Local increase in IgE and class switch recombination to IgE in nasal polyps in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis with nasal polyps is generally characterized by local Th2 inflammation and is categorized into two subtypes in Japan: eosinophilic chronic rhinosinusitis (similar to chronic rhinosinusitis with nasal polyps in western countries) and non-eosinophilic chronic rhinosinusitis (characterized by Th1-dominant inflammation). OBJECTIVE: To investigate local IgE production and class switch recombination to IgE in these two subtypes of chronic rhinosinusitis with nasal polyps. METHODS: The identity of IgE-positive cells was determined using double-immunofluorescent staining for IgE and cell-type-specific molecular markers. To investigate the local class switch recombination to IgE and IgE synthesis in the mucosa, we performed real-time polymerase chain reaction to examine the mRNA expression of Th2 cytokines and class-switch-related molecules, including IL-4, IL-5, IL-13, ε germline gene transcripts, IgE mature transcript, IgG mature transcript, RAG1, RAG2 and activation-induced cytidine deaminase in eosinophilic polyps, non-eosinophilic polyps and controls. RESULTS: The concentrations of total IgE and number of IgE-positive cells were significantly higher in the eosinophilic polyps compared with control and non-eosinophilic polyps. IgE-positive cells were predominantly mast cells in eosinophilic polyps and significantly correlated with the number of FcεR1-positive cells in the subepithelial layer. IL-5 and IL-13 mRNA and ε germline gene transcripts expression levels were significantly higher in eosinophilic polyps compared with control and non-eosinophilic polyps. In contrast, the number of plasma cells and the expression of IgG mature transcripts were increased in non-eosinophilic polyps compared with eosinophilic polyps. RAG2 mRNA was significantly increased in both eosinophilic and non-eosinophilic polyps compared with control mucosa. CONCLUSION AND CLINICAL RELEVANCE: The current study suggests local class switching to IgE, production of IgE and IgE localization to the surface of mast cells in eosinophilic chronic rhinosinusitis in the Japanese population. The difference in the IgE-related profiles between eosinophilic chronic rhinosinusitis and non-eosinophilic chronic rhinosinusitis suggests heterogeneity in the pathogenesis of chronic rhinosinusitis with nasal polyps.

Tumour necrosis factor inhibitor-associated sinusitis.

AIM: To describe the features of chronic sinusitis associated with the use of tumour necrosis factor (TNF) inhibitors. METHODOLOGY: A retrospective review of the medical records between 2003 and 2011 revealed that five patients had developed chronic sinusitis after the start of TNF inhibitor administration and required rhinological evaluation and treatment. RESULTS: The incidence of refractory sinusitis associated with TNF inhibitors was approximately 2%. Of the five patients identified, four patients were medicated with etanercept and one with infliximab. The maxillary sinus was most commonly involved and cultures of the sinus discharge revealed Pseudomonas aeruginosa in three cases. Two patients showed improvement of sinusitis with antibiotic medication, despite the continuous use of TNF inhibitor, while in two other patients, sinusitis was resistant to antibiotic medication. Another patient who had developed recurrence of sinusitis after complete remission of previous chronic sinusitis by endoscopic sinus surgery showed remission only after cessation of TNF inhibitor. CONCLUSION: Chronic sinusitis associated with TNF inhibitors is considered to be a new disease entity, and it will become more common due to the increasing use of TNF inhibitors.

Topical application of the antiapoptotic TAT-FNK protein prevents aminoglycoside-induced ototoxicity.

We previously demonstrated that an artificial protein, TAT-FNK, has antiapoptotic effects against cochlear hair cell (HC) damage caused by ototoxic agents when applied systemically. To examine the feasibility of topical protein therapy for inner ear disorders, we investigated whether gelatin sponge soaked with TAT-FNK and placed on the guinea pig round window membrane (RWM) could deliver the protein to the cochlea and attenuate aminoglycoside (AG)-induced cochlear damage in vivo. First, we found that the immunoreactivity of TAT-myc-FNK was distributed throughout the cochlea. The immunoreactivity was observed from 1-24 h after application. When Tat-FNK was applied 1 h before ototoxic insult (a combination of kanamycin sulfate and ethacrynic acid), auditory brainstem response threshold shifts and the extent of HC death were significantly attenuated. When cochlear organotypic cultures prepared from P5 rats were treated with kanamycin, TAT-FNK significantly reduced the extent of caspase-9 activation and HC death. These findings indicate that TAT-FNK topically applied on the RWM can enter the cochlea by diffusion and effectively prevent AG-induced apoptosis of cochlear HCs by suppressing the mitochondrial caspase-9 pathway.

Visceral obesity is associated with the metabolic syndrome and elevated plasma retinol binding protein-4 level in obstructive sleep apnea syndrome.

Obstructive sleep apnea syndrome (OSAS) is related to the increased prevalence of cardiovascular disease and metabolic syndrome (MS). A novel adipokine, retinol binding protein-4 (RBP4), was reported to be associated with insulin resistance and the prevalence of type 2 diabetes. To examine whether plasma RBP4 is associated with insulin resistance and MS development in OSAS, we measured plasma RBP4 levels in 181 Japanese men (24 healthy controls and 40 mild, 64 moderate, and 53 severe OSAS) of whom 26 had mild glucose intolerance with HbA1c < or = 6.0%. After a full polysomnography, blood was collected between 06:00 and 07:00 AM. Plasma RBP4 levels in moderate/severe OSAS patients were higher than in control subjects. Plasma RBP4 was not correlated with apnea variables, HOMA-IR, or blood pressure. However, it was positively correlated with visceral fat areas and plasma triglyceride levels. The prevalence of MS was higher in severe OSAS patients than in mild/moderate OSAS and control subjects. Plasma RBP4 was higher in OSAS patients with MS than in those without MS. This study indicates that plasma RBP4 is associated with dyslipidemia, but not with insulin resistance, glucose intolerance, or hypertension in patients with OSAS. Visceral obesity may play key roles in increasing the plasma RBP4 level and MS development in OSAS.

Activating mutations in NOTCH1 in acute myeloid leukemia and lineage switch leukemias.

Activating mutations in NOTCH1 are found in over 50% of human T-cell lymphoblastic leukemias (T-ALLs). Here, we report the analysis for activating NOTCH1 mutations in a large number of acute myeloid leukemia (AML) primary samples and cell lines. We found activating mutations in NOTCH1 in a single M0 primary AML sample, in three (ML1, ML2 and CTV-1) out of 23 AML cell lines and in the diagnostic (myeloid) and relapsed (T-lymphoid) clones in a patient with lineage switch leukemia. Importantly, the ML1 and ML2 AML cell lines are derived from an AML relapse in a patient initially diagnosed with T-ALL. Overall, these results demonstrate that activating mutations in NOTCH1 are mostly restricted to T-ALL and are rare in AMLs. The presence of NOTCH1 mutations in myeloid and T-lymphoid clones in lineage switch leukemias establishes the common clonal origin of the diagnostic and relapse blast populations and suggests a stem cell origin of NOTCH1 mutations during the molecular pathogenesis of these tumors.

Characterization of a patient with glycoprotein (GP) VI deficiency possessing neither anti-GPVI autoantibody nor genetic aberration.

BACKGROUND: There have been only seven reported cases of glycoprotein (GP) VI deficiency. However, the pathogenesis of this disorder has not been well-elucidated. OBJECTIVES: We characterized a novel patient with GPVI deficiency and used these platelets to investigate the role of GPVI in normal hemostasis. PATIENT: A 31-year-old female with immune thrombocytopenic purpura who had been suffering from mild bleeding diathesis even after recovery from thrombocytopenia. RESULTS AND CONCLUSION: The patient's platelets did not aggregate in response to either convulxin or collagen-related peptide. Immunoblotting revealed complete absence of the GPVI molecule, whereas a significantly reduced but substantial amount of Fc receptor (FcR) gamma-chain was expressed. Platelet stimulation with convulxin did not induce tyrosine-phosphorylation of FcR gamma-chain, indicating a defect in GPVI-mediated signaling. Concerning the underlying pathogenesis, we found normal level of GPVI-mRNA expression, no aberration of the sequence of the entire coding region of GPVI, and presence of degraded GPVI in her plasma. However, no anti-GPVI autoantibody was detected either by the binding assay to GPVI-Fc2 fusion protein or by immunoblotting/immunoprecipitation using the patient's immunoglobulin. We thus consider that either a short-time exposure to anti-GPVI autoantibody or a continuous exposure to low titers of the autoantibody has resulted in persistent GPVI deficiency. Under high shear flow, the patient's platelets could not form large aggregates, although initial platelet attachment was obviously observed. These results suggest that GPVI deficiency in this patient resulted in defective platelet thrombi development, manifesting as bleeding diathesis. Furthermore, our observations indicate that coordination of GPVI with integrin alpha2beta1 is essential for physiological platelet thrombus formation.

EVI-1 zinc finger protein works as a transcriptional activator via binding to a consensus sequence of GACAAGATAAGATAAN1-28 CTCATCTTC.

Previously, the DNA-binding consensus sequences for domains 1 (GACAAGATAAGATAA) and 2 (GAAGATGAG) of the EVI-1 protein were identified using GST fusion proteins of each domain in binding and amplification reactions. We have utilized full-length EVI-1 protein to confirm these consensus sequences and determine the spacial and orientation requirements for binding. Our data demonstrate that full-length EVI-1 can independently bind the consensus sequences in gel mobility shift assays. In binding and amplification reactions only the domain 1 consensus sequence (D1-CONS) was obtained with full-length EVI-1 protein. However, by using constructs in which D1-CONS was anchored, products were obtained in which the domain 2 consensus sequence (D2-CONS) was observed with the spacing and orientation of GACAAGATAATATAAN1-28 CTCATCTTC. Using this consensus sequence we show that EVI-1 can activate transcription from reporter constructs. No transcriptional activation was seen with the reporter construct containing D1-CONS alone while activation was seen with the construct-containing D2-CONS alone. These results indicate that the EVI-1 protein works as a transcriptional activator and the binding of the domain 2 with D2-CONS is essential for its activation.

Fusion of the MLL gene with two different genes, AF-6 and AF-5alpha, by a complex translocation involving chromosomes 5, 6, 8 and 11 in infant leukemia.

We analysed a complex translocation involving chromosomes 5, 6, 8 and 11 in a case of infant leukemia. Molecular analysis of the MLL gene revealed that MLL was fused with two different genes, AF-6 on chromosome 6q27 and AF-5alpha. AF-5alpha, the 11th partner gene fused with MLL, is a novel gene mapped to chromosome 5q12, which encodes a 31 kDa protein of 269 amino acids and contains a possible nuclear targeting sequence, a potential leucine zipper dimerization motif and an alpha-helical coiled-coil domain. In situ hybridization and molecular cloning analyses demonstrated that two different types of chromosomal recombination had occurred in the cells. One was a three-way translocation among chromosomes 6, 8 and 11, and the other was an insertion of a chromosome 5-derived segment into the breakpoint of chromosomes 8 and 11. Accordingly, the karyotype was defined as del(5)(q11.2q12), der(6)t(6;8) (q27;q11.2), der(8)(8pter-->8q11.2::5q11.2-->5q12::11q23-->++ +11qter), der(11)t(6;11) (q27;q23). Thus, the MLL gene created two different fusion mRNAs, since the chromosome 11 split into two different chromosomes 5 and 6. This is the first report demonstrating fusion of the MLL gene with two different genes by a complex translocation.

The human thrombopoietin gene is located on chromosome 3q26.33-q27, but is not transcriptionally activated in leukemia cells with 3q21 and 3q26 abnormalities (3q21q26 syndrome).

We previously demonstrated that the EVI-1 gene was transcriptionally activated in the 3q21q26 syndrome and chromosomal breakpoints at 3q26 were clustered within 400 Kb of the EVI-1 gene. Since thrombocytosis is often observed in the 3q21q26 syndrome, we first mapped the thrombopoietin (TPO) gene and then we examined for transcriptional activation and chromosomal rearrangement of the TPO gene in four cases of the 3q21q26 syndrome. The TPO gene was assigned to chromosome 3q26.33-q27 by fluorescence in situ hybridization analysis. Although the TPO gene was mapped to the same locus as the EVI-1 gene, the distance between the TPO gene and the EVI-1 gene at 3q26 was more than 600 Kb and no gross chromosomal rearrangements of the TPO gene were detected by Southern blot analysis and pulsed field gel electrophoresis (PFGE) analysis. TPO transcripts were not detected in these leukemia cells by Northern blot analysis. These results indicate that activation of the TPO gene is not the main cause of thrombocytosis in the 3q21q26 syndrome.

Establishment of a new natural killer (NK) cell line, TKS-1, from a patient with aggressive type of large granular lymphocyte (LGL) leukemia.

A new NK cell line, designated as TKS-1, was established from the peripheral blood of a patient with large granular lymphocyte (LGL) leukemia which showed highly aggressive clinical course. The original leukemic cells, which had a surface phenotype of CD2+, CD3-, CD16-, and CD56+, proliferated well in the presence of 200 U/ml of interleukin-2 (IL-2). Morphologically, TKS-1 cells had immature nuclei and abundant cytoplasm with fine azulophilic granules. Surface phenotype of the cell line was CD2+, CD3-, CD16-, CD57-, and CD56-, CD56 antigen disappearing during the culture. Analyses of T-cell receptor (TCR) beta- and gamma-chain genes showed germ line configurations. Functionally, TKS-1 cells showed significant cytotoxicity against K562, Raji, and Daudi target cells. We consider that TKS-1 cells originated from CD16-negative immature type of peripheral blood NK cells. This cell line will be useful not only for the investigation of NK cell leukemia, but also for that of the mechanism of cytotoxicity mediated by normal NK cells.

Activation of EVI1 transcripts with chromosomal translocation joining the TCRVbeta locus and the EVI1 gene in human acute undifferentiated leukemia cell line (Kasumi-3) with a complex translocation of der(3)t(3;7;8).

A cell line (Kasumi-3) established from acute myeloid leukemia (AML-M0) had unique phenotypes of undifferentiated leukemia cells with expression of both T cell and myeloid antigens. Kasumi-3 cells with t(3;7)(q26;q22) highly expressed a 6 kb transcript of EVI1, which is located on chromosome 3q26. Therefore, we further characterized the chromosomal breakpoint by pulsed-field gel electrophoresis near EVI1. We identified and isolated the chromosomal breakpoint at approximately 80 kb upstream from the 5' end of EVI1. Sequence analysis of the breakpoint revealed that the whole Vbeta region from T cell receptor beta (TCRbeta) at 7q35 was translocated to the upstream of EVI1. A 1.0 kb TCRbeta transcript was expressed in the Kasumi-3 cells, suggesting that TCRbeta rearrangement occurred as Dbeta-Jbeta joining events. Fluorescence in situ hybridization analysis revealed that the inverted chromosome 7q22-q35 segment between TCRbeta and the region proximal to the erythropoietin gene at 7q22 was translocated to the region distal to EVI1 in der(3). Since the telomeric region of chromosome 8 q was also translocated to the inverted chromosome 7q22-q35 segment in der(3), the chromosomal abnormalities of der(3) were defined as being der(3)t(3;7;8)(3pter-3q26::7q35-7q22::8q22 -8qter). It is suggested that a translocated enhancer element in the TCRbeta locus and/or loss of a negative regulatory element near EVI1 might function to enhance the EVI1 expression. Therefore, the enhanced EVI1 expression may contribute to the development of a subset of undifferentiated leukemia.

EVI1 expression associated with a 3q26 anomaly in a leukemia cell line derived from the blast crisis of chronic myeloid leukemia.

Two leukemia cell lines, TS9;22 and YS9;22, were established from different individuals with Philadelphia chromosome (Ph)-positive chronic myeloid leukemia in blast crisis. The reverse transcript-polymerase chain reaction (RT-PCR) technique revealed that both cell lines expressed GATA-1, GATA-2, and the stem cell leukemia (SCL) gene, consistent with a megakaryocyte lineage. Chromosome analysis revealed that TS9;22 cells show the Ph translocation without abnormality of chromosome 3. In contrast, YS9;22 cells show the Ph translocation and dic(3)(q26;p12). Northern analysis revealed that YS9;22 cells express the EVI1 (ecotropic virus integration-1) gene, possibly because of the chromosomal translocation in the 3q26 region; TS9;22 cells do not express EVI1. However, no rearrangements were detected over 600 kb upstream or over 900 kb downstream of EVI1 in the YS9;22 cell line, suggesting a different mechanism of EVI1 activation from that in leukemia cells with either a t(3;3)(q21;q26) or inv(3)(q21q26). These results indicate that EVI1 expression in YS9;22 cells is linked to the 3q26 abnormality and that EVI1 activation plays an oncogenic role in the blastic transformation of chronic myeloid leukemia.

Immature megakaryocytes undergo apoptosis in the absence of thrombopoietin.

We examined withdrawal effects of recombinant mouse Tpo (rm-Tpo) on the apoptosis of mature and immature megakaryocytes in in vitro experiments. Apoptotic megakaryocytes were detected by double staining for acetylcholinesterase and by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method. When the purified mature megakaryocytes were cultured with or without rm-Tpo, the numbers of viable megakaryocytes, apoptotic megakaryocytes, and megakaryocytes with cytoplasmic processes were not significantly different between the two groups. In contrast, purified immature megakaryocytes underwent apoptosis when rm-Tpo was absent from the culture system. Murine bone marrow cells were cultured with rm-Tpo (50 U/mL) on days 1-7 to generate immature megakaryocytes and subsequently were cultured with different concentrations of rm-Tpo (0-50 U/mL) on days 8-14. The number of viable megakaryocytes was decreased and that of apoptotic megakaryocytes was increased by rm-Tpo in a dose-dependent manner. These results indicated a clear relation between the rm-Tpo level and the apoptosis of immature megakaryocytes.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Nasal natural killer cell lymphoma in a post-renal transplant patient.

Posttransplant lymphoproliferative disorders in organ allograft recipients are most commonly of B-cell origin and only occasionally of T-cell origin. We present here a case of nasal natural killer cell lymphoma associated with Epstein-Barr virus that occurred in a recipient of a renal transplant 4 years posttransplantation. Immunohistochemically, the lymphoma cells showed CD2-, surface CD3-, cytoplasmic CD3E+, CD56+, CD57-, CD16-, and CD43+ phenotype. Analyses of T-cell receptor beta and gamma genes showed germ line configurations. EBER-1 was detectable in the lymphoma cells. The patient was diagnosed as having natural killer cell lymphoma and was treated with six courses of combination chemotherapy for non-Hodgkin's lymphoma He has been in remission for more than 3 years thereafter. To the best of our knowledge, this is the first report of a posttransplant NK cell lymphoma associated with Epstein-Barr virus.

Chorioamnionitis caused by Serratia marcescens in a non-immunocompromised host.

A 26 year old pregnant woman with antithrombin III deficiency developed recurrent septicaemia with Serratia marcescens. In spite of the administration of antibiotics, high grade fever persisted. She subsequently manifested lower abdominal pain, and spontaneous abortion occurred. After the abortion, she became completely afebrile. The amnion was turbid, and microscopic examination of the placenta showed haemorrhage and massive infiltration of neutrophils, suggestive of infectious chorioamnionitis. Pulsed field gel electrophoresis showed that isolates from the blood, urine, and vaginal discharge were genetically identical. Intravenous pyelography revealed that she had a bilateral completed double ureter. It was thought that a urinary tract anomaly caused infection with S marcescens, and the pathogen spread to the chorioamnion via the bloodstream. This is the first report of chorioamnionitis caused by S marcescens in a non-immunocompromised host. In addition, these findings indicate that the chorioamnion can serve as a site for persistent infection in normal pregnancies.

High-dose chemotherapy with peripheral blood stem cell rescue in blastoid natural killer cell lymphoma.

A 25-year-old man was referred because of skin rash, lymphadenopathy and anemia. Laboratory examinations revealed severe anemia (Hb, 4.8 g/dl) and elevated levels of GOT, GPT, LDH and soluble interleukin-2 receptor. Work-up studies disclosed the involvement of lymphoma cells in lymph nodes, skin, bilateral kidneys and bone marrow. Lymph node biopsy revealed diffuse proliferation of medium- to large-sized lymphoblastic cells. Bone marrow aspiration showed massive infiltration of large blastic cells with no cytoplasmic granules. The lymphoma cells in bone marrow and lymph node showed surface CD3-, cytoplasmic CD3epsilon+, CD4+, CD8-, CD56+, CD57-, CD16- and CD43 (MT-1)+ phenotype. Analyses of T cell receptor beta and gamma genes showed germ line configurations. EBER-1 was not detectable in the lymphoma cells. He was diagnosed as having blastoid natural killer (NK) cell lymphoma. In spite of several courses of combination chemotherapy, the lymphoma was progressive. He was then treated with high-dose chemotherapy and peripheral blood stem cell rescue, achieving remission which has now lasted for more than 12 months. We consider that blastoid NK cell lymphoma is an extremely aggressive subtype of CD56-positive lymphomas, and high-dose chemotherapy with peripheral blood stem cell rescue should be included for the choice of the treatment.

Cardiac malignant fibrous histiocytoma metastasizing to the brain: development of multiple neoplastic cerebral aneurysms.

We report a case in which bilateral occipital brain metastases and neoplastic cerebral aneurysms developed from primary cardiac malignant fibrous histiocytoma. The origin of metastases was confirmed at autopsy. The clinical presentation, radiographic features, and autopsy findings are presented along with a histopathologic analysis of the tumor.

Demonstration of the deposition of hemosiderin in the kidneys of patients with paroxysmal nocturnal hemoglobinuria by magnetic resonance imaging.

Hemosiderinuria caused by intravascular hemolysis is a characteristic clinical feature of an acquired hemolytic disorder, paroxysmal nocturnal hemoglobinuria (PNH). We examined the deposition of hemosiderin (iron) in the kidneys of 6 patients with PNH using magnetic resonance imaging (MRI). Three patients with autoimmune hemolytic anemia (AIHA), a hemolytic disorder showing extravascular hemolysis, served as controls. In five of the six patients with PNH, a characteristic T2-weighted MRI of the kidneys, suggesting the deposition of iron (hemosiderin) predominantly in the renal cortex, was obtained. Hemosiderin-deposition was not revealed in the kidneys of any of the patients with AIHA. We conclude that MRI is a sensitive means of detecting hemosiderin deposited in the renal cortex of patients with PNH and that this feature is considerably specific for diseases showing intravascular hemolysis, as represented by PNH.

Evaluation of the transcranial approach to pituitary adenomas based on quantitative analysis of pre- and postoperative visual function.

Over a 16-year period, 105 patients with pituitary adenoma accompanied by visual disturbance underwent transcranial intracapsular removal of the tumor followed by radiotherapy. Postoperative recovery of visual function in these patients was compared with the results obtained in other institutions after trans-sphenoidal surgery. The severity of preoperative visual impairment was correlated with the duration of visual impairment, the degree of optic atrophy, the extent of suprasellar tumor infiltration, and age. Trans-sphenoidal surgery appears more effective in patients with mild preoperative visual disturbance, whereas the transcranial approach yields better results in patients with moderate to severe preoperative visual deficits.