduct was absent in the six control cases. The 7-(deoxyadenosin-N6-yl)-aristolactam I adduct levels in CHN ranged from 0.7 to 5.3/10(7) nucleotides. Our data demonstrate that AA is implicated in CHN. They suggest a mechanism for the urothelial atypia and cancers observed in this disease and raise the possibility that a DNA mutation is responsible for the kidney-destructive fibrotic process.

[Evaluation of bone mineral density in children with sickle cell disease]. / Evaluación de la densidad mineral ósea en pacientes con enfermedad de células falciformes.

OBJECTIVE: To evaluate bone mineral density (BMD) in children with sickle cell disease (SCD) in the Community of Madrid. MATERIAL AND METHODS: The BMD was estimated in 40 children with SCD, and with an age range between 3 and 16 years, using densitometry (DXA), as recommended by the International Society for Clinical Densitometry (ISCD). RESULTS: The mean age at the time of the study was 7.97±3.95 years, the mean value of the DXA expressed in Z -score was -0.91±1.46 with a range of minimum values - 5.30 and 2.30 maximum. More than half (57.5%) of all the children had normal BMD (Z>-1), 25% had low BMD (Z between -1 and -2), and 17.5% showed an abnormal Z -score values of osteoporosis (Z -score<-2). The Pearson linear correlation was statistically significant between Z -score value and the haemoglobin level (r=0.368, p=.019), finding no correlation with the levels of 25 (OH) vitamin D. CONCLUSION: Prospective studies are needed with a larger number of patients to understand the future implications of bone densitometry changes and associated risk factors.

Aristolochic acid nephropathy in a Chinese patient: time to abandon the term "Chinese herbs nephropathy"?

The causal role of aristolochic acid (AA) in the so-called Chinese herbs nephropathy (CHN) has been conclusively demonstrated only in the Belgian epidemic. We report a biopsy-proven hypocellular interstitial fibrosing nephropathy in a Chinese patient who had ingested a Chinese herbal preparation bought in Shanghai. The identification of AA in the preparation and of AA-DNA adducts in the kidney tissue unequivocally demonstrates, for the first time, the causal role of AA outside the Belgian epidemic. Because the ingested preparation is very popular in China as an over-the-counter product, our observation raises the possibility that many such cases due to AA might be currently unrecognized in China. AA should be banned from herbal preparations worldwide. All cases of the so-called CHN, in which the causal role of AA has been thoroughly documented, should be further identified as aristolochic acid nephropathy (AAN). The term phytotherapy-associated interstitial nephritis (PAIN) might refer to the other cases associated with phytotherapy without identification, as yet, of the causal agent.

The anticancer agent ellipticine on activation by cytochrome P450 forms covalent DNA adducts.

Ellipticine is a potent antitumor agent whose mechanism of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Using [3H]-labeled ellipticine, we observed substantial microsome (cytochrome P450)-dependent binding of ellipticine to DNA. In rat, rabbit, minipig, and human microsomes, in reconstituted systems with isolated cytochromes P450 and in Supersomes containing recombinantly expressed human cytochromes P450, we could show that ellipticine forms a covalent DNA adduct detected by [32P]-postlabeling. The most potent human enzyme is CYP3A4, followed by CYP1A1, CYP1A2, CYP1B1, and CYP2C9. Another minor adduct is formed independent of enzymatic activation. The [32P]-postlabeling analysis of DNA modified by activated ellipticine confirms the covalent binding to DNA as an important type of DNA modification. The DNA adduct formation we describe is a novel mechanism for the ellipticine action and might in part explain its tumor specificity.

DNA adduct formation from the mutagenic air pollutant 3-nitrobenzanthrone.

The environmental contaminant 3-nitrobenzanthrone (3-nitro-7H-benz[d, e]anthracen-7-one) was recently shown to be a very strong bacterial mutagen, suggesting a new class of mutagenic compounds present in airborne particulate matter and diesel exhaust. Using the 32P-postlabeling assay, we investigated the capacity for 3-nitrobenzanthrone to form DNA adducts in vitro. Calf thymus DNA was incubated with 3-nitrobenzanthrone and either xanthine oxidase, a mammalian nitroreductase or rat liver S9 or zinc. Under these conditions 3-nitrobenzanthrone formed a total of seven adducts detectable by 32P-postlabeling. Using enrichment by butanol extraction the highest level of DNA adduct formation was found with activation by zinc (RAL: 88.4+/-32 per 108 nucleotides) followed by activation with xanthine oxidase (RAL: 75.5+/-12) and activation by rat liver S9 (RAL: 48.6+/-8). Three of the seven adduct spots were detected in all activation systems, however different amounts of individual spots were obtained in the different in vitro systems. The adduct pattern observed for the enzymatic incubations consisted of three major spots and was essentially identical. Chemical reduction of 3-nitrobenzanthrone by zinc resulted in five adduct spots whose formation was found to be concentration dependent. All adducts of 3-nitrobenzanthrone observed in this study migrated primarily along a diagonal zone, typical for DNA adducts derived from extracts of airborne particulate matter. When butanol enrichment was compared with nuclease P1 enrichment one adduct was clearly sensitive to the 3'-monophosphatase activity of nuclease P1. Our results demonstrate that 3-nitrobenzanthrone binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vitro all of which are reduction products. These adducts may contribute to the known genotoxicity and carcinogenicity of extracts from airborne particulates.

Aristolactam I a metabolite of aristolochic acid I upon activation forms an adduct found in DNA of patients with Chinese herbs nephropathy.

Aristolochic acid (AA) a naturally occuring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). We identified AA-specific DNA adducts in kidneys and in a ureter obtained from CHN patients after renal transplantation. AA is a plant extract of aristolochia species containing AA I as the major component. Aristolactams are the principal detoxication metabolites of AA, which were detected in urine and faeces from animals and humans. They are activated by cytochrome P450 (P450) and peroxidase to form DNA adducts. Using the 32P-postlabelling assay we investigated the formation of DNA adducts by aristolactam I in these two activation systems. A combination of two independent chromatographic systems (ion-exchange chromatography TLC and reversed-phase HPLC) with reference compounds was used for the identification of adducts. Aristolactam I activated by peroxidase led to the formation of several adducts. Two major adducts were identical to adducts previously observed in vivo. 7-(deoxyguanosin-N2-yl)aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)aristolactam I (dA-AAI) were formed in DNA during the peroxidase-mediated one-electron oxidation of aristolactam I. Aristolactam I activated by P450 led to one major adduct and four minor ones. Beside the principal AA-DNA adducts identified recently in the ureter of one patient with CHN, an additional minor adduct was detected, which was found to have indistinguishable chromatographic properties on TLC and HPLC from the major adduct formed from aristolactam I by P450 activation. Thus, this minor AA-adduct might be evolved from the AAI detoxication metabolite (aristolactam I) by P450 activation. These results indicate a potential carcinogenic effect of aristolactam I in humans.

[Infantile vascular tumors]. / Tumores vasculares en la infancia.

The use in the past of an imprecise terminology to designate vascular tumors has contributed to its incorrect diagnosis, and as a consequence, to inadequate treatment. In childhood, different types of vascular tumors may be present. Hemangiomas of infancy are by far the most frequent, and other less common types are congenital hemangiomas (rapidly involuting or RICH and non-involuting or NICH), kaposiform hemangioendothelioma, angioblastoma or tufted angioma and pyogenic granuloma. The correct knowledge and diagnosis, always in a multidisciplinary setting, is required to reduce incorrect diagnosis, unnecessary complementary examinations and invasive tests, and for the patient to receive the most effective and precise treatment in each case. This article reviews the historical evolution, nomenclature and classification of vascular lesions, the different clinical and pathological characteristics of each vascular tumor, the complementary examinations required correct diagnosis, the differential diagnosis, as well as highlighting the treatment options currently available for different vascular tumors and related clinical conditions.

DNA adduct formation by the ubiquitous environmental contaminant 3-nitrobenzanthrone in rats determined by (32)P-postlabeling.

Diesel exhaust is known to induce tumors in animals and is suspected of being carcinogenic in humans. Of the compounds found in diesel exhaust and in airborne particulate matter, 3-nitrobenzanthrone (3-NBA), is a particularly powerful mutagen. We investigated the capacity of 3-NBA to form DNA adducts in vivo that could be used as agent-specific biomarkers of exposure. Female Sprague-Dawley rats were treated orally with 2 mg/kg body weight of 3-NBA, and DNA from various organs was analyzed by (32)P-postlabeling. High levels of 3-NBA-specific adducts were detectable in all organs. Both enrichment versions nuclease P1 digestion and n-butanol extraction resulted in patterns consisting of either 3 or 4 adducts remarkably similar in all tissues examined. The highest level of DNA adducts was found in the small intestine (38 adducts per 10(8) nucleotides) followed by forestomach, glandular stomach, kidney, liver, lung and bladder. To provide information on the nature of the adducts formed in vivo in rats, DNA adducts were cochromatographed in 2 independent systems with standardized deoxyguanosine adducts and deoxyadenosine adducts produced by reaction of 3-NBA in the presence of xanthine oxidase with deoxyribonucleoside 3'-monophosphates in vitro. In both systems, each of the rat adducts comigrated either with a deoxyguanosine or a deoxyadenosine-derived 3-NBA adduct. Our results demonstrate that 3-NBA binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vivo, all of which are products derived from reductive metabolites bound to the purine bases (deoxyguanosine 60% and deoxyadenosine 40%).

32P-post-labelling analysis of DNA adducts formed by aristolochic acid in tissues from patients with Chinese herbs nephropathy.

Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post-labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients.

Chinese herbs nephropathy-associated slimming regimen induces tumours in the forestomach but no interstitial nephropathy in rats.

Chinese herbs nephropathy (CHN), a rapidly progressive interstitial fibrosis of the kidney, has been described in approximately 100 young Belgian women who had followed a slimming regimen containing some Chinese herbs. In 4 patients multifocal transitional cell carcinomas (TCC) were observed. Aristolochic acid (AA), suspected as the causal factor of CHN, is a well known carcinogen but its ability to induce fibrosis has never been demonstrated. The objective of this study was to evaluate the latter using doses of AA, durations of intoxication and delays of sacrifice known to yield tumours in rats. We also tested the hypothesis that a possible fibrogenic role of AA was enhanced by the other components of the slimming regimen. Male and female rats were treated orally with 10 mg isolated AA/kg per day for 5 days/week, or with approximately 0.15 mg AA/ kg per day 5 days/week contained in the herbal powder together with the other components prescribed in the slimming pills for 3 months. The animals were killed respectively 3 and 11 months later. At sacrifice, animals in both groups had developed the expected tumours but not fibrosis of the renal interstitium. Whether the fibrotic response observed in man is due to species and/or strain related differences in the response to AA or to other factors, remains to be determined. Interestingly, despite the addition of fenfluramine and diethylpropion, two drugs incriminated in the development of valvular heart disease, no cardiac abnormalities were observed.

Urothelial carcinoma associated with the use of a Chinese herb (Aristolochia fangchi)

BACKGROUND: Chinese-herb nephropathy is a progressive form of renal fibrosis that develops in some patients who take weight-reducing pills containing Chinese herbs. Because of a manufacturing error, one of the herbs in these pills (Stephania tetrandra) was inadvertently replaced by Aristolochia fangchi, which is nephrotoxic and carcinogenic. METHODS: The diagnosis of a neoplastic lesion in the native urinary tract of a renal-transplant recipient who had Chinese-herb nephropathy prompted us to propose regular cystoscopic examinations and the prophylactic removal of the native kidneys and ureters in all our patients with end-stage Chinese-herb nephropathy who were being treated with either transplantation or dialysis. Surgical specimens were examined histologically and analyzed for the presence of DNA adducts formed by aristolochic acid. All prescriptions written for Chinese-herb weight-reducing compounds during the period of exposure (1990 to 1992) in these patients were obtained, and the cumulative doses were calculated. RESULTS: Among 39 patients who agreed to undergo prophylactic surgery, there were 18 cases of urothelial carcinoma (prevalence, 46 percent; 95 percent confidence interval, 29 to 62 percent): 17 cases of carcinoma of the ureter, renal pelvis, or both and 1 papillary bladder tumor. Nineteen of the remaining patients had mild-to-moderate urothelial dysplasia, and two had normal urothelium. All tissue samples analyzed contained aristolochic acid-related DNA adducts. The cumulative dose of aristolochia was a significant risk factor for urothelial carcinoma, with total doses of more than 200 g associated with a higher risk of urothelial carcinoma. CONCLUSIONS: The prevalence of urothelial carcinoma among patients with end-stage Chinese-herb nephropathy (caused by aristolochia species) is a high.