Yuko Kawano

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 β2-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.

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.

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 (AUC0–24h). In allo-BMT, high AUC0–24h (>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, AUC0–24h less than 30 μg h/ml was sufficient for low incidence of acute/chronic GVHD and high survival. Strong correlation between AUC0–24h and C2h, plasma MPA concentration at 2 h after administration was observed. Single point assessment of C2h was shown to provide a useful surrogate of AUC0–24h 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.

Extended Mycophenolate Mofetil Administration Beyond Day 30 in Allogeneic Hematopoietic Stem Cell Transplantation as Preemptive Therapy for Severe Graft-Versus-Host Disease

To prevent acute graft-versus-host disease (GVHD), mycophenolate mofetil (MMF) combined with calcineurin inhibitors have been used in allogeneic hematopoietic stem cell transplantation (allo-SCT). Previous studies commonly utilize MMF treatment until day 30 after allo-SCT. However, the feasibility of continuous administration after day 30 has not been well evaluated. We retrospectively assessed the safety and efficacy of extended drug administration. Twenty-five patients ceased MMF at day 30 (group A); whereas, 16 patients (group B) received extended regimens depending on individual risk factors for GVHD. No severe adverse events were observed in either group. Although the cumulative incidence (CI) of grade I to IV GVHD at day 100 was comparable between the 2 groups, the CI of grade II to IV GVHD was less among group B (12.5%) compared with group A (42.3%). Extended MMF administration may be safe and beneficial as preemptive therapy to reduce the development of moderate-to-severe acute GVHD.

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.

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 β-adrenergic stimulation, but not by G-CSF, which was inhibited by an NSAID. Although neutrophils expressed all β-adrenergic receptors, only β3-agonist induced this phenomenon. Liquid chromatography-tandem mass spectrometry traced β-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.

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.

Therapy-related, mixed phenotype acute leukemia with t(1;21)(p36;q22) and RUNX1 rearrangement

We describe here a new case of therapy-related acute leukemia with t(1;21)(p36;q22). A 25-year-old man was admitted because of anemia and thrombocytopenia. Four years before, he had received combination chemotherapy including etoposide for seminoma. Bone marrow was hypercellular, with 49% myeloperoxidase (MPO) staining-negative blasts. Chromosome analysis showed 46,XY,t(1;21)(p36.3;q22)[11]/49,sl,+8,+16,+20[9]. Fluorescence in situ hybridization demonstrated that RUNX1 signals at 21q22 were split onto the der(1)t(1;21) and der(21)t(1;21). Immunophenotypic analyses revealed that blasts were positive for CD19, CD79a, and cytCD22, as well as MPO, CD13, and CD33, fulfilling the diagnostic criteria of mixed phenotype acute leukemia, B/myeloid. The patient died of disease progression after 10 months. Thus, acute leukemia with t(1;21) and RUNX1 rearrangement could be associated with B/myeloid mixed phenotype as well as previous topoisomerase II inhibitor therapy and poor prognoses.

Recurrence of abdominal large-vessel vasculitis and development of severe Sweet syndrome after a single cycle of 5-azacytidine in a patient with myelodysplastic syndrome.

To the Editor: Various types of autoimmune disorders have been reported as manifestations of myelodysplastic syndromes (MDS) (1). They include vasculitis, neutrophilic dermatosis, and immune thrombocytopenia (2). Recently, it has been reviewed that vasculitis, including large-vessel vasculitis (LVV) associated with MDS, could be treated with corticosteroid treatment, but the treatment is associated with increased risk of fatal infections (3, 4). The hypomethylating agent 5-azacytidine (5-aza), an analogue of cytidine, is increasingly used for the treatment of MDS (5). 5-aza has also been evaluated for its immunomodulatory effects including induction of regulatory T cells and interference with cytokine production on some autoimmune disorders (6–8). However, the effectiveness and safety in patients with autoimmune diseases are controversial. We describe here the first case of MDS patient with relapsed LVV after 5-aza treatment. A 65-yr-old Japanese man with pancytopenia was referred to our hospital. His medical history was pertinent for epilepsy and hypertension. Bone marrow aspiration showed hyperplastic marrow, trilineage dysplasia with 5.4% blasts, and complex karyotypic abnormalities. After 1 month, the patient was hospitalized due to severe left lower back pain with lowgrade fever. The laboratory data were as follows: WBC 3.5 9 10/L without blasts, Hb 62 g/L, Plt 46 9 10/L, and C-reactive protein 12.5 mg/dL. Serology was negative for rapid plasma reagin, antinuclear antibody, rheumatoid factor, and antineutrophil cytoplasmic antibodies. Repetitive culture revealed negative for infection. Computed tomography (CT) revealed abdominal aorta with enhanced periaortic tissue (Fig. 1A). An 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) scan demonstrated abdominal aorta with FDG uptake in the vascular wall (Fig. 1B). The patient was diagnosed with abdominal LVV and started on methylprednisolone 62.5 mg intravenous daily then prednisolone (PSL) 50 mg oral daily. Although the arteritis was resolved after 3 wks, he subsequently suffered from severe pneumonia. After 2 months, he recovered and then began to receive chemotherapy with 5-aza (50 mg/m/d intravenous for 5 d, in consideration of his comorbidities). At this time, the dose of PSL was 17.5 mg/d, and the arteritis was clinically resolved. However, 3 wks after one cycle of 5-aza, the LVV relapsed with the same symptoms as the first episode. The PSL dose was increased to 50 mg/d, and the symptoms gradually improved. However, he subsequently developed febrile systemic painful erythematous rash pathologically diagnosed with neutrophilic dermatosis, Sweet syndrome, which was severe and refractory to treatments, including steroid and potassium iodide (Figure S1). The patient finally expired due to acute heart failure. At autopsy, invasion of lymphocytes was observed in the tunica adventitia of the involved aorta (Fig. 1C,D), and the increase in blasts in bone marrow was not observed. It is implied that a pathologic clone of stem cells in an MDS patient could trigger the expansion and activation of cytotoxic T cells involving in autoimmune disorders (9). Thus, it is reasonable that LVV could be involved in the pathophysiology of MDS (3). Although there is no report on microscopic evaluation, we found that the tunica adventitia was mainly involved in our case and did not fulfill any of the diagnostic critera for Takayasu’s arteritis (10). In giant cell arteritis, the most common form of LVV, Th1 cells contribute to the corticosteroid-dependent pathophysiology because Th1 cells are refractory and continue to support Adv

Unusual hepatic involvement with significant fibrosis in adult T cell leukemia.

Dear Editor, Adult T cell leukemia (ATL) is a heterogeneous disease characterized by a broad spectrum of cytological features, which presents with variable clinical features [1]. We describe a patient with acute ATL who presented with distinctive hepatic involvement with prominent fibrosis. In 2009, a 58-year-old Japanese woman presented with a 3-month history of abdominal discomfort and back pain. She had no previous chronic hepatic diseases. Physical examination revealed no lymphadenopathy and cutaneous lesions. Her CBC was as follows: WBC 13.8×10/L with abnormal lymphocyte 11 %, hemoglobin 12.8 g/dL, and platelet 152×10/L. Serum biochemistry studies were performed as follows: AST 315 U/L, ALT 318 U/L, LDH 601 U/L (115–217), total bilirubin 3.0 mg/dL, and sIL-2R 14,014 U/ml. Calcium level was normal. Hepatitis virus serology and autoantibody were negative. HTLV-1 was serologically positive, and monoclonal integration of HTLV-I proviral genome was confirmed with southern blot analysis. Flow cytometric analysis for bone marrow showed surface CD2+, CD3−, CD4+, CD5+, CD7−, CD8−, and CD25+ abnormal T lymphocytes (25 %), and CD30 was expressed on larger population of ATL cells. Computed tomography revealed hepatosplenomegaly (spleen, 10.0×11.5×5.5 cm in size) and lymphadenopathy (1.0 cm to 1.8 cm in size) on paraaortic, periportal, mesenteric, and mediastinal areas. Fluorodeoxyglucose (FDG)-positron emission tomography showed elevated FDG uptake only on these lymph nodes. The histology revealed infiltration of numerous medium to large-sized atypical lymphoid cells characterized by nuclear irregularities and nucleolar prominence in bone marrow, liver, and lymph nodes. Interestingly, atypical lymphoid cells infiltrated in the widening portal area with remarkable fibrosis although hepatocytes were almost preserved (Fig. 1). Immunohistochemistry revealed that the atypical lymphoid cells were positive for CD3, CD4, CCR4, and CD30 (Fig. 1). Although splenomegaly was mildly improved after one cycle of CHOP chemotherapy, the patient developed diureticrefractory pleural effusion and ascites due to portal hypertension. She finally succumbed to disease progression 5 months after diagnosis. Although skin lesions are frequently observed in patients with ATL, the liver is also known as one of the invasive sites, in which ATL cells usually invades in the portal area without fibrosis, as well as in the sinusoids [1]. Interestingly, our case showed hepatotrophic feature without skin involvement and superficial lymphadenopathy. CXCR3 on T cell has been implied as a bile duct homing chemokine in patients with primary biliary cirrhosis, an autoimmune disorder H. Kawano (*) :K. Wakahashi : S. Ishii : T. Suzuki :Y. Kawano : A. Sada :K. Minagawa :Y. Katayama Hematology, Department of Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe, Hyogo 650-0017, Japan e-mail: hkawano@med.kobe-u.ac.jp