Development of a novel cell line-derived xenograft model of primary herpesvirus 8-unrelated effusion large B-cell lymphoma and antitumor activity of birabresib in vitro and in vivo.

BACKGROUND: Primary human herpesvirus 8 (HHV8)-unrelated effusion large B-cell lymphoma is a clinical disease entity distinct from HHV8-positive primary effusion lymphoma (PEL). However, the lack of experimental HHV8-unrelated effusion large B-cell lymphoma models continues to hinder the pathophysiologic and therapeutic investigations of this disorder. METHODS: The lymphoma cells were obtained from the pleural effusion of a patient with primary HHV8-unrelated effusion large B-cell lymphoma and cultured in vitro. RESULTS: We established a novel HHV8-unrelated effusion large B-cell lymphoma cell line, designated Pell-1, carrying a c-MYC rearrangement with features distinct from those of HHV8-positive PEL. Moreover, we developed an HHV8-unrelated effusion large B-cell lymphoma cell line-derived xenograft model. Pell-1 cells induced profuse lymphomatous ascites and subsequently formed intra-abdominal tumors after intraperitoneal implantation into irradiated nonobese diabetic/severe combined immunodeficient mice. Thus, this xenograft mouse model mimicked the clinical phenomena observed in patients and recapitulated the sequential stages of aggressive HHV8-unrelated effusion large B-cell lymphoma. The bromodomain and extraterminal domain (BET) inhibitors JQ1 and birabresib (MK-8628/OTX015) reduced the proliferation of Pell-1 cells in vitro through the induction of cell cycle arrest and apoptosis. The antitumor effect of BET inhibition was also demonstrated in vivo, as birabresib significantly reduced ascites and suppressed tumor progression without apparent adverse effects in the xenografted mice. CONCLUSION: These preclinical findings suggest the therapeutic potential of targeting c-MYC through BET inhibition in HHV8-unrelated effusion large B-cell lymphoma.

Epstein-Barr virus-positive pyothorax-associated lymphoma expresses CCL17 and CCL22 chemokines that attract CCR4-expressing regulatory T cells.

Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphomas associated with chronic inflammation (DLBCL-CI) develop in patients with chronic inflammation but without any predisposing immunodeficiency. Given the expression of the EBV latent genes, DLBCL-CI should have mechanisms for evasion of host antitumor immunity. EBV-positive pyothorax-associated lymphoma (PAL) is a prototype of DLBCL-CI and may provide a valuable model for the study of immune evasion by DLBCL-CI. This study demonstrates that PAL cell lines express and secrete CCL17 and/or CCL22 chemokines, the ligands of C-C motif chemokine receptor 4 (CCR4), in contrast to EBV-negative DLBCL cell lines. Accordingly, culture supernatants of PAL cell lines efficiently attracted CCR4-positive regulatory T (Treg) cells in human peripheral blood mononuclear cells. PAL cells injected into mice also attracted CCR4-expressing Treg cells. Furthermore, this study confirmed that CCR4-expressing Treg cells were abundantly present in primary PAL tissues. Collectively, these findings provide new insight into the mechanisms of immune evasion by PAL, and further studies are warranted on whether such mechanisms eventually lead to the development of DLBCL-CI.

Generation and characteristics of a novel "double-hit" high grade B-cell lymphoma cell line DH-My6 with MYC/IGH and BCL6/IGH gene arrangements and potential molecular targeted therapies.

"Double-hit" lymphoma (DHL) is a high-grade B-cell lymphoma that harbors concurrent MYC and BCL2 or BCL6 rearrangements. Because cases of MYC/BCL6 DHL are uncommon, most reported conclusions have been based on cases of MYC/BCL2 DHL. Lack of experimental MYC/BCL6 DHL models continues to hinder the pathophysiologic and therapeutic investigations of this disorder. We herein describe a novel MYC/BCL6 DHL cell line, designated DH-My6, carrying both the MYC-IGH and BCL6-IGH fusion genes. Interruptions of MYC and BCL6 expressions using short interfering RNAs and chemical inhibitors led to significant attenuation of DH-My6 cell growth. Greater antitumor effects were found when the cells were treated with a combination of MYC and BCL6 inhibitors. Moreover, the PLK1 inhibitor volasertib and the HDAC inhibitor vorinostat synergized strongly when combined with the bromodomain inhibitor JQ1. DH-My6 is a new well-validated MYC/BCL6 DHL cell line that will provide a useful model for studies of the pathogenesis and therapeutics for the less common DHL tumor type. The rationale for approaches targeting both MYC and BCL6, and in combination with PLK1 or HDAC inhibitors for superior suppression of the aggressive MYC/BCL6 DHL warrants further in vivo testing in a preclinical model.

Differential gene expression profiling linked to tumor progression of splenic marginal zone lymphoma.

The genetic events that lead to aggressive transformation of cases of splenic marginal zone lymphoma (SMZL) after the chronic clinical stage have not been well understood. We aimed to find candidate genes associated with aggressive features of SMZL. We have successfully established two SMZL cell lines, designated SL-15 and SL-22, derived from the same patient's tumor clone in chronic and aggressive phases, respectively. Microarray analysis identified cell cycle-associated genes-specifically PLK1-as the most significantly upregulated in primary aggressive SMZL cells compared with cells from chronic phase. EPHA4 and MS4A1 (CD20) were found to be downregulated dramatically. These gene expression patterns were reproduced in both cell lines. Genetic knockdown of PLK1 resulted in inhibition of cell proliferation and induction of apoptosis in SL-22 cells, which expressed higher levels of PLK1 than SL-15 cells. SL-22 cells needed higher concentrations of chemical PLK1 inhibitors to achieve greater effects. In addition, we found homozygous deletion of the MS4A1 gene as a newly identified molecular mechanism of CD20-negative conversion. Our findings are expected to stimulate further studies on whether PLK1 could be a potential therapeutic target for this tumor. Furthermore, cases with CD20-negatively converted lymphomas should be screened for the genomic loss of MS4A1.

Epstein-Barr Virus-Positive Pyothorax-Associated Lymphoma Arising from a Posttraumatic Empyema.

Pyothorax-associated lymphoma (PAL) develops from a pyothorax caused by an artificial pneumothorax created during the treatment of pulmonary tuberculosis or tuberculous pleuritis. We report the first case of Epstein-Barr virus (EBV)-positive PAL arising from a posttraumatic empyema. A 75-year-old woman with chronic posttraumatic empyema presented with a tumor, which was connected to the wall of a pyothorax in the right thoracic cavity. She had a history of trauma to the right chest, which had occurred at the age of 45 years and had caused the chronic posttraumatic empyema. Pathological features of the resected tumor were conclusive for a diagnosis of EBV-positive PAL. Although neither postoperative chemotherapy nor radiotherapy was performed, remission was maintained for 3 years until recurrence in the liver. Combination chemotherapy led to complete remission, and 9 years after the initial diagnosis of PAL, the patient is still alive. An intriguing finding is the phenotypic alteration during the disease course. Although the primary tumor was negative for CD20 and CD3, the recurrent tumor expressed both of these molecules. We discuss this case of PAL, which was not a complication of lung tuberculosis, and the aberrant chronological phenotypic change observed in the lymphoma cells, and compare it with a usual case of PAL.

Prevalence of human cytomegalovirus, polyomaviruses, and oncogenic viruses in glioblastoma among Japanese subjects.

BACKGROUND: The association between human cytomegalovirus (HCMV) and glioblastoma multiforme (GBM) is becoming a new concept. However, information on the geographic variability of HCMV prevalence in GBM remains scarce. Moreover, the potential roles of various viruses, such as polyomaviruses and oncogenic viruses, in gliomagenesis remain unclear. Our aim was to investigate the prevalence of HCMV in GBM among Japanese patients. Furthermore, this was the first study that evaluated infection with four new human polyomaviruses in GBMs. This study also provided the first data on the detection of human papillomavirus (HPV) in GBM in the Eastern world. METHODS: We measured the number of various viral genomes in GBM samples from 39 Japanese patients using real-time quantitative PCR. The tested viruses included HCMV, Merkel cell polyomavirus, human polyomavirus (HPyV) 6, HPyV7, HPyV9, Epstein-Barr virus, human herpesvirus 8, and HPV. Our quantitative PCR analysis led to the detection of eight copies of the HCMV DNA mixed with DNA extracted from 10(4) HCMV-negative cells. The presence of HCMV and HPV genomes was also assessed by nested PCR. Immunohistochemical study was also carried out to detect HPV-derived protein in GBM tissues. RESULTS: The viral DNAs were not detectable, with the exception of HPV, which was present in eight out of 39 (21%) GBMs. All HPV-positive cases harbored high-risk-type HPV (HPV16 and HPV18). Moreover, the HPV major capsid protein was detected in GBM tumor cells. CONCLUSIONS: In contrast with previous reports from Caucasian patients, we did not obtain direct evidence in support of the association between HCMV and GBM. However, high-risk-type HPV infection may play a potential etiological role in gliomagenesis in a subset of patients. These findings should prompt further worldwide epidemiological studies aimed at defining the pathogenicity of virus-associated GBM.

[Successful eculizumab treatment for multiple coronary thrombosis complicated in paroxysmal nocturnal hemoglobinuria].

We herein report a rare case of paroxysmal nocturnal hemoglobinuria (PNH) who repeatedly developed coronary arterial thromboembolism. Anticoagulant therapies including heparin, aspirin as an antiplatelet agent and even drug-eluting stent placement in the coronary artery failed to prevent the recurrence of ischemic heart disease. Of note, initiating the administration of a humanized anti-C5 antibody, eculizumab, achieved prompt thrombolysis and maintenance treatment with eculizumab prevented the recurrence of thromboembolic disease in this patient. Taking these observations together, we suggest that the use of eculizumab be considered for treatment or prevention of arterial thrombosis complicated by PNH, although arterial thrombosis is an extremely rare event in the Japanese population.

Phylogenetic analysis of Merkel cell polyomavirus based on full-length LT and VP1 gene sequences derived from neoplastic tumours in Japanese patients.

Most Merkel cell polyomavirus (MCPyV) gene sequences have been reported from Western countries and few data are available for the virus sequences from other geographical areas, especially Asia. Thus, we performed phylogenetic analyses based on the nucleotide sequences of the full-length large T-antigen (LT) and viral protein 1 (VP1) genes derived from a variety of cancers in Japanese patients and compared them with sequences from Caucasians. The LT and VP1 gene-based phylogenetic trees identified two main genetic clades. One clade comprised strains isolated from Caucasians, whereas all of the Japanese tumour-derived MCPyV strains belonged to another clade. These findings confirm that most of the MCPyV strains present in Japan form a clade, distinct from Caucasian strains.

Effect of recombinant human soluble thrombomodulin on clinical outcomes of patients with coagulopathy after hematopoietic stem cell transplantation.

From 2001 to 2012, 71 individuals with hematological diseases received HSCT in our institution. Of these, 41 developed disseminated intravascular coagulation (DIC) in association with various underlying conditions. The patients who developed DIC after 2008 (n = 23) were treated by recombinant human soluble thrombomodulin (rTM), and the others (n = 11) were treated by either heparin and/or antithrombin III concentrate. Seven patients did not receive any anticoagulant therapy. Of note, treatment for coagulopathy by rTM significantly improved clinical outcomes of patients at day 100 and dramatically prolonged their overall survival (P = 0.044). Taken together, rTM is useful to improve clinical outcomes of transplant recipients with coagulopathy.

Pyothorax-associated lymphoma (PAL) with biclonal Epstein-Barr virus infection: characterization of a novel PAL cell line with unique features.

Pyothorax-associated lymphoma (PAL) is a representative form of diffuse large B-cell lymphoma associated with chronic inflammation, in which the Epstein-Barr virus (EBV) genome is consistently detectable in the lymphoma cells of all PAL cases. Cell lines and animal models would be useful for understanding better this rare lymphoma, but reports of PAL-derived cell lines are scarce. We report a new PAL cell line, designated Pal-2, with unique phenotypic expression. Pal-2 is the first PAL cell line that carries a biclonal EBV infection with abundant viral genome and that exhibits tumorigenic capacity once injected into nude mice.

Recombinant human soluble thrombomodulin safely and effectively rescues acute promyelocytic leukemia patients from disseminated intravascular coagulation.

We treated individuals for disseminated intravascular coagulation (DIC) caused by acute promyelocytic leukemia (APL) (n=9) using human soluble thrombomodulin (rTM) in combination with all-trans retinoic acid (ATRA) and chemotherapy, and compared the clinical outcomes with historical control patients (n=8) treated with ATRA and/or chemotherapy. Two control patients developed intracranial vascular incidents. On the other hand, no bleeding related mortality was noted in rTM-treated patients. Notably, treatment with rTM rescued patients from DIC earlier than historical controls (log rank test, p=0.019). These results suggest that administration of rTM should be considered for the treatment of individuals with DIC associated with APL.

Novel human polyomaviruses, Merkel cell polyomavirus and human polyomavirus 9, in Japanese chronic lymphocytic leukemia cases.

BACKGROUND: Chronic lymphocytic leukemia (CLL) is the rarest adult leukemia in Japan, whereas it is the most common leukemia in the Western world. Recent studies from the United States and Germany suggest a possible etiological association between Merkel cell polyomavirus (MCPyV) and CLL, although no data have been reported from Eastern countries. To increase the volume of relevant data, this study investigated the prevalence and DNA loads of MCPyV and human polyomavirus 9 (HPyV9), another lymphotropic polyomavirus, in Japanese CLL cases. FINDINGS: We found that 9/27 CLL cases (33.3 %) were positive for MCPyV using quantitative real-time polymerase chain reaction analysis. The viral DNA loads ranged from 0.000017 to 0.0012 copies per cell. All cases were negative for HPyV9. One MCPyV-positive CLL case was evaluated by mutational analysis of the large T (LT) gene, which indicated the presence of wild-type MCPyV without a nucleotide deletion. DNA sequence analysis of the entire small T (ST) gene and the partial LT gene revealed that a Japanese MCPyV isolate, designated CLL-JK, had two nucleotide gaps when compared with the reference sequence of the North American isolate MCC350. CONCLUSIONS: This study provides the first evidence that MCPyV is present in a subset of Japanese CLL cases with low viral DNA loads. MCPyV and HPyV9 are unlikely to contribute directly to the development of CLL in the majority of Japanese cases. MCPyV isolated from the Japanese CLL cases may constitute an Asian group and its pathogenicity needs to be clarified in future studies.

Thrombomodulin enhances the antifibrinolytic and antileukemic effects of all-trans retinoic acid in acute promyelocytic leukemia cells.

This study found that levels of thrombomodulin (TM) were downregulated in freshly isolated leukemia cells from patients with acute promyelocytic leukemia (APL, n = 7) and acute myelogenous leukemia (n = 14), as compared with CD34(+)/CD38(-) hematopoietic stem/progenitor cells and CD34(-)/CD33(+)/CD11b(-) promyelocytes isolated from healthy volunteers (n = 3). Exposure of APL NB4 cells to recombinant human soluble TM (rTM, 1500 ng/mL) inhibited clonogenic growth of these cells by approximately 30%, and induced expression of CD11b, a marker of myeloid differentiation, on their surfaces, in association with upregulation of nuclear levels of myeloid-specific transcription factor CCAAT/enhancer binding protein ε. These antileukemic effects of rTM were mediated by thrombin/activated protein C-dependent mechanisms, as hirudin, an inhibitor of thrombin and a blocking antibody against endothelial receptor for protein C to which activated protein C binds, hampered the ability of rTM to induce expression of CD11b in NB4 cells. This study also found that rTM downregulated expression of Annexin II, a receptor for both plasminogen and tissue plasminogen activator, and inhibited plasmin activity in APL cells. Interestingly, rTM significantly enhanced the ability of all-trans retinoic acid to induce growth arrest, differentiation and apoptosis, and inhibited plasmin activity in APL cells. Taken together, these results suggest that administration of rTM should be considered for treatment of individuals with disseminated intravascular coagulation associated with APL.

Promoter methylation of the bone morphogenetic protein 6 gene in multiple myeloma.

Bone morphogenetic proteins (BMPs), which belong to the transforming growth factor-ß superfamily, are multifunctional signaling molecules that have become of increasing interest in cancer research. Recent observations suggest that alterations in BMPs and BMP signaling are associated with tumorigenesis and disease progression in various types of malignancies. This study investigated the methylation status of the BMP6 gene promoter in various types of plasma cell proliferative disorders by combined bisulfite restriction analysis. While BMP6 methylation was not detected in any samples from monoclonal gammopathies of undetermined significance, intramedullary multiple myeloma (MM), plasma cell leukemia or solitary plasmacytoma, both case studies and cell line studies showed that multiple extramedullary plasmacytoma (MEP) consistently carried a methylated BMP6 promoter. The BMP6 methylation-positive MEP was an aggressive form of MM with extremely high levels of serum lactate dehydrogenase (LDH). Bisulfite sequencing analysis confirmed intensive methylation at CpG sites of the BMP6 promoter region. The methylation of BMP6 was correlated with decreased levels of mRNA transcripts. Expression of BMP6 was restored by the demethylating agent 5-aza-2'-deoxycytidine, suggesting that the methylation is associated with transcriptional silencing. Our study implied that BMP6 promoter methylation is not a common event in MMs, but occurs in aggressive MEP. These findings warrant further investigation to clarify whether BMP6 methylation together with elevated LDH could be a marker of poor prognosis in MEP patients who should be considered for early intensive treatment.

Epstein-Barr virus-infected subcutaneous panniculitis-like T-cell lymphoma associated with methotrexate treatment.

Methotrexate-associated lymphoproliferative disorder (MTX-LPD) is a lymphoid proliferation or lymphoma in patients treated with MTX. We report a case of Epstein-Barr virus (EBV)-positive subcutaneous panniculitis-like T-cell lymphoma (SPTCL) in a 60-year-old Japanese man treated with MTX for rheumatoid arthritis (RA). SPTCL is a rare cytotoxic T-cell lymphoma characterized by involvement of subcutaneous fat mimicking panniculitis. The patient, who had been on MTX therapy for RA, manifested high fever and lumbago. Physical examination showed multiple subcutaneous nodules on the trunk including axillary and inguinal regions. Biopsy of the inguinal nodule showed profuse infiltration of CD8(+) T-cell lymphoma cells in the subcutaneous adipose tissues. A diagnosis of SPTCL was made according to the diagnostic criteria of World Health Organization classification. EBV-encoded small RNA in situ hybridization revealed that the lymphoma cells contained EBV genome. The cells were positive for EBV latent membrane protein 1, but not for EBNA2. After discontinuation of MTX, the nodules regressed spontaneously. Studies have reported that most MTX-LPDs are B-cell type lymphomas and Hodgkin lymphoma. To the best of our knowledge, EBV-positive SPTCL has not been reported in patients receiving MTX. Our case emphasizes the importance of clinical and virological characterization of MTX-associated SPTCL.

Promoter methylation of the bone morphogenetic protein-6 gene in association with adult T-cell leukemia.

Bone morphogenetic proteins (BMP), belonging to the transforming growth factor-beta superfamily, are multifunctional regulators of cell proliferation, differentiation and apoptosis in various types of malignant cells. In this study, we investigated BMP-6 promoter methylation in patients with various types of leukemias. The BMP-6 methylation was found preferentially in adult T-cell leukemia (ATL) (49 of 60, 82%) compared with other types of leukemias studied including acute myeloid leukemia (3 of 67, 5%), acute lymphoblastic leukemia (6 of 38, 16%) and chronic lymphocytic leukemia (1 of 21, 5%). Among subtypes of ATL, the BMP-6 gene was more frequently methylated in aggressive ATL forms of acute (96%) and lymphoma (94%) types than less malignant chronic ATL (44%) and smoldering ATL (20%). We also analyzed the methylation status of peripheral blood mononuclear cells from healthy donors and nonmalignant lymph nodes with reactive lymphadenopathy, none of which showed detectable BMP-6 methylation in this study. The BMP-6 methyaltion was correlated with decreased mRNA transcript and protein expression. Expression of BMP-6 was restored by the demethylating agent 5-aza-2'-deoxycytidine, suggesting that methylation was associated with the transcriptional silencing. Serial analysis demonstrated an increasing methylation of CpG sites in the BMP-6 promoter and the resultant suppression of BMP-6 expression as ATL progressed. These findings suggested that BMP-6 promoter methylation is likely to be a common epigenetic event at later stages of ATL and that the methylation profiles may be useful for the staging of ATL as well as for evaluation of the individual risk of developing the disease.

Blockade of MEK/ERK signaling enhances sunitinib-induced growth inhibition and apoptosis of leukemia cells possessing activating mutations of the FLT3 gene.

The FMS-like tyrosine kinase 3 (FLT3) is a cell surface receptor tyrosine kinase. Activating mutations of this gene occur in nearly 30% of acute myelogenous leukemia (AML) patients. These mutations, in part, result in activation of mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways. In this study, we found that AZD6244 (ARRY-142886), a novel inhibitor of MEK1/2 kinases, effectively inhibited the proliferation of acute biphenotypic leukemia MV4-11 and acute monocytic leukemia MOLM13 cells. The concentrations that inhibited 50% growth were approximately 0.3 and 1.2 microM, respectively, as measured by thymidine uptake on day 2 of culture. AZD6244 potently down-regulated the levels of phospho-ERK1/2 and its downstream effector, p-p70S6K, in the MV4-11 and MOLM13 cells as measured by Western blot analysis. Interestingly, when AZD6244 was combined with sunitinib, a FLT3 kinase inhibitor, growth inhibition and apoptosis of both MV4-11 and MOLM13 cells were synergistically enhanced in association with further down-regulation of phospho-ERK1/2 and p-p70S6K in these cells. Taken together, concomitant blockade of FLT3 and MEK signaling represents a promising treatment strategy for individuals with leukemia who possess activating mutations of FLT3.

Histone deacetylase inhibitors induce growth arrest and apoptosis of HTLV-1-infected T-cells via blockade of signaling by nuclear factor kappaB.

Adult T-cell leukemia/lymphoma (ATL) is a highly aggressive disease with a poor prognosis in which nuclear factor kappa B (NF-kappaB) is thought to play a role. This study explored the effects of histone deacetylase inhibitors (HDACIs) MS-275, suberoylanilide hydroxamic acid (SAHA), and LBH589 on both human T-cell lymphotropic virus type I (HTLV-1)-infected T cells (MT-1, -2, -4, and HUT102) and freshly isolated ATL cells harvested from patients. HDACIs effectively inhibited the proliferation of these cells. For example, MS-275, SAHA, and LBH589 effectively inhibited the proliferation of MT-1 cells with ED(50s) of 6microM, 2.5microM, and 100nM, respectively, as measured by 3-(4,5-dimethylithiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay on day 2 of culture. In addition, HDACIs induced cell cycle arrest at the G2/M phase and apoptosis of HTLV-1-infected T-cells in conjunction with regulation of apoptosis-related proteins. Electrophoretic mobility shift assay showed that exposure of HTLV-1-infected T-cells to HDACIs for 48h inhibited formation of the NF-kappaB/DNA binding complex. Moreover, we found that HDACIs accumulated NF-kappaB and inhibitory subunit of NF-kappaB in the cytoplasm in conjunction with the down-regulation of NF-kappaB in the nucleus, suggesting that HDACIs blocked nuclear translocation of NF-kappaB. Based on these findings, we believe HDACIs can be useful for treating patients with ATL or other types of cancer in which NF-kappaB plays a role.

A novel treatment strategy targeting Aurora kinases in acute myelogenous leukemia.

The Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. Aberrant expression of these kinases occurs in solid tumors and is associated with aneuploidy and carcinogenesis. We found in this study that Aurora kinase A and B were aberrantly expressed in a variety of types of human leukemia cell lines (n = 15, e.g., PALL-1, PALL-2, HL-60, NB4, MV4-11, etc.), as well as freshly isolated leukemia cells from individuals with acute myelogenous leukemia (n = 44) compared with bone marrow mononuclear cells from healthy volunteers (n = 11), as measured by real-time PCR. ZM447439 is a novel selective Aurora kinase inhibitor. The compound induced growth inhibition, caused accumulation of cells with 4N/8N DNA content, and mediated apoptosis of human leukemia cells as measured by thymidine uptake, cell cycle analysis, and annexin V staining, respectively. Especially profound growth inhibition occurred with the PALL-1 and PALL-2 cells, which possess wild-type p53 gene. In contrast, ZM447439 did not inhibit clonogenic growth of myeloid committed stem cells harvested from healthy normal volunteers. Taken together, inhibition of Aurora kinases may be a promising treatment strategy for individuals with leukemia.