ABSTRACT
INTRODUCTION: Although Hematopoietic Stem Cell Transplantation (HSCT) is the only curative option for children with certain non-malignant disorders, a proportion of these children are admitted to the Pediatric Intensive Care Unit (PICU) due to treatment related life-threatening complications. AIMS: To analyze risk factors for ICU hospitalizations, morbidity and mortality in children with genetic diseases who have undergone HSCT and were admitted to intensive care units. METHODS: This retrospective study is based on the collection and analysis of clinical and laboratory data from the medical records of patients from the departments of Bone Marrow Transplantations and Intensive Care, from 2 hospitals, Hadassah and Rambam Medical Centers. RESULTS: Over the course of 15 years (2005-2019), 463 HSCT were performed for pediatric patients with non-malignant diseases, 68 of them (15%) required hospitalization in Intensive Care Units (ICU), 41% of the patients survived. The PICU mortality rate has decreased over the last years. Factors found to have a significant negative impact on PICU survival were severe neutropenia at admission to ICU, mechanical ventilation, inotropic support, and Multi Organ Failure (MOF). CONCLUSIONS: Our results showed low incidence of ICU admissions and relatively high survival rate for pediatric patients with non-malignant disorders post HSCT, comparing with literature data.
Subject(s)
Hematopoietic Stem Cell Transplantation , Child , Humans , Retrospective Studies , Hematopoietic Stem Cell Transplantation/adverse effects , Intensive Care Units, Pediatric , Hospitalization , Risk FactorsABSTRACT
Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is an efficient treatment for numerous malignant and nonmalignant conditions affecting children. This procedure can result in infectious and noninfectious neurological complications (NCs). Objective: The objective of the study is to examine the incidence, risk factors, and outcomes of NCs in pediatric patients following allogeneic HSCT. Methods: We performed a retrospective study of 746 children who underwent 943 allogeneic HSCTs in two large pediatric hospitals in Israel from January 2000 to December 2019. Results: Of the pediatric patients 107 (14.3%) experienced 150 NCs. The median follow-up was 55 months. Noninfectious NCs were more common than infectious NCs (81.3% vs. 18.7%). Factors significantly associated with type of NC (infectious vs. noninfectious) were underlying disease (immunodeficiency vs. malignant and metabolic/hematologic disease) (p-value = 0.000), and use of immunosuppressive agent, either Campath or ATG (p-value = 0.041). Factors with a significant impact on developing neurological sequelae post-NC were number of HSCT >1 (p-value = 0.028), the use of alemtuzumab as an immunosuppressive agent (p-value = 0.003), and infectious type of NC (p-value = 0.046). The overall survival rate of whole NC-cohort was 44%; one-third of all mortality cases were attributed to the NC. The strongest prognostic factors associated with mortality were older age at HSCT (p-value = 0.000), the use of alemtuzumab as an immunosuppressive agent (p-value = 0.004), and the existence of neurological sequelae (p-value = 0.000). Abnormal central nervous system imaging (p-value = 0.013), the use of alemtuzumab as an immunosuppressive agent (p-value = 0.019), and neurological sequelae (p-value = 0.000) had statistically significant effects on neurological cause of death. Conclusion: Infectious and noninfectious NCs are a significant cause of morbidity and mortality following allogeneic HSCT in children. Further research is required to better understand the risk factors for different NCs and their outcomes regarding sequelae and survival.
ABSTRACT
BACKGROUND: Although hematopoietic stem cell transplantation (HSCT) is the only curative option for some children with malignant and nonmalignant disorders, the procedure itself carries a high risk of complications. A proportion of children undergoing HSCT develop severe transplant-related complications requiring hospitalization in the pediatric intensive care unit (PICU). METHODS: A retrospective cohort study included 793 children with malignant and nonmalignant diseases that underwent 963 HSCTs in two large pediatric hospitals over 15 years. Ninety-one patients needed 105 (11%) PICU admissions. The objective of the study was to analyze the risk factors associated with morbidity and mortality in children post HSCT who were admitted to the PICU. RESULTS: Survival rate of a single PICU hospitalization was 43%. Long-term survival rate (classified as 1 year and 3 years) was 29.1% and 14.9% among PICU hospitalized patients compared with 74.6% and 53.3% among patients who had undergone HSCT and did not require PICU hospitalization. Factors found to have a significant negative association with PICU survival were respiratory failure as indication for PICU admission, neutropenia, graft-versus-host disease, mechanical ventilation, inotropic support, need for dialysis, and multiple-organ failure (MOF) with more than one systemic intensive intervention. The strongest prognostic factors associated with mortality were MOF (p < .001) and the need for inotropic support (p = .004). CONCLUSIONS: Neutropenia was found to be negatively associated with survival, suggesting non-engraftment and late engraftment are important risk factors for HSCT patients hospitalized in PICU. MOF and inotropic support were found to be the main negatively associated predictive factors with survival.
Subject(s)
Hematopoietic Stem Cell Transplantation , Neutropenia , Child , Hematopoietic Stem Cell Transplantation/adverse effects , Hospitalization , Humans , Infant , Intensive Care Units, Pediatric , Multiple Organ Failure/etiology , Neutropenia/etiology , Prognosis , Retrospective Studies , Risk FactorsABSTRACT
We report the results of national retrospective study of 45 children with hemophagocytic lymphohistiocytosis (HLH) who underwent allogeneic hematopoietic stem-cell transplantation (HSCT) in Israel between the years 2000-2018. Donors were either HLA-matched (n = 26), partially mismatched (n = 7), haploidentical (n = 8), or cord-blood (n = 4). Myeloablative conditioning (MAC) was used in 20 procedures, and reduced-intensity conditioning (RIC) in 25. Forty-two patients engrafted, two had primary graft failure (one successfully retransplanted), one died prior to engraftment, and two developed secondary graft failure. Of the eight patients who had mixed donor chimerism at day 30 (5-95%), five achieved stable mixed or full donor chimerism. The 5-year probabilities of overall survival and event-free survival (EFS) were 86% and 82%, respectively. Five-year EFS was lower for patients receiving RIC compared to MAC (72% vs. 100%, p = 0.018) and following alternative-donor transplant (68% vs. 92% for HLA-matched donors, p = 0.034), mostly due to increased transplant-related mortality (TRM). Thus, both HLA-matched and alternative donor transplant procedures may benefit form a myeloablative conditioning regimen.
Subject(s)
Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Lymphohistiocytosis, Hemophagocytic , Humans , Lymphohistiocytosis, Hemophagocytic/therapy , Myeloablative Agonists , Retrospective Studies , Transplantation ConditioningABSTRACT
BACKGROUND: Mucormycosis is a life-threatening infection with a tendency for angioinvasion that may lead to progressive dissemination. Disseminated mucormycosis, defined as the involvement of two or more non-contiguous sites, is rare in children, and data concerning its management and outcome are scarce. The aim of this study was to assess the contemporary management strategies and outcomes of disseminated mucormycosis in the pediatric population. METHODS: We conducted a retrospective search in six large tertiary medical centers for all cases of disseminated mucormycosis that occurred between 2009-2020 in patients aged 1-20 years. RESULTS: Twelve cases were identified. Underlying conditions included hematological malignancies (n = 10), solid tumor (post-autologous hematopoietic stem cell transplantations; n = 1), and solid organ (liver) transplantation (n = 1). In all cases, amphotericin B formulations were administered as first-line therapy; in eight cases, they were also administered in combination with an echinocandin or triazole. Seven patients underwent surgical debridement procedures. The six-week mortality was 58%. Among the patients diagnosed between 2009-2015, one of the six survived, and of those diagnosed between 2016-2020, four of the six were salvaged. CONCLUSIONS: Disseminated mucormycosis is a life-threatening and often fatal disease, and improved diagnostic and therapeutic strategies are needed. Nevertheless, in this population-based study, five patients (42%) were salvaged through combined liposomal amphotericin/triazole treatment and extensive surgical interventions.
ABSTRACT
BACKGROUND: Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a progressive autosomal recessive disorder characterized by cachexia, gastrointestinal (GI) dysmotility, ptosis, peripheral neuropathy, and brain magnetic resonance imaging (MRI) white matter changes. Bi-allelic TYMP mutations lead to deficient thymidine phosphorylase (TP) activity, toxic accumulation of plasma nucleosides (thymidine and deoxyuridine), nucleotide pool imbalances, and mitochondrial DNA (mtDNA) instability. Death is mainly due to GI complications: intestinal perforation, peritonitis, and/or liver failure. Based on our previous observations in three patients with MNGIE that platelet infusions resulted in a transient 40% reduction of plasma nucleoside levels, in 2005 we performed the first hematopoietic stem cell transplantation (HSCT) worldwide as a life-long source of TP in a patient with MNGIE. PROCEDURE: HSCT was performed in a total of six patients with MNGIE. The multiple factors involved in the prognosis of this cohort were analyzed and compared to the literature experience. RESULTS: Cell source was bone marrow in five patients and peripheral stem cells in one, all from fully human leukocyte antigen (HLA)-matched related donors, including four who were TYMP mutation carriers. Four of six (66%) survived compared to the 37% survival rate in the literature. Reduced intensity conditioning regimen contributed to secondary graft failure in two patients. Fifteen years post HSCT, the first transplanted patient is seemingly cured. Severe GI symptoms before transplantation were mostly irreversible and were poor prognostic factors. CONCLUSIONS: Allogenic HSCT could constitute a curative therapeutic option for carefully selected, young, presymptomatic, or mildly affected patients. Timing, donor selection, and optimal conditioning protocol are major determinants of outcome. HSCT is inadvisable in patients with advanced MNGIE disease.
Subject(s)
Hematopoietic Stem Cell Transplantation/methods , Intestinal Pseudo-Obstruction/therapy , Muscular Dystrophy, Oculopharyngeal/therapy , Ophthalmoplegia/congenital , Adolescent , Adult , Child , Cohort Studies , Female , Humans , Male , Ophthalmoplegia/therapy , Pedigree , Prognosis , Retrospective Studies , Treatment Outcome , Young AdultABSTRACT
BACKGROUND: Autologous hematological stem cell transplantation (HSCT) is a novel therapy for systemic sclerosis (SSc) that has been validated in three randomized controlled trials. OBJECTIVES: To report the first Israeli experience with HSCT for progressive SSc and review the current literature. METHODS: Five SSc patients who were evaluated in our department and were treated by HSCT were included. Medical records were evaluated retrospectively. Demographic, clinical, and laboratory data were recorded. Continuous data are presented as the mean ± standard deviation. Categorical variables are presented as frequencies and percentages. RESULTS: Five SSc patients were treated with HSCT. Four patients were adults (mean age 53 ± 12 years) and one was a 12-year-old pediatric patient. All patients were female. HSCT was initiated 1.4 ± 0.8 years after diagnosis. Two patients were RNA POLIII positive, two were anti-topoisomerase 1 positive, and one only antinuclear antibodies positive. All patients had skin and lung involvement. The mean modified Rodnan Skin Score was 29 ± 4.7 before HSCT, which improved to 10.4 ± 9.6 after HSCT. The forced vital capacity improved from 68 ± 13% to 90 ± 28%. Diffusing capacity of the lungs for carbon monoxide increased by 6%. Among severe adverse events were cyclophosphamide-related congestive heart failure, antithymocyte globulin-related capillary leak syndrome, and scleroderma renal crisis. All symptoms completely resolved with treatment without sequela. No treatment related mortality was recorded. CONCLUSIONS: HSCT is an important step in the treatment of progressive SSc in Israel. Careful patient selection reduces treatment related morbidity and mortality.
Subject(s)
Cyclophosphamide , Hematopoietic Stem Cell Transplantation , Scleroderma, Systemic , Adult , Autoantibodies/blood , Autoantibodies/classification , Child , Cyclophosphamide/administration & dosage , Cyclophosphamide/adverse effects , Female , Hematopoietic Stem Cell Transplantation/adverse effects , Hematopoietic Stem Cell Transplantation/methods , Humans , Immunosuppressive Agents/administration & dosage , Immunosuppressive Agents/adverse effects , Israel/epidemiology , Lung/pathology , Monitoring, Physiologic/methods , Outcome and Process Assessment, Health Care , Respiratory Function Tests/methods , Retrospective Studies , Scleroderma, Systemic/diagnosis , Scleroderma, Systemic/epidemiology , Scleroderma, Systemic/immunology , Scleroderma, Systemic/therapy , Skin/pathology , Transplantation, AutologousABSTRACT
(1) Background: Refractory acute graft-versus-host disease (R-aGvHD) remains a leading cause of death after allogeneic stem cell transplantation. Survival rates of 15% after four years are currently achieved; deaths are only in part due to aGvHD itself, but mostly due to adverse effects of R-aGvHD treatment with immunosuppressive agents as these predispose patients to opportunistic infections and loss of graft-versus-leukemia surveillance resulting in relapse. Mesenchymal stromal cells (MSC) from different tissues and those generated by various protocols have been proposed as a remedy for R-aGvHD but the enthusiasm raised by initial reports has not been ubiquitously reproduced. (2) Methods: We previously reported on a unique MSC product, which was generated from pooled bone marrow mononuclear cells of multiple third-party donors. The products showed dose-to-dose equipotency and greater immunosuppressive capacity than individually expanded MSCs from the same donors. This product, MSC-FFM, has entered clinical routine in Germany where it is licensed with a national hospital exemption authorization. We previously reported satisfying initial clinical outcomes, which we are now updating. The data were collected in our post-approval pharmacovigilance program, i.e., this is not a clinical study and the data is high-level and non-monitored. (3) Results: Follow-up for 92 recipients of MSC-FFM was reported, 88 with GvHD ≥°III, one-third only steroid-refractory and two-thirds therapy resistant (refractory to steroids plus ≥2 additional lines of treatment). A median of three doses of MSC-FFM was administered without apparent toxicity. Overall response rates were 82% and 81% at the first and last evaluation, respectively. At six months, the estimated overall survival was 64%, while the cumulative incidence of death from underlying disease was 3%. (4) Conclusions: MSC-FFM promises to be a safe and efficient treatment for severe R-aGvHD.
Subject(s)
Cell- and Tissue-Based Therapy , Graft vs Host Disease/therapy , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Adolescent , Adult , Aged , Bone Marrow Transplantation/adverse effects , Cell- and Tissue-Based Therapy/adverse effects , Cell- and Tissue-Based Therapy/methods , Child , Child, Preschool , Female , Graft vs Host Disease/diagnosis , Graft vs Host Disease/etiology , Graft vs Host Disease/prevention & control , Humans , Infant , Male , Mesenchymal Stem Cell Transplantation/adverse effects , Mesenchymal Stem Cell Transplantation/methods , Middle Aged , Transplantation Conditioning , Treatment Outcome , Young AdultABSTRACT
Alemtuzumab, fludarabine, and melphalan reduced-intensity conditioning (RIC) regimens are increasingly used for the hematopoietic cell transplantation (HCT) of pediatric and young adult patients with nonmalignant diseases. Early experience suggests that these regimens are associated with good survival but a high incidence of mixed chimerism, which we have previously shown to be influenced by the alemtuzumab schedule. We hypothesized that the underlying diagnosis and donor graft source would also affect the development of mixed chimerism and that the majority of patients would survive RIC HCT without graft loss. To examine this, we conducted a retrospective study of 206 patients with metabolic diseases, non-Fanconi anemia marrow failure disorders, and primary immune deficiencies who underwent 210 consecutive RIC HCT procedures at Cincinnati Children's Hospital. Ninety-seven percent of the patients engrafted. Mixed donor and recipient chimerism developed in 46% of patients. Patients with marrow failure had a low risk of mixed chimerism (hazard ratio [HR], .208; 95% confidence interval [CI], .061 to .709; P = .012). The risk of mixed chimerism was high in patients who received a cord blood graft (HR, 3.122; 95% CI, 1.236 to 7.888; P = .016). As expected, patients who received a proximal or higher dose per kilogram of alemtuzumab schedule also experienced higher rates of mixed chimerism (all HR > 2, all P < .05). At the time of last follow-up (median, 654 days; range, 13 to 3337), over 75% of patients had greater than 90% whole blood donor chimerism. A second transplantation was performed in 5% of patients. Three-year survival without retransplantation was 84% (95% CI, 71% to 98%) for patients who underwent transplantation with an HLA-matched sibling donor. Survival without retransplantation was negatively affected by lack of a matched related donor, increasing age, and development of grades III and IV acute graft-versus-host disease. We conclude that alemtuzumab, fludarabine, and melphalan RIC HCT offers good results for many patients and that the risk of developing mixed chimerism is influenced by underlying diagnosis, graft source, and alemtuzumab dosing.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Melphalan/therapeutic use , Vidarabine/analogs & derivatives , Adolescent , Adult , Alemtuzumab , Antibodies, Monoclonal, Humanized/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Child , Child, Preschool , Chimerism , Female , Hematopoietic Stem Cell Transplantation/methods , Humans , Male , Melphalan/administration & dosage , Transplantation Conditioning/methods , Treatment Outcome , Vidarabine/administration & dosage , Vidarabine/therapeutic use , Young AdultABSTRACT
Both transplanted and leukemia patients are at high risk (HR) for invasive pulmonary aspergillosis (IPA). Methods for rapid diagnosis are crucial. Our objective was to investigate the impact of serial serum galactomannan assay (GMA) screening on IPA diagnosis in children. Between January 2010 and December 2011, all children following stem cell transplantation (SCT) or with HR leukemia were prospectively included. Serum samples for GMA were taken once-twice weekly. Results >.5 were considered positive. Patients suspected of having IPA were stratified as possible, probable, and definite. Forty-six children (median age, 8 years) were included, 38 after SCT (32 allogeneic), 8 with HR leukemia. A total of 510 samples were taken; screening period was 1-6 months for 34 patients. GMA was negative in 28 patients, all but one without suspicion of IPA. Eighteen patients had positive GMA: while four (22%) were upgraded to probable IPA, fourteen (78%) were considered as false positives (FP), some associated with piperacillin-tazobactam treatment. GMA sensitivity and specificity were 0.8 and 0.66, respectively; positive- and negative-predictive values (PPV, NPV) were 0.22 and 0.96, respectively. GMA may have a role in evaluating HR children for IPA. Both NPV and FP rates are high. The cost benefit of early detection versus over-diagnosis should be further studied.
Subject(s)
Hematopoietic Stem Cell Transplantation , Invasive Pulmonary Aspergillosis , Leukemia , Mannans/blood , Penicillanic Acid/analogs & derivatives , Piperacillin/administration & dosage , Adolescent , Adult , Allografts , Autografts , Child , Child, Preschool , Female , Galactose/analogs & derivatives , Humans , Infant , Infant, Newborn , Invasive Pulmonary Aspergillosis/blood , Invasive Pulmonary Aspergillosis/diagnosis , Invasive Pulmonary Aspergillosis/drug therapy , Invasive Pulmonary Aspergillosis/etiology , Leukemia/blood , Leukemia/therapy , Male , Penicillanic Acid/administration & dosage , Prospective Studies , TazobactamABSTRACT
BACKGROUND: Pharmacokinetics (PK), pharmacodynamics and optimal dosing of vancomycin in obese children is not known. Higher trough levels of vancomycin may improve outcomes. This prospective study evaluated the appropriateness of twice-daily regimen for the adherence to guidelines, among obese and non obese children. METHODS: Children receiving vancomycin, (20 mg/kg BID) were included. Patients were divided into 3 groups. Adequacy was defined as trough level ≥ 10mg/L and AUC/MIC > 400. An alternative-dosing regimen was calculated based on individual PK parameters. RESULTS: Seventy-seven pairs (trough, peak) were taken from 51 children. Mean trough level was 3.36 ± 2.58, only 3% fell in therapeutic range, no statistical difference was observed between obese, normal weight or underweight groups. One child had an AUC/MIC > 400. All children recovered. CONCLUSION: PK properties of all weight groups were similar. More frequent and higher doses are needed to achieve the goals of current guidelines.
Subject(s)
Anti-Bacterial Agents/administration & dosage , Body Weight , Vancomycin/administration & dosage , Adolescent , Area Under Curve , Child , Child, Preschool , Drug Administration Schedule , Female , Humans , Infant , Male , Microbial Sensitivity Tests , Prospective Studies , Vancomycin/pharmacokineticsABSTRACT
Legionnaires' disease is a rare cause of community acquired pneumonia in children and is exceedingly rare in infants and neonates. We describe a case of a 15 years old female, with lymphoblastic leukemia and legionella pneumophila pneumonia. Diagnosis was made by specific culture and polymerase chain reaction method from sputum and bronchoalveolar ravage specimens. Treatment was prolonged because of slow resolution and the fact that she underwent bone marrow transplantation. This emphasizes the importance of considering this rare entity in cases of severe pneumonia, especially in the immunocompromised host.
Subject(s)
Legionella pneumophila/isolation & purification , Legionnaires' Disease/etiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/therapy , Adolescent , Bone Marrow Transplantation/methods , Bronchoalveolar Lavage Fluid/microbiology , Female , Humans , Immunocompromised Host , Legionnaires' Disease/diagnosis , Polymerase Chain Reaction , Sputum/microbiologyABSTRACT
In Xenopus embryos, XMeis3 protein activity is required for normal hindbrain formation. Our results show that XMeis3 protein knock down also causes a loss of primary neuron and neural crest cell lineages, without altering expression of Zic, Sox or Pax3 genes. Knock down or inhibition of the Pax3, Zic1 or Zic5 protein activities extinguishes embryonic expression of the XMeis3 gene, as well as triggering the loss of hindbrain, neural crest and primary neuron cell fates. Ectopic XMeis3 expression can rescue the Zic knock down phenotype. HoxD1 is an XMeis3 direct-target gene, and ectopic HoxD1 expression rescues cell fate losses in either XMeis3 or Zic protein knock down embryos. FGF3 and FGF8 are direct target genes of XMeis3 protein and their expression is lost in XMeis3 morphant embryos. In the genetic cascade controlling embryonic neural cell specification, XMeis3 lies below general-neuralizing, but upstream of FGF and regional-specific genes. Thus, XMeis3 protein is positioned at a key regulatory point, simultaneously regulating multiple neural cell fates during early vertebrate nervous system development.
Subject(s)
Cell Lineage , Homeodomain Proteins/metabolism , Nervous System/cytology , Nervous System/embryology , Paired Box Transcription Factors/metabolism , Transcription Factors/metabolism , Xenopus Proteins/metabolism , Xenopus laevis/embryology , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Biomarkers/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/metabolism , Fibroblast Growth Factor 3/metabolism , Fibroblast Growth Factor 8/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Genes, Dominant/genetics , Homeodomain Proteins/genetics , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nervous System/metabolism , Neural Crest/cytology , Neural Crest/embryology , Neural Crest/metabolism , Neurons/cytology , Neurons/metabolism , PAX3 Transcription Factor , Paired Box Transcription Factors/genetics , Phenotype , Transcription Factors/genetics , Xenopus Proteins/genetics , Xenopus laevis/geneticsABSTRACT
Xenopus oocytes generate pigment granules (melanosomes) that predominantly localize to the animal hemisphere cortex. During embryonic development, these granules are located near the membranes of outer layer ectoderm cells. We report a novel phenotype found during an expression cloning screen in Xenopus laevis embryos. The phenotype is characterized by dissociation of pigment granules from the cell membrane to form large central aggregates. This phenomenon was induced by a truncated form of the Xenopus Del1 (XDel1) protein that contains only the C-terminal discoidin (D2) domain. This truncated form of XDel1 localized to membranes as shown by a chimeric enhanced green fluorescent protein construct. Although a similar localization occurred in immature oocytes, dissociation of pigment granules was limited to the oocyte vegetal hemisphere. The full-length XDel1 cDNA was cloned, and XDel1 mRNA expression was found to be ubiquitous and continuous from early oocyte to tail bud stages, with a transient enrichment in the cement gland. Ectopic expression of various deletion or full-length constructs or antisense morpholino oligonucleotides did not induce any significant developmental phenotypes.