Kohshi Ohishi

Delta-1 enhances marrow and thymus repopulating ability of human CD34 + CD38 – cord blood cells

We investigated the effect of Notch signaling, a known regulator of cell fate in numerous developmental systems, on human hematopoietic precursors. We show that activation of endogenous Notch signaling in human CD34(+)CD38(-) cord blood precursors with immobilized Delta-1 in serum-free cultures containing fibronectin and hematopoietic growth factors inhibited myeloid differentiation and induced a 100-fold increase in the number of CD34(+) cells compared with control cultures. Immobilized Delta-1 also induced a multifold expansion of cells with the phenotype of common lymphoid precursors (CD34(+)CD7(+)CD45RA(+)) and promoted the development of cytoplasmic CD3(+) T/NK cell precursors. IL-7 enhanced the promotion of T/NK cell differentiation by immobilized Delta-1, but granulocytic differentiation occurred when G-CSF was added. Transplantation into immunodeficient mice showed a substantial increase in myeloid and B cell engraftment in the marrow and also revealed thymic repopulation by CD3(+) T cells due to cells being cultured for a longer period with immobilized Delta-1. These data suggest that Delta-1 can enhance myeloid and lymphoid marrow-repopulating ability and promote the generation of thymus-repopulating T cell precursors.

Activity of interleukin 6 in the differentiation of monocytes to macrophages and dendritic cells

Peripheral blood monocytes are common precursor cells of dendritic cells (DCs) and macrophages. We have searched for factors with the potential to regulate the differentiation of monocytes to DCs and macrophages. When CD14+ monocytes are cultured with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL) 4, the CD14+CD1a− population, which consists of macrophages, was found in the serum‐containing cultures but not in the serum‐free cultures. Addition of IL‐6 receptor‐neutralizing monoclonal antibody (mAb) or gp130‐neutralizing mAb to the serum‐containing cultures resulted in a decreased population of CD14+CD1a− cells. An increase in the CD14+CD1a− population with reduction in CD14−CD1a+ DCs was observed with the addition of IL‐6 to cultures, whereas IL‐11, leukaemia inhibitory factor, oncostatin M or macrophage colony‐stimulating factor did not affect the differentiation of monocytes in the presence of GM‐CSF plus IL‐4. This effect of IL‐6 was blocked by tumour necrosis factor α (TNF‐α), lipopolysaccharide (LPS), IL‐1β, CD40 ligand (CD40L) and transforming growth factor β1 (TGF‐β1). Among these factors, TNF‐α was most potent in interfering with the action of IL‐6. These results suggest that IL‐6 inhibits the differentiation of monocytes to DCs by promoting their differentiation toward macrophages, which is modulated by factors such as TNF‐α, LPS, IL‐1β, CD40L and TGF‐β1.

The notch pathway: modulation of cell fate decisions in hematopoiesis.

The hematopoietic system is maintained by a rare population of hematopoietic stem cells (HSC) that are thought to undergo self-renewal as well as continuously produce progeny that differentiate into the various hematopoietic lineages. However, the mechanisms regulating cell fate choices by HSC and their progeny have not been understood. Results of most studies support a stochastic model of cell fate determination in which growth factors support only the survival or proliferation of the progeny specified along a particular lineage. In other developmental systems, however, Notch signaling has been shown to play a central role in regulating fate decisions of numerous types of precursors, often inhibiting a particular (default) pathway while permitting self-renewal or differentiation along an alternative pathway. There is also accumulating evidence that the Notch pathway affects survival, proliferation, and cell fate choices at various stages of hematopoietic cell development, including the decisions of HSC to self-renew or differentiate and of common lymphoid precursors to undergo T- or B-cell differentiation. These data suggest that the Notch pathway plays a fundamental role in the development and maintenance of the hematopoietic system.

A novel role for Notch ligand Delta-1 as a regulator of human Langerhans cell development from blood monocytes.

Human Langerhans cells (LCs) are of hematopoietic origin, but cytokine regulation of their development is not fully understood. Notch ligand Delta‐1 is expressed in a proportion of the skin. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and transforming growth factor‐β1 (TGF‐β1) are also secreted in the skin. We report here that Delta‐1, in concert with GM‐CSF and TGF‐β1, induces the differentiation of human CD14+ blood monocytes into cells that express LC markers: CD1a, Langerin, cutaneous lymphocyte‐associated antigen, CC chemokine receptor 6, E‐cadherin, and Birbeck granules. The resulting cells display phagocytic activity and chemotaxis to macrophage inflammatory protein‐1α (MIP‐1α). In response to CD40 ligand and tumor necrosis factor α, the cells acquire a mature phenotype of dendritic cells that is characterized by up‐regulation of human leukocyte antigen (HLA)‐ABC, HLA‐DR, CD80, CD86, CD40, and CD54 and appearance of CD83. These cells in turn show chemotaxis toward MIP‐1β and elicit activation of CD8+ T cells and T helper cell type 1 polarization of CD4+ T cells. Thus, blood monocytes can give rise to LCs upon exposure to the skin cytokine environment consisting of Delta‐1, GM‐CSF, and TGF‐β1, which may be, in part, relevant to the development of human epidermal LCs. Our results extend the functional scope of Notch ligand δ‐1 in human hematopoiesis.

Adoptive Transfer of MAGE-A4 T-cell Receptor Gene-Transduced Lymphocytes in Patients with Recurrent Esophageal Cancer

Purpose: Preparative lymphodepletion, the temporal ablation of the immune system, has been reported to promote persistence of transferred cells along with increased rates of tumor regression in patients treated with adoptive T-cell therapy. However, it remains unclear whether lymphodepletion is indispensable for immunotherapy with T-cell receptor (TCR) gene–engineered T cells. Experimental Design: We conducted a first-in-man clinical trial of TCR gene-transduced T-cell transfer in patients with recurrent MAGE-A4–expressing esophageal cancer. The patients were given sequential MAGE-A4 peptide vaccinations. The regimen included neither lymphocyte-depleting conditioning nor administration of IL2. Ten patients, divided into 3 dose cohorts, received T-cell transfer. Results: TCR-transduced cells were detected in the peripheral blood for 1 month at levels proportional to the dose administered, and in 5 patients they persisted for more than 5 months. The persisting cells maintained ex vivo antigen-specific tumor reactivity. Despite the long persistence of the transferred T cells, 7 patients exhibited tumor progression within 2 months after the treatment. Three patients who had minimal tumor lesions at baseline survived for more than 27 months. Conclusions: These results suggest that TCR-engineered T cells created by relatively short-duration in vitro culture of polyclonal lymphocytes in peripheral blood retained the capacity to survive in a host. The discordance between T-cell survival and tumor regression suggests that multiple mechanisms underlie the benefits of preparative lymphodepletion in adoptive T-cell therapy. Clin Cancer Res; 21(10); 2268–77. ©2015 AACR.

Clinical utility of a panfungal polymerase chain reaction assay for invasive fungal diseases in patients with haematologic disorders.

Invasive fungal diseases (IFDs) are life‐threatening events in patients with haematologic disorders, and the spectrum of the aetiological pathogens continues to expand. This study aimed to evaluate the clinical utility of a panfungal polymerase chain reaction (PCR) assay for the management of IFDs in such patients.

Plasma ADAMTS13, von Willebrand factor (VWF) and VWF propeptide profiles in patients with DIC and related diseases.

ADAMTS13, endothelial von Willebrand factor (VWF) and related proteins are involved in the pathogenesis of some life threatening systemic thrombotic coagulopathies. Changes of plasma ADAMTS13 activity in thrombotic thrombocytopenic purpura (TTP) is well known but is also involved in septic disseminated intravascular coagulation (DIC). Here we investigated the ADAMTS13 activity, VWF and VWF propeptide (VWFpp) antigens in 69 patients with DIC, 143 with non-DIC, 21 with thrombotic thrombocytopenic purpura (TTP) and 23 with atypical hemolytic uremic syndrome (aHUS) for diagnosis of DIC. The plasma ADAMTS13 activity was significantly low in patients with DIC, and the plasma levels of VWF and VWFpp antigens, were the highest in these patients, but there were no significant differences in the plasma VWFpp levels between the patients with DIC and those with aHUS. The difference in the plasma ADAMTS13 activity, the VWF and VWFpp antigens between DIC and non-DIC cases was significant in those with infectious and malignant diseases, but the difference in the VWFpp/ VWF ratio were significant only in subjects with infectious diseases. As an indicator for prognosis, the plasma levels of VWFpp were significantly higher in non-survivors than in survivors. Then, VWFpp/ VWF ratio and VWFpp/ADAMATS13 ratio will be potent informative indicators in DIC. These findings suggest that ADAMTS13/VWF profiles may have important roles in the pathogenesis of DIC, and that ADAMTS13 and VWFpp are useful indicators for the diagnosis and prognosis of DIC.

Activity of the ligand for c-mpl, thrombopoietin, in early haemopoiesis.

We examined the role of the ligand for c‐mpl, thrombopoietin (TPO), in murine early haemopoiesis, using a serum‐free culture system. TPO in combination with the ligand for c‐kit (SF) or interleukin‐3 (IL‐3) supported colony formation by marrow cells of 5‐fluorouracil (5‐FU)‐treated mice, whereas TPO alone yielded no colony. When blast cell colonies grown in the presence of TPO plus SF or TPO plus IL‐3 were individually replated in suspension cultures containing serum and several growth factors, various combinations of myeloid lineages were seen, indicating that the progenitors supported by TPO plus SF or TPO plus IL‐3 are multipotential. Delayed addition experiments demonstrated that TPO has the potential to effectively support the survival of haemopoietic progenitors. We then studied the effects of TPO on proliferative kinetics of cycling progenitors. TPO hastened IL‐3‐dependent growth of progenitors by shortening the time required for cell cycling. These results suggest that TPO, as a single factor, can support the survival of haemopoietic progenitors and TPO synergizes with SF or IL‐3 to act on early multipotential haemopoietic progenitors.

Diagnostic Value of PCR Analysis of Bacteria and Fungi from Blood in Empiric-Therapy-Resistant Febrile Neutropenia

ABSTRACT This study aimed to assess the clinical utility of PCR for the analysis of bacteria and fungi from blood for the management of febrile neutropenic patients with hematologic malignancies. Using a PCR system able to detect a broad range of bacteria and fungi, we conducted a prospective pilot study of periodic analyses of blood from patients following intensive chemotherapy. When fever occurred, it was treated with empirical antibiotic therapy, basically without knowledge of the PCR results. In 23 febrile episodes during the neutropenic period, bacteria were detected by PCR in 11 cases, while the same species were identified by blood culture in 3 cases. In 10 out of 11 PCR-positive cases, fever could be managed by empirical therapy. In the empirical-therapy-resistant case, the identification of Stenotrophomonas maltophilia by PCR led to improvement of fever. No fungi were detected by PCR in febrile cases, while Aspergillus fumigatus was detected in one afebrile patient, several days before a clinical diagnosis was made. In subsequent sporadic PCR analyses in 15 cases of febrile neutropenia, bacteria were detected by both PCR and blood culture in 7 cases and by PCR alone in 6. Fungi were not detected. While fever was improved by empirical therapy in 12 out of the 13 PCR-positive cases, the identification of Pseudomonas aeruginosa by PCR in one therapy-resistant case contributed to the successful treatment of persistent fever. Our results indicate that PCR analysis of bacteria from blood provides essential information for managing empirical-therapy-resistant febrile neutropenia.

Efficacy and safety of ruxolitinib in Asian patients with myelofibrosis

Abstract Myelofibrosis is characterized by progressive cytopenias, bone marrow fibrosis, splenomegaly and severe constitutional symptoms. In the phase 3 Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment (COMFORT) studies, ruxolitinib, a potent Janus kinase 1 (JAK1)/JAK2 inhibitor, provided substantial improvements in splenomegaly, symptoms, quality-of-life measures and overall survival compared with placebo or best available therapy. No assessments of the efficacy and safety of ruxolitinib have been conducted in Asian patients. Here, we describe results from an open-label, single-arm, phase 2 trial evaluating ruxolitinib in Asian patients with myelofibrosis (n = 120). The primary endpoint was met, with 31.7% of patients achieving a ≥ 35% reduction from baseline spleen volume at week 24. As measured by the 7-day Myelofibrosis Symptom Assessment Form v2.0, 49% of patients achieved a ≥ 50% reduction from baseline in total symptom score. Adverse events were consistent with those seen in the COMFORT studies. Ruxolitinib was well tolerated in Asian patients with myelofibrosis and provided substantial reductions in splenomegaly and improvements in symptoms.