Tfl deletion induces extraordinary Cxcl13 secretion and cachexia in VavP-Bcl2 transgenic mice.

STATEMENT OF SIGNIFICANCE: Loss of TFL, found in several types of lymphoma, induces excessive CXCL13 secretion through RNA dysregulation contributing to body weight loss and early death in lymphoma model mice. Follicular lymphoma (FL) is associated with overexpressed BCL-2 and other genetic aberrations, including 6q-. We identified a novel gene on 6q25, "Transformed follicular lymphoma (TFL)," from a transformed FL. TFL regulates several cytokines via mRNA degradation, which has been suggested to underlie resolving inflammation. Fluorescence in situ hybridization revealed a deletion of TFL occurred in 13.6% of various B-cell lymphoma samples. We developed VavP-bcl2 transgenic, TFL deficit mice (Bcl2-Tg/Tfl -/-) to seek how TFL affects disease progression in this lymphoma model. While Bcl2-Tg mice developed lymphadenopathy and died around 50 weeks, Bcl2-Tg/Tfl -/- mice lost body weight around 30 weeks and died about 20 weeks earlier than Bcl2-Tg mice. Furthermore, we found a unique B220-IgM+ cell population in the bone marrow of Bcl2-Tg mice. cDNA array in this population revealed that Cxcl13 mRNA in Bcl2-Tg/Tfl -/- mice expressed significantly higher than Bcl2-Tg mice. In addition, bone marrow extracellular fluid and serum showed an extremely high Cxcl13 concentration in Bcl2-Tg/Tfl -/- mice. Among bone marrow cells, the B220-IgM+ fraction was the main producer of Cxcl13 in culture. A reporter assay demonstrated TFL regulates CXCL-13 via induction of 3'UTR mRNA degradation in B lineage cells. These data suggest Tfl regulates Cxcl13 in B220-IgM+ cells in the bone marrow, and a very high concentration of serum Cxcl13 arising from these cells may contribute to early death in lymphoma-bearing mice. Since several reports have suggested the association of CXCL13 expression with lymphoma, these findings provide new insights into cytokine regulation via TFL in lymphoma.

Mobilization efficiency is critically regulated by fat via marrow PPARδ.

The mobilization efficiency of hematopoietic stem/progenitor cells from bone marrow (BM) to circulation by granulocyte colony-stimulating factor (G-CSF) is dramatically dispersed in humans and mice with no mechanistic lead for poor mobilizers. The regulatory mechanism for mobilization efficiency by dietary fat was assessed in mice. Fat-free diet (FFD) for 2 weeks greatly increased mobilization compared to normal diet (ND). The BM mRNA level of peroxisome proliferator-activated receptor δ (PPARδ), a receptor for lipid mediators, was markedly up-regulated by G-CSF in mice fed with ND and displayed strong positive correlation with widely scattered mobilization efficiency. It was hypothesized that BM fat ligand for PPARδ might inhibit mobilization. The PPARδ agonist inhibited mobilization in mice fed with ND and enhanced mobilization by FFD. Treatment with the PPARδ antagonist and chimeric mice with PPARδ+/- BM showed enhanced mobilization. Immunohistochemical staining and flow cytometry revealed that BM PPARδ expression was enhanced by G-CSF mainly in mature/immature neutrophils. BM lipid mediator analysis revealed that G-CSF treatment and FFD resulted in the exhaustion of ω3-polyunsaturated fatty acids such as eicosapentaenoic acid (EPA). EPA induced the up-regulation of genes downstream of PPARδ, such as carnitine palmitoyltransferase-1α and angiopoietin-like protein 4 (Angptl4), in mature/immature neutrophils in vitro and inhibited enhanced mobilization in mice fed with FFD in vivo. Treatment of wild-type mice with the anti-Angptl4 antibody enhanced mobilization together with BM vascular permeability. Collectively, PPARδ signaling in BM mature/immature neutrophils induced by dietary fatty acids negatively regulates mobilization, at least partially, via Angptl4 production.

FGF-23 from erythroblasts promotes hematopoietic progenitor mobilization.

Fibroblast growth factor 23 (FGF-23) hormone is produced by bone-embedded osteocytes and regulates phosphate homeostasis in kidneys. We found that administration of granulocyte colony-stimulating factor (G-CSF) to mice induced a rapid, substantial increase in FGF-23 messenger RNA in bone marrow (BM) cells. This increase originated mainly from CD45-Ter119+CD71+ erythroblasts. FGF-23 protein in BM extracellular fluid was markedly increased during G-CSF-induced hematopoietic progenitor cell (HPC) mobilization, but remained stable in the blood, with no change in the phosphate level. Consistent with the BM hypoxia induced by G-CSF, low oxygen concentration induced FGF-23 release from human erythroblast HUDEP-2 cells in vitro. The efficient mobilization induced by G-CSF decreased drastically in both FGF-23-/- and chimeric mice with FGF-23 deficiency, only in hematopoietic cells, but increased in osteocyte-specific FGF-23-/- mice. This finding suggests that erythroblast-derived, but not bone-derived, FGF-23 is needed to release HPCs from BM into the circulation. Mechanistically, FGF-23 did not influence CXCL-12 binding to CXCR-4 on progenitors but interfered with their transwell migration toward CXCL-12, which was canceled by FGF receptor inhibitors. These results suggest that BM erythroblasts facilitate G-CSF-induced HPC mobilization via FGF-23 production as an intrinsic suppressor of chemoattraction.

Vitamin D receptor-mediated skewed differentiation of macrophages initiates myelofibrosis and subsequent osteosclerosis.

Myelofibrosis in myeloproliferative neoplasms (MPNs) with mutations such as JAK2V617F is an unfavorable sign for uncontrollable disease progression in the clinic and is complicated with osteosclerosis whose pathogenesis is largely unknown. Because several studies have revealed that macrophages are an indispensable supporter for bone-forming osteoblasts, we speculated that macrophages might play a significant role in the proliferation of collagen-producing myofibroblasts in marrow fibrotic tissues. Here, we show that myelofibrosis critically depends on macrophages whose differentiation is skewed by vitamin D receptor (VDR) signaling. In our novel myelofibrosis model established by transplantation of VDR+/+ hematopoietic stem/progenitor cells into VDR-/- mice, donor-derived F4/80+ macrophages proliferated together with recipient-derived α-smooth muscle actin-positive myofibroblasts, both of which comprised fibrotic tissues with an indistinguishable spindle-shaped morphology. Interfering VDR signals, such as low vitamin D diet and VDR deficiency in donor cells as well as macrophage depletion prevented myelofibrosis in this model. These interventions also ameliorated myelofibrosis in JAK2V617F-driven murine MPNs likely in a transforming growth factor-ß1- or megakaryocyte-independent manner. These results suggest that VDR and macrophages may be novel therapeutic targets for MPNs with myelofibrosis.

G-CSF-induced sympathetic tone provokes fever and primes antimobilizing functions of neutrophils via PGE2.

Granulocyte colony-stimulating factor (G-CSF) is widely used for peripheral blood stem/progenitor mobilization. G-CSF causes low-grade fever that is ameliorated by nonsteroidal anti-inflammatory drugs (NSAIDs), suggesting the activation of arachidonic acid (AA) cascade. How G-CSF regulated this reaction was assessed. G-CSF treatment in mice resulted in fever, which was canceled in prostaglandin E synthase (mPGES-1)-deficient mice. Mobilization efficiency was twice as high in chimeric mice lacking mPGES-1, specifically in hematopoietic cells, suggesting that prostaglandin E2 (PGE2) from hematopoietic cells modulated the bone marrow (BM) microenvironment. Neutrophils from steady-state BM constitutively expressed mPGES-1 and significantly enhanced PGE2 production in vitro by ß-adrenergic stimulation, but not by G-CSF, which was inhibited by an NSAID. Although neutrophils expressed all ß-adrenergic receptors, only ß3-agonist induced this phenomenon. Liquid chromatography-tandem mass spectrometry traced ß-agonist-induced PGE2 synthesis from exogenous deuterium-labeled AA. Spontaneous PGE2 production was highly efficient in Gr-1high neutrophils among BM cells from G-CSF-treated mice. In addition to these in vitro data, the in vivo depletion of Gr-1high neutrophils disrupted G-CSF-induced fever. Furthermore, sympathetic denervation eliminated both neutrophil priming for PGE2 production and fever during G-CSF treatment. Thus, sympathetic tone-primed BM neutrophils were identified as one of the major PGE2 producers. PGE2 upregulated osteopontin, specifically in preosteoblasts, to retain progenitors in the BM via EP4 receptor. Thus, the sympathetic nervous system regulated neutrophils as an indispensable PGE2 source to modulate BM microenvironment and body temperature. This study provided a novel mechanistic insight into the communication of the nervous system, BM niche components, and hematopoietic cells.

A novel FOXP1-PDGFRA fusion gene in myeloproliferative neoplasm with eosinophilia.

We identified a novel fusion gene, FOXP1-PDGFRA, in a patient with myeloproliferative neoplasm (MPN) with eosinophilia, harboring the chromosome abnormality t(3;4)(p13;q12). The patient responded well to imatinib and has remained in molecular remission for 3 years. This is the seventh fusion gene involving PDGFRA in MPN with eosinophilia. PDGFRA was truncated in its autoinhibitory domain, as in other PDGFRA-related MPNs, and was fused to FOXP1 at its functional forkhead domain. Comparing genomic DNA with mRNA sequences provides the possibility that the splicing process near the breakpoint junction in the FOXP1-PDGFRA fusion gene may use the normal splice donor site for intron 23a of FOXP1 and the cryptic splice acceptor site in exon 12 of PDGFRA. This is the first report to describe the FOXP1-PDGFRA fusion gene in MPN.

[Thrombopoietin receptor agonists administration for acute exacerbation of chronic idiopathic thrombocytopenic purpura and subsequent anticoagulant therapy for accompanying deep venous thrombosis of the lower limbs].

We report two patients (70- and 49-year-old Japanese men) with acute exacerbation of chronic idiopathic thrombocytopenic purpura (ITP) and deep venous thrombosis of the lower extremities. Both were successfully managed with thrombopoietin receptor agonist (TPO-RA) administration. Both had ITP refractory to steroid treatment. Their immature platelet fraction (absolute-IPF) counts were increased and paralleled the platelet recoveries after TPO-RA (eltrombopag and romiplostim, respectively) without progression of thrombosis. Although ITP has recently been evaluated as a thrombophilic disorder, reports on acute exacerbation of ITP with newly diagnosed thrombosis are limited, and the pathophysiology and association between ITP and thrombosis remain to be elucidated. Moreover, the influences of TPO-RA on thrombosis are still controversial. To our knowledge, this is the first case report describing patients with exacerbation of ITP who developed thrombosis and were treated with TPO-RA. The outcomes of our cases underscore the importance of monitoring thrombosis and not delaying the initiation of anticoagulation treatment during the use of TPO-RA.

Hes1 upregulation contributes to the development of FIP1L1-PDGRA-positive leukemia in blast crisis.

We have previously shown that elevated expression of Hairy enhancer of split 1 (Hes1) contributes to blast crisis transition in Bcr-Abl-positive chronic myelogenous leukemia. Here we investigate whether Hes1 is involved in the development of other myeloid neoplasms. Notably, Hes1 expression was elevated in only a few cases of 65 samples with different types of myeloid neoplasms. Interestingly, elevated expression of Hes1 was found in two of five samples of Fip1-like1 platelet-derived growth factor receptor-α (FIP1L1-PDGFA)-positive myeloid neoplasms associated with eosinophilia. Whereas FIP1L1-PDGFRα alone induced acute T-cell leukemia or myeloproliferative neoplasms in mouse bone marrow transplantation models, mice transplanted with bone marrow cells expressing both Hes1 and FIP1L1-PDGFRα developed acute leukemia characterized by an expansion of myeloid blasts and leukemic cells without eosinophilic granules. FIP1L1-PDGFRα conferred cytokine-independent growth to Hes1-transduced common myeloid progenitors, interleukin-3-dependent cells. Imatinib inhibited the growth of common myeloid progenitors expressing Hes1 with FIP1L1-PDGFRα, but not with imatinib-resistant FIP1L1-PDGFRα mutants harboring T674I or D842V. In contrast, ponatinib efficiently eradicated leukemic cells expressing Hes1 and the imatinib-resistant FLP1L1-PDGFRΑ mutant in vitro and in vivo. Thus, we have established mouse models of FIP1L1-PDGFRA-positive leukemia in myeloid blast crisis, which will help elucidate the pathogenesis of the disease and develop a new treatment for it.

Posttranscriptional modulation of cytokine production in T cells for the regulation of excessive inflammation by TFL.

Posttranscriptional machinery regulates inflammation and is associated with autoimmunity as well as tumorigenesis in collaboration with transcription factors. We previously identified the tumor suppressor gene transformed follicular lymphoma (TFL) on 6q25 in a patient with follicular lymphoma, which transformed into diffuse large B cell lymphoma. TFL families have a common RNase domain that governs macrophage-mediated inflammation. In human peripheral blood, TFL is dominantly expressed at the glycine- and tryptophan-rich cytoplasmic processing bodies of T lymphocytes, and it is persistently upregulated in activated T cells. To address its physiological role, we established TFL(-/-) mice in which TFL(-/-) lymphocytes proliferated more rapidly than TFL(+/+) upon stimulation with inappropriate cytokine secretion, including IL-2, IL-6, and IL-10. Moreover, TFL inhibited the synthesis of cytokines such as IL-2, IL-6, IL-10, TNF-α, and IL-17a by 3' untranslated region RNA degradation. Experimental autoimmune encephalitis induced in TFL(-/-) mice demonstrated persistent severe paralysis. CNS-infiltrated CD4(+) T cells in TFL(-/-) mice contained a higher proportion of Th17 cells than did those in TFL(+/+) mice during the resolution phase, and IL-17a mRNA levels were markedly increased in TFL(-/-) cells. These results suggest that TFL may play an important role in attenuating local inflammation by suppressing the infiltration of Th17 cells in the CNS during the resolution phase of experimental autoimmune encephalitis. TFL is a novel gradual and persistent posttranscriptional regulator, and the TFL-driven attenuation of excessive inflammation could contribute to recovery from T cell-mediated autoimmune diseases.

Eosinophilia and bone lesion as clinical manifestations of aggressive systemic mastocytosis.

We report a patient with aggressive systemic mastocytosis (SM), who exhibited eosinophilia and unusual destructive bone lesions. A 43-year-old female was referred to our hospital because of a vertebral compression fracture, multiple lytic bone lesions, and eosinophilia in February 2011. A diagnosis of aggressive SM was made on the basis of abnormal mast cells in the bone marrow, high serum tryptase levels, and multiple lytic bone lesions including vertebral compression fractures. Polymerase chain reaction and subsequent sequencing of its products to identify mutations of c-kit yielded negative results and imatinib mesylate failed to improve the SM of the patient. She was then treated with interferon-α, with considerable improvement of the disease, although severe myelosuppression prevented the continued administration of a sufficient dose of this agent. In August 2011, the patient suddenly developed paraplegia of the lower extremities. Magnetic resonance imaging demonstrated epidural mass lesions at the levels from Th9 to Th11, compressing the spinal cord. Emergent laminectomy and subsequent irradiation of the tumors were performed without improvement of the paraplegia. Histopathologic examination of the epidural tumors, from samples obtained intraoperatively, confirmed the diagnosis of SM. She was further treated with dasatinib and then cladribine without obvious improvement, although the latter reduced the eosinophilia to some extent ; however, she died of sepsis in September 2011.

Osteocytes regulate primary lymphoid organs and fat metabolism.

Osteocytes act as mechanosensors to control local bone volume. However, their roles in the homeostasis of remote organs are largely unknown. We show that ablation of osteocytes in mice (osteocyte-less [OL] mice) leads to severe lymphopenia, due to lack of lymphoid-supporting stroma in both the bone marrow and thymus, and complete loss of white adipose tissues. These effects were reversed when osteocytes were replenished within the bone. In contrast, neither in vivo supply of T cell progenitors and humoral factors via shared circulation with a normal parabiotic partner nor ablation of specific hypothalamic nuclei rescued thymic atrophy and fat loss in OL mice. Furthermore, ablation of the hypothalamus in OL mice led to hepatic steatosis, which was rescued by parabiosis with normal mice. Our results define a role for osteocytes as critical regulators of lymphopoiesis and fat metabolism and suggest that bone acts as a central regulator of multiple organs.

Matrix-embedded osteocytes regulate mobilization of hematopoietic stem/progenitor cells.

The bone marrow (BM) niche comprises multiple cell types that regulate hematopoietic stem/progenitor cell (HSPC) migration out of the niche and into the circulation. Here, we demonstrate that osteocytes, the major cellular component of mature bone, are regulators of HSPC egress. Granulocyte colony-stimulating factor (G-CSF), used clinically to mobilize HSPCs, induces changes in the morphology and gene expression of the osteocytic network that precedes changes in osteoblasts. This rapid response is likely under control of the sympathetic nervous system, since osteocytes express the ß2-adrenergic receptor and surgical sympathectomy prevents it. Mice with targeted ablation of osteocytes or a disrupted osteocyte network have comparable numbers of HSPCs in the BM but fail to mobilize HSPCs in response to G-CSF. Taken together, these results indicate that the BM/bone niche interface is critically controlled from inside of the bone matrix and establish an important physiological role for skeletal tissues in hematopoietic function.

Severe inhibitor-negative acquired factor XIII/13 deficiency with aggressive subdural haemorrhage.

Acquired factor XIII (FXIII) deficiency is a common disease and seldom causes bleeding. However, severe FXIII deficiency may result in life-threatening bleeding. Although the inhibitor against FXIII has recently been focused as the cause of haemorrhagic acquired FXIII deficiency, the pathophysiology of inhibitor-negative cases could also be involved. We report a case of an 85-year-old Japanese man with serious subdural haemorrhage showing a remarkable decreased level of FXIII activity. He also manifested complications of compensated disseminated intravascular coagulation (DIC) with chronic renal failure, abdominal aortic aneurysm (AAA) and right renal carcinoma. Despite the successful evacuation of the haemorrhage, acute subdural haemorrhage subsequently developed that necessitated further craniotomies. Plasma cross-mixing studies and dot blot assay revealed no inhibitors against FXIII. We speculated that the decreased FXIII activity could be mainly due to hyperconsumption by DIC and surgery. Because plasma-derived FXIII concentrates are available to stop bleeding, clinicians should be aware of severe acquired inhibitor-negative FXIII deficiency in cases of unexplained excessive bleeding.

Successful management of obstructive jaundice due to gallstones with eculizumab in a patient with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) makes patients susceptible to intravascular hemolysis and thrombosis, and it can be life-threatening in stressful situations. Eculizumab, a humanized monoclonal antibody that inhibits the complement protein C5, has been evaluated as a novel therapy for PNH. We herein describe the case of a 59-year-old Japanese woman with classic PNH, who had been successfully treated with eculizumab, but who later developed acute cholecystitis/cholangitis from gallstones. Although the severe obstructive jaundice requiring endoscopic therapy following cholecystectomy was complicated, critical intravascular hemolysis and thrombosis were not observed. Therefore, utilizing eculizumab during the peri-operative management of PNH patients should be carefully taken into consideration.

[Diminished expression of CD5 and/or CD7 surface antigens as the first clue of diagnosis for monoclonal T lymphocytosis].

The significance of precursor status for mature T-cell neoplasms has not been elucidated. Diagnosis of T-cell neoplasms is often challenging and sometimes overlooked when leukocytosis is not evident and neoplastic cell morphology is hardly distinguishable. Here, we report two cases of monoclonal T lymphocytosis (MTL) without evident lymphocytosis and lymphadenopathies that show slightly diminished CD5 and/or CD7 surface antigens as the first clue of diagnosis. Recently, some reports have demonstrated the possibility that monoclonal lymphocytosis precedes leukemia/lymphoma. Although its clinical significance is unknown, flow cytometric analysis of peripheral blood is useful for detection of occult MTL and careful follow-up is required.

Hyper-eosinophilia in granular acute B-cell lymphoblastic leukemia with myeloid antigen expression.

Acute lymphoblastic leukemia with eosinophilia (ALLEo) is a rare but a distinctive clinical entity. Clinical features of idiopathic hyper-eosinophilic syndrome (HES) can be seen in patients with ALLEo. We report a 10-year-old girl, in whom HES was initially suspected but further investigation confirmed the diagnosis of acute B-cell lymphoblastic leukemia with myeloid antigen expression. Clinical response to chemotherapy was excellent with achievement of complete remission for 4 years. Serum interleukin-3 and -5 were elevated at presentation and normalized with disappearance of eosinophilia after induction therapy, supporting the reactive nature of eosinophilia in ALLEo. Hematologic malignancy should be considered in patients with hyper-eosinophilia, before attributing it to HES.

[Idiopathic hypereosinophilic syndrome in a child with fever and hepatomegaly].

Idiopathic hypereosinophilic syndrome (IHES) in children is a rare disorder. A 1-year-old girl presented to our hospital for evaluation of eosinophilia. At the onset, her white blood cell count in peripheral blood was 70,600/µl with 74% eosinophils. She had a high fever and mild hepatomegaly but had no remarkable evidence of organ involvement by CT, MRI and ultrasonography. She was diagnosed with IHES without any evidence of secondary eosinophilia, expression of the FIP1L1-PDGFRα fusion transcript, chromosomal abnormalities, and aberrant T-cell populations. The serum IgE, vitamin B12, IL-5 and TARC levels were normal. Systemic administration of corticosteroid and suplatast tosilate resolved the symptoms promptly and resulted in improvement of eosinophilia.

Pharmacokinetics-based optimal dose prediction of donor source-dependent response to mycophenolate mofetil in unrelated hematopoietic cell transplantation.

Mycophenolate mofetil (MMF) has been widely used for prophylaxis against graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (allo-SCT). However, no clear advantage over methotrexate has been reported, other than reduced incidence of mucositis. We speculated that the wide inter-individual variation of plasma mycophenolic acid (MPA) levels veiled the benefits of MMF. Data from 36 unrelated allogeneic bone marrow (allo-BMT) and cord blood transplantation (CBT) were analyzed retrospectively based on MPA area under the curve (AUC(0-24h)). In allo-BMT, high AUC(0-24h) (>30 µg h/ml) resulted in no incidence of grade II-IV acute/extensive chronic GVHD and tended to show higher overall and disease-free survival, lower relapse rates, and non-relapse mortality. In CBT, AUC(0-24h) less than 30 µg h/ml was sufficient for low incidence of acute/chronic GVHD and high survival. Strong correlation between AUC(0-24h) and C(2h), plasma MPA concentration at 2 h after administration was observed. Single point assessment of C(2h) was shown to provide a useful surrogate of AUC(0-24h) to predict GVHD incidence. The results of this study suggest that individualized MMF dosing in a donor source-dependent fashion may be important for maximizing the benefit of MMF in allo-SCT.