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[Introduction] AKAP13 (Protein Kinase A Anchoring Protein 13) is a guanine nucleotides exchange factor (GEF) that activates RhoA as well as a scaffold protein integrating multiple signaling cascades. Previous study has shown that Akap13 hetero knockout mice develop osteoporotic changes and result in decreased osteogenesis and we believe AKAP13 is involved in the early stages of osteogenesis. On the other hand, the involvement of Wnt signaling in bone metabolism has been extensively studied, however the detailed mechanism has not been elucidated. Recent study showed that focal adhesion kinase (FAK)-integrin contributes to bone formation via Wnt signaling pathway. Thus, it is suggested that Akap13 may influence osteogenesis via Wnt signaling through FAK mediated integrin pathway that is linked to Rho GTPase. In this study, we investigated the involvement of AKAP13 in bone formation and the relationship with Wnt signaling. [Methods] uCT analyses of femurs of Akap13 heterozygous-conventional knock-out mice were performed. We transfected the Akap13 or control siRNA into osteoblastic cells and examined the Wnt-related genes expression and osteogenic markers by real time RT-PCR. Osteogenic marker expression was examined after application of RhoA expression vector with or without Akap13 siRNA to osteoblastic cells. [Results] Mice haploinsufficient for Akap13 (Akap13+/-) displayed reduced bone mineral density resembling the changes observed in osteoporotic bone. Colony forming unit-fibroblast numbers were diminished in Akap13+/- mice. Osteoblast numbers in periosteum and extracellular matrix production in Akap13+/- mice was reduced compared to control littermates. Runx2 and Alp mRNAs were both reduced in femora of Akap13+/- mice. Akap13 knockdown showed a significant decrease of Lef1 mRNA expression in MC3T3-E1 cells as well as a reduction of Runx2 and ALP mRNA expression in immortalized bone marrow stem cells (BMSCs). RhoA overexpression in BMSCs showed Alp mRNA increment in RhoA induction dose dependent manner. Reduction of Alp mRNA by AKAP13 knockdown was amplified in higher RhoA overexpression. [Discussion] The Akap13 knockdown showed decreased expression of Wnt-related genes in osteoblastic cells, suggesting the possibility that AKAP13 plays an important role in regulating osteogenesis through Wnt signaling. AKAP13 may be involved in or contribute directly to Wnt signaling through interaction with RhoA by mechanical stress or stimulation of extracellular matrix.

Adrenal vein (AV) sampling (AVS) is the diagnostic gold standard for primary aldosteronism (PA), but right-sided AVS is difficult. We compared detection of AVs by selective retrograde CT adrenal venography (SRCTAV) with digital subtraction angiography (DSA).Data on 29 subjects (11 males, mean age 55 y) with increased serum aldosterone concentrations (SAC) and a diagnosed right or left aldosterone-producing tumor (APT) by AVS who underwent laparoscopic adrenalectomy were retrospectively analyzed. Before AVS, visualizing AVs was attempted by DSA and SRCTAV (Aquilion). If after the adrenocorticotropic hormone loading test serum cortisol concentration (SCC) from either AV was200 μg/dl, AVS was considered successful. If the SAC/SCC ratio for one side was ≥4 times higher than the other side, we diagnosed a one-sided APT.Left and right AV, respectively, were visualized in 29 (100%) and 22 subjects (76%) by DSA and 29 (100%) and 28 subjects (97%) by SRCTAV, with right-AV detection significantly higher by SRCTAV (p0.05). Cannulations were regarded successful in 28 subjects having both AVs observed on SRCTAV but not in the remaining subject whose adrenocortical scintigram was positive, however. Adrenalectomy was performed with a diagnosis of adenoma. Among 28 subjects with successful AVS, histopathological diagnoses included adenoma (25), nodular hyperplasia (2) and normal (1). After adrenalectomy, antihypertensive drug usage in 28 patients was reduced or stopped with decreases in SAC (97%).Detection of AV was significantly higher by SRCTAV than DSA, especially on the right side, in 29 subjects diagnosed with one-sided APT.

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

Deficiency of Smad3, an intracellular mediator of TGF-β, was shown to significantly accelerate re-epithelialization of the colonic mucosa. This study was performed to investigate the molecular mechanisms by which Smad3 controls colonic epithelial cell proliferation and crypt formation. Smad3(ex8/ex8) C57BL/6 mice were used in this study and wild-type littermates served as controls. The number of proliferating cells in the isolated colonic epithelium of Smad3(-/-) mice was significantly increased compared to that in wild-type littermates. Protein levels of the cell cycle inhibitors p21 and p27 were significantly decreased, while that of c-Myc was increased in the isolated colonic epithelium from Smad3(-/-) mice. In the colonic tissue of wild-type mice, cell proliferation was restricted to the bottom of the crypts in accordance with nuclear β-catenin staining, whereas proliferating cells were located throughout the crypts in Smad3(-/-) mice in accordance with nuclear β-catenin staining, suggesting that Smad3 is essential for locating proliferating cells at the bottom of the colonic crypts. Notably, in Smad3(-/-) mice, there was loss of EphB2 and EphB3 receptor protein expression, critical regulators of proliferating cell positioning, while EphB receptor protein expression was confirmed at the bottom of the colonic crypts in wild-type mice. These observations indicated that disturbance of the EphB/ephrin B system brings about mispositioning of proliferating cells in the colonic crypts of Smad3(-/-) mice. In conclusion, Smad3 is essential for controlling number and positioning of proliferating cells in the colonic crypts and contributes to formation of a "proliferative zone" at the bottom of colonic crypts in the normal colon.

Epidemiological studies have shown that the incidence of coronary artery disease increases as the LDL- C, TC1-9), and TG10, 11) levels rise and the HDL-C level lowers5, 8, 12) both in Japan and in Western countries (Fig.1). At present, prevalence of coronary artery disease in Japan is much lower than that in Western countries13-16). However, recent increases in the LDL-C and TC levels in Japanese associated with so-called Westernization of diet implies future increases in coronary artery disease. In this guideline, therefore, criteria for the diagnosis of dyslipidemia were defined as in Table 1, with a greater emphasis on the prevention of coronary artery disease.The first step in this diagnostic procedure is to measure TC, TG, and HDL-C levels after overnight fasting. LDL-C level is then calculated by use of the Friedewald equation (LDL-C=TC – HDL-C – TG/5). The LDL-C level may be measured by a homogenous method especially in the case of postprandial examination or when the TG level is 400 mg/dL or higher.

Successful treatment with rituximab and donor lymphocyte infusions for fulminant EBV-associated lymphoproliferative disorder that developed 14 years after unrelated BMT

CD4(+) T cells play a central role in the development of inflammatory bowel disease (IBD) via high-level production of effector cytokines such as IFN-γ and TNF-α. To better characterize the colitogenic CD4(+) T cells, we examined their expression of CXCR6, a chemokine receptor that is expressed by T cells upon activation and is upregulated in several inflammatory diseases. We found that 80% of colonic lamina propria CD4(+) T cells expressed CXCR6 in the CD45RB(high) T cell-transferred colitis model. CXCR6 expression was similarly upregulated in inflamed mucosa of patients with Crohn's disease. Although surface marker analysis demonstrated that both CXCR6(+) and CXCR6(-) CD4(+) T-cell subsets consist of the cells with effector and effector-memory cells, the more cells in the CXCR6(+) subset produced IFN-γ and TNF-α compared to CXCR6(-) subset, and only the CXCR6(+) subset produced IL-17A. Nevertheless, adoptive retransfer of lamina propria CXCR6(+) T cells into Rag1 (-/-) recipients failed to induce the disease due to limited expansion of the transferred cells. By contrast, retransfer of CXCR6(-) cells evoked colitis similar to that observed in CD4(+)CD45RB(high) T cell-transferred mice, and resulted in their conversion into CXCR6(+) cells. Collectively, these observations suggest that the CXCR6(+)CD4(+) T-cell subset consists of terminally differentiated effector cells that serve as the major source of effector cytokines in the inflamed tissue, whereas CXCR6(-)CD4(+) T-cell subset serves as a colitogenic memory compartment that retains the ability to proliferate and differentiate into CXCR6(+)CD4(+) T cells.

Low-dose trimethoprim–sulfamethoxazole for Pneumocystis jiroveci pneumonia prophylaxis after allogeneic hematopoietic SCT

Abstract 2673 Introduction: Follicular Lymphoma International Prognostic Index 2 (FLIPI-2) is a widely accepted tool for risk assessment of follicular lymphoma (FL), which is based on age, hemoglobin level, presence of bone marrow (BM) invasion, tumor size, and b2-microgloblin levels. Although it is easy to evaluate in clinical practice, it is a combination of tumor burden and patient physical condition, and a simple and powerful biomarker reflecting the tumor burden and its character is still not established. LR11 (also called SorLA or SORL1) was identified and characterized as a regulator of uPAR function through complex formation with uPAR. We have identified that serum soluble LR11 (sLR11) levels are significantly elevated in patients with acute leukemia and B cell lymphomas, and are associated with tumor burden and BM invasion (Sakai et al 2012). We have also found that high sLR11 levels had a significant negative prognostic impact on progression-free survival (PFS) in FL. Therefore, we have retrospectively evaluated the clinical characteristics of sLR11 and its prognostic impact on FL, in a larger patient cohort. Patients and Methods: Sixty-one patients with FL treated at Chiba University Hospital and affiliated hospitals from 2002 to 2012 were evaluated. The majority of patients were treated by the R-CHOP regimen (rituximab 375 mg/m2 on day 1; cyclophosphamide, 750 mg/m2 on day 1; adriamycin, 50 mg/m2 on day 1; vincristine, 1.4 mg/m2 on day 1; and prednisolone, 100 mg/body on day 1–5). Serum sLR11 levels were measured by ELISA method. Patient laboratory data and treatment outcome were obtained retrospectively. Results: Serum sLR11 levels of patients with lymphoma were significantly increased (mean ± SD: 19.4 ± 17.1 ng/ml) compared with those of normal control subjects (8.8 ± 1.79 ng/ml, P15.4 ng/ml at diagnosis compared with those with ≤15.4 ng/ml (Figure 1, 9.7 % vs 100 %, P15.4 ng/ml at diagnosis was a significant prognostic factor for PFS (hazard ratio: 2.65 × 107, 95% CI: NA). Conclusions: Serum sLR11 levels in FL patients were associated with BM invasion, lower Hb levels and elevated b2-microglobulin, which are 3 of 5 variables in FLIPI-2. This suggests that serum sLR11 is a useful tool to predict “high” FLIPI-2 score, especially highlighting those with high tumor burden by simple serum evaluation and can be a promising biomarker in patients with FL. Disclosures: No relevant conflicts of interest to declare.

Abstract 2416 TET2 mutations are frequently observed in myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN. Several groups have already reported that deletion of Tet2 in mice leads to dysregulated hematopoietic stem cell self-renewal and subsequent development of myeloid malignancies. Of note, mice hypomorphic or heterozygous for the Tet2 allele have been reported to show similar phenotypes as those of Tet2-null mice, suggesting that haploinsufficiency of Tet2 plays a role in the development of myeloid malignancies. However, little is known about long-term cell autonomous effects of Tet2 loss in hematopoietic cells: most of the reports were based on relatively short-term observations or did not exclude the influence of Tet2 loss in the niche cells. To study long-term cell autonomous effect of Tet2 loss in hematopoietic cells, we analyzed the hematopoiesis of wild-type recipient mice reconstituted with fetal liver cells from Tet2 hypomorphic mice for a longer period up to 1 year. Tet2 gene trap mice (Tet2trap/trap), in which the gene trap vector was inserted into the exon 2 of Tet2 just before the first coding exon, express Tet2 mRNA at the level approximately 20% of that of the wild-type (WT) mice (Shide et al. Leukemia 2012). We transplanted fetal liver cells from E14.5 WT or Tet2trap/trap mice into lethally irradiated recipient mice. At 4 months after transplantation, the recipient mice reconstituted with Tet2trap/trap cells showed a significantly increased proportion of monocytes in peripheral blood (PB) compared with those with WT cells (WT=5.59±2.57%, Tet2trap/trap=12.67±7.45%, p=0.01). While there were no significant differences between the two groups in the bone marrow (BM) compartments including the numbers of Lineage−Sca-1+c-Kit+ (LSK) hematopoietic stem/progenitor cells, extramedullary hematopoiesis in the spleen was markedly enhanced in the recipients with Tet2trap/trap cells. The proportion of LSK, granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythroid progenitors (MEPs) in the spleen of recipient mice reconstituted with WT and Tet2trap/trap cells were 0.002±0.001% vs 0.006±0.001% (p Of note, after a long observation period, particularly after 9 months post-transplantation, mice reconstituted with Tet2trap/trap cells developed advanced hematological disease and 53.3% (8 of 15) died or were killed because of their moribund condition by 11 months after transplantation. Detailed analysis on moribund as well as surviving mice reconstituted with Tet2trap/trap cells (n=3 each) revealed that the 2/3 of the mice developed CMML-like disease with monocytosis in PB and evident extramedullary hematopoiesis harboring increased number of LSK and GMPs in spleen, whereas the remaining 1/3 of mice developed MDS/MPN-like disease with severe anemia. The latter mice did not show monocytosis in PB and BM, but displayed dyserythropoiesis accompanied by massive extramedullary erythropoiesis, as we saw increased number of LSK and MEPs, but not GMPs, in spleen. The proportion of Annexin V+ cells in Ter119highCD71higherythroblasts in the BM of WT mice, CMML-like disease-carrying mice and MDS/MPN-like disease-carrying mice were 9.46±0.53%, 10.71±0.36%, and 19.04±0%, respectively, suggesting that the enhanced apoptosis led to the severe anemia seen in the MDS/MPN-like disease-carrying mice. Thus, decreased expression of Tet2 is sufficient to promote not only CMML-like disease, but also an MDS/MPN-like disease as well. Our findings confirmed a long-term cell autonomous effect from insufficient function of Tet2 in the development of hematological diseases. Interested in the molecular mechanism of disease progression, we have identified 1,642 differentially methylated regions (DMRs) in Tet2trap/trap GMPs compared with WT GMPs by ChIP-sequencing. We are now working to understand how these DMRs are involved in the development of the two distinct diseases associated with hypomorphic Tet2. Disclosures: No relevant conflicts of interest to declare.

Abstract 5104 Introduction: Intravascular large B-cell lymphoma (IVLBCL) is a rare disease entity of malignant lymphoma, characterized by the selective growth of lymphoma cells within the lumina of vessels in various organs. Although recent reports showed that prompt and accurate diagnosis lead to better outcomes, absence of typical clinical manifestations and its aggressive behavior frequently makes it difficult. LR11 (also called SorLA or SORL1) is a type I membrane protein, from which a soluble form (sLR11) is released by proteolytic shedding. sLR11 is originally known to be a biomarker of carotid intima-media thickness. We have recently found that LR11 is expressed on human leukemia cell lines, and sLR11 is released in its culture medium. Serum sLR11 levels are significantly elevated in patient serum samples with acute leukemia and B cell lymphomas, and are associated with tumor burden and bone marrow invasion. Based on these findings, we hypothesized that LR11 may be also expressed and released from IVLBCL cells; therefore we evaluated its clinical importance in IVLBCL. Materials and methods: Serum samples and paraffin embedded tumor specimens were obtained from 6 patients who were histologically diagnosed as IVLBCL from 2009 to 2012. Specimens were subjected to immunostaining using anti-LR11 antibody, and serum sLR11 levels were measured by ELISA method. Patient laboratory and clinical data were collected retrospectively. Also, serum samples from 75 healthy volunteers, and 10 patients with collagen diseases presenting similar clinical manifestations as IVLBCL were analyzed. Results: Tissue samples of IVLBCL were obtained by bone marrow biopsy (N=2), transbronchial lung biopsy (N=2), and random skin biopsy (N=2). Biopsy specimens showed that cytoplasm of IVLBCL cells were specifically immunoreacted against the anti-LR11 antibody (Figure 1). Median serum sLR11 level of IVLBCL patients was 86. 0 ng/ml (mean ± SD: 201. 8 ± 260. 0 ng/ml), which was significantly elevated than those of healthy volunteers (median: 8. 4 ng/ml, mean±SD: 8. 8 ± 1. 8 ng/ml, p Conclusions: We have identified LR11 as a novel molecule which was proven to be expressed in IVLBCL cells and circulated in patients' serum. LR11 immunostaining clearly highlights the tumor cells and sLR11 enables to distinguish IVLBCL from other differential diagnosis by serum evaluation. These findings suggest that LR11 is a useful diagnostic tool for IVLBCL, and serum sLR11 is a sensitive biomarker for diagnosis and disease monitoring of this challenging disease. Disclosures: No relevant conflicts of interest to declare.

Mutations of epigenetic regulators are often found in patients with myelodysplastic syndrome (MDS). Furthermore, DNA methylation inhibitors have a therapeutic impact on MDS. However, it remains unknown how altered DNA methylation promotes the development of MDS. We have shown that concurrent depletion of Tet2 and Ezh2 in hematopoietic cells significantly promotes the development of MDS in vivo by utilizing hypomorphic Tet2 (Tet2KD/KD) mice and Ezh2 conditional knockout mice (Cre-ERT;Ezh2fl/fl)(Muto T, et al. J Exp Med 2014). In order to determine how DNA methylation contributes to the formation of MDS in Tet2KD/KDEzh2Δ/Δ mice, we transplanted wild type (WT), Tet2KD/KD, Cre-ERT;Ezh2fl/fl, and Cre-ERT;Tet2KD/KDEzh2fl/fl fetal liver cells in lethally irradiated CD45.1+ recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. We then performed reduced representation bisulfite sequence (RRBS) in Lin-Sca1+Kit+ (LSK) cells isolated from Tet2KD/KD and Ezh2Δ/Δ mice at 3 and 7 months post-deletion and WT and Tet2KD/KDEzh2Δ/Δ mice at 5 months post-deletion. We defined ≥10% difference of methylation in test cells compared with that in WT cells as hyper- or hypo-differentially methylated regions (DMRs) (p-value To determine an association between levels of differential DNA methylation and transcription of genes, we next performed DNA microarray analysis by using LSK cells. As several studies reported, we observed no significant association between levels of DNA methylation and gene expression (R2=0.003). However, 27 hypermethylated genes showed reduced levels of gene expression in Tet2KD/KDEzh2Δ/Δ-MDS LSK cells. Those included key transcription factors and epigenetic modifiers for hematopoiesis, such as Gata2, Gata3, Evi1, and Bcor. Finally, we examined whether decitabine, a demethylating agent, reverses the DNA hypermethylation of Tet2KD/KDEzh2Δ/Δ-MDS LSK cells in vivo. Although the administration of decitabine into secondary recipients of MDS BM cells did not improve the survival of mice, platelet counts were significantly increased in decitabine-treated MDS mice, compared to the control MDS mice. After 4 weeks of treatment, a partial restoration of DNA hypermethylation was observed in the decitabine-treated MDS mice (34 out of 124 CGIs; p Taken together, our work unveiled the biological function of DNA hypermethylation in the pathogenesis of MDS, and will give a clue to understanding how demethylating agents improve the outcome of patients with MDS. Disclosures No relevant conflicts of interest to declare.