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Figure S1. Skin exposure to UVB induces splenic erythroid cell proliferation in mice. Figure S2. Skin exposure to UVB results in expansion of splenic cell populations with erythroid colony-forming potential. Figure S3. Administration of an anti-EPO antibody prevents UVB-responsive splenic erythroid expansion. Figure S4. Tumor growth induces splenic erythroid cell proliferation in mice. Figure S5. CD235A immunostaining identifies tumor-associated erythroid cells in human cancer tissues.

Hematopoietic-derived cells are integral components of the tumor microenvironment and serve as critical mediators of tumor–host interactions. Host cells derived from myeloid and lymphoid lineages perform well-established functions linked to cancer development, progression, and response to therapy. It is unclear whether host erythroid cells also contribute to shaping the path that cancer can take, but emerging evidence points to this possibility. Here, we show that tumor-promoting environmental stress and tumor-induced hemodynamic changes trigger renal erythropoietin production and erythropoietin-dependent expansion of splenic erythroid cell populations in mice. These erythroid cells display molecular features indicative of an immature erythroid phenotype, such as the expression of both CD71 and TER119 and the retention of intact nuclei, and express genes encoding immune checkpoint molecules. Nucleated erythroid cells with similar properties are present in mouse and human tumor tissues. Antibody-mediated erythropoietin blockade reduces tumor-responsive erythroid cell induction and tumor growth. These findings reveal the potential of tumor-induced erythropoietin and erythroid cells as targets for cancer treatment.Implications: Our study identifies erythropoietin and erythroid cells as novel players in tumor–host interactions and highlights the involvement of multiorgan signaling events in their induction in response to environmental stress and tumor growth.

Keratinocytes, the epithelial cells of the skin, reprogram their gene expression and produce immune effector molecules when exposed to environmental and endogenous triggers of inflammation. It remains unclear how keratinocytes process physiological signals generated during skin irritation and switch from a homeostatic to an inflammatory state. In this article, we show that the stress-activated protein kinase p38α is crucial for keratinocytes to prompt changes in their transcriptome upon cytokine stimulation and drive inflammation in allergen-exposed skin. p38α serves this function by phosphorylating p63, a transcription factor essential for the lineage identity and stemness of the skin epithelium. Phosphorylation by p38α alters the activity of p63 and redeploys this developmental transcription factor to a gene expression program linked to inflammation. Genetic ablation and pharmacological inhibition of p38α or the p38α–p63 target gene product MMP13 attenuate atopic dermatitis–like disease in mice. Our study reveals an epithelial molecular pathway promoting skin inflammation and actionable through treatment with topical small-molecule therapeutics.

Coordinated induction, but also repression, of genes are key to normal differentiation. Although the role of lineage-specific transcription regulators has been studied extensively, their functional integration with chromatin remodelers, one of the key enzymatic machineries that control chromatin accessibility, remains ill-defined. Here we investigate the role of Mi-2β, a SNF-2-like nucleosome remodeler and key component of the nucleosome remodeling and histone deacetylase (NuRD) complex in early B cells. Inactivation of Mi-2β arrested differentiation at the large pre-B-cell stage and caused derepression of cell adhesion and cell migration signaling factors by increasing chromatin access at poised enhancers and chromosome architectural elements. Mi-2β also supported IL-7R signaling, survival, and proliferation by repressing negative effectors of this pathway. Importantly, overexpression of Bcl2, a mitochondrial prosurvival gene and target of IL-7R signaling, partly rescued the differentiation block caused by Mi-2β loss. Mi-2β stably associated with chromatin sites that harbor binding motifs for IKAROS and EBF1 and physically associated with these transcription factors both on and off chromatin. Notably, Mi-2β shared loss-of-function cellular and molecular phenotypes with IKAROS and EBF1, albeit in a distinct fashion. Thus, the nucleosome remodeler Mi-2β promotes pre-B-cell differentiation by providing repression capabilities to distinct lineage-specific transcription factor-based regulatory networks.

The cohesin complex participates in the organization of 3D genome through generating and maintaining DNA loops. Stromal antigen 2 (STAG2), a core subunit of the cohesin complex, is frequently mutated in various cancers. However, the impact of STAG2 inactivation on 3D genome organization, especially the long-range enhancer-promoter contacts and subsequent gene expression control in cancer, remains poorly understood. Here we show that depletion of STAG2 in melanoma cells leads to expansion of topologically associating domains (TADs) and enhances the formation of acetylated histone H3 lysine 27 (H3K27ac)-associated DNA loops at sites where binding of STAG2 is switched to its paralog STAG1. We further identify Interferon Regulatory Factor 9 (IRF9) as a major direct target of STAG2 in melanoma cells via integrated RNA-seq, STAG2 ChIP-seq and H3K27ac HiChIP analyses. We demonstrate that loss of STAG2 activates IRF9 through modulating the 3D genome organization, which in turn enhances type I interferon signaling and increases the expression of PD-L1. Our findings not only establish a previously unknown role of the STAG2 to STAG1 switch in 3D genome organization, but also reveal a functional link between STAG2 and interferon signaling in cancer cells, which may enhance the immune evasion potential in STAG2-mutant cancer.

Objective There are few reports on the outcomes of 12-week paritaprevir, ombitasvir, and ritonavir (PTV/OBV/r) treatment in real-world clinical settings. We aimed to evaluate the efficacy and safety of 12-week treatment with ritonavir-boosted paritaprevir and ombitasvir in patients with hepatitis C virus (HCV) genotype 1 infection in a real-world setting. Methods Fifty-eight patients with chronic hepatitis or compensated hepatic cirrhosis and genotype-1 HCV infection were treated with PTV/OBV/r and followed for 24 weeks after the completion of treatment in 10 centers in northern Tohoku. The efficacy and safety of this 12-week treatment regimen was analyzed. Results Among the 58 treated patients, 18 (31%) had compensated liver cirrhosis, while 11 (19%) patients had experienced treatment failure with another treatment regimen. NS5A resistance-associated variants (RAVs) were detected at baseline in 3 patients (5.2%), including Y93H in two patients and L31M in two patients. One patient had NS5A RAVs at both positions 93 and 31. The overall sustained virological response (SVR) 24 rate was 96.6%. Three patients with NS5A RAVs also achieved an SVR24. The SVR24 rate was not significantly affected by age, sex, prior treatment, prior history of HCC, or liver stiffness. The mean alanine aminotransferase (ALT) levels decreased significantly during this treatment. Adverse events occurred in 15 patients (26%), 26% of which were grade 1 or 2. No severe adverse events occurred. Conclusion In this real-world study, 12-week PTV/OBV/r treatment was effective and safe for treating patients with HCV-1 infection who had chronic hepatitis or compensated hepatic cirrhosis.

IKAROS is required for the differentiation of highly proliferative pre-B-cell precursors, and loss of IKAROS function indicates poor prognosis in precursor B-cell acute lymphoblastic leukemia (B-ALL). Here we show that IKAROS regulates this developmental stage by positive and negative regulation of superenhancers with distinct lineage affiliations. IKAROS defines superenhancers at pre-B-cell differentiation genes together with B-cell master regulators such as PAX5, EBF1, and IRF4 but is required for a highly permissive chromatin environment, a function that cannot be compensated for by the other transcription factors. IKAROS is also highly enriched at inactive enhancers of genes normally expressed in stem-epithelial cells. Upon IKAROS loss, expression of pre-B-cell differentiation genes is attenuated, while a group of extralineage transcription factors that are directly repressed by IKAROS and depend on EBF1 relocalization at their enhancers for expression is induced. LHX2, LMO2, and TEAD-YAP1, normally kept separate from native B-cell transcription regulators by IKAROS, now cooperate directly with them in a de novo superenhancer network with its own feed-forward transcriptional reinforcement. Induction of de novo superenhancers antagonizes Polycomb repression and superimposes aberrant stem epithelial cell properties in a B-cell precursor. This dual mechanism of IKAROS regulation promotes differentiation while safeguarding against a hybrid stem epithelial B-cell phenotype that underlies high-risk B-ALL. Funding Agencies|National Institutes of Health [R01CA162092, R21AI124326, R01CA090576, R01CA190964]

Purpose of review Loss of IKAROS in committed B cell precursors causes a block in differentiation while at the same time augments aberrant cellular properties, such as bone marrow stromal adhesion, self-renewal and resistance to glucocorticoid-mediated cell death. B cell acute lymphoblastic leukaemias originating from these early stages of B cell differentiation and associated with IKAROS mutations share a high-risk cellular phenotype suggesting that deregulation of IKAROS-based mechanisms cause a highly malignant disease process. Recent studies Recent studies show that IKAROS is critical for the activity of super-enhancers at genes required for pre-B cell receptor (BCR) signalling and differentiation, working either downstream of or in parallel with B cell master regulators such as EBF1 and PAX5. IKAROS also directly represses a cryptic regulatory network of transcription factors prevalent in mesenchymal and epithelial precursors that includes YAP1, TEAD1/2, LHX2 and LMO2, and their targets, which are not normally expressed in lymphocytes. IKAROS prevents not only expression of these 'extra-lineage' transcription factors but also their cooperation with endogenous B cell master regulators, such as EBF1 and PAX5, leading to the formation of a de novo for lymphocytes super-enhancer network. IKAROS coordinates with the Polycomb repression complex (PRC2) to provide stable repression of associated genes during B cell development. However, induction of regulatory factors normally repressed by IKAROS starts a feed-forward loop that activates de-novo enhancers and elevates them to super-enhancer status, thereby diminishing PRC2 repression and awakening aberrant epithelial-like cell properties in B cell precursors. Summary Insight into IKAROS-based transcriptional circuits not only sets new paradigms for cell differentiation but also provides new approaches for classifying and treating high-risk human B-ALL that originates from these early stages of B cell differentiation.

The Ikaros family of DNA-binding proteins are critical regulators of lymphocyte differentiation. In multipotent, hematopoietic progenitors, Ikaros supports transcriptional priming of genes promoting lymphocyte differentiation. Ikaros targets the Nucleosome Remodeling Deacetylase (NuRD) complex to lymphoid lineage genes, thereby increasing chromatin accessibility and transcriptional priming. After lymphoid lineage specification, Ikaros expression is raised to levels characteristic of intermediate B cell and T cell precursors, which is necessary to support maturation and prevent leukemogenesis. Loss of Ikaros in T cell precursors allows the NuRD complex to repress lymphocyte genes and extends its targeting to genes that support growth and proliferation, causing their activation and triggering a cascade of events that leads to leukemogenesis. Loss of Ikaros in B cell precursors blocks differentiation and perpetuates stromal adhesion by enhancing integrin signaling. The combination of integrin and cytokine signaling in Ikaros-deficient pre-B cells promotes their survival and self-renewal. The stages of lymphocyte differentiation that are highly dependent on Ikaros are underscored by changes in Ikaros transcription, supported by a complex network of stage-specific regulatory networks that converge upon the Ikzf1 locus. It is increasingly apparent that understanding the regulatory networks that operate upstream and downstream of Ikaros is critical not only for our understanding of normal lymphopoiesis, but also in placing the right finger on the mechanisms that support hematopoietic malignancies in mouse and human.

The ability of somatic stem cells to self-renew and differentiate into downstream lineages is dependent on specialized chromatin environments that keep stem cell-specific genes active and key differentiation factors repressed but poised for activation. The epigenetic factors that provide this type of regulation remain ill-defined. Here we provide the first evidence that the SNF2-like ATPase Mi-2β of the Nucleosome Remodeling Deacetylase (NuRD) complex is required for maintenance of and multilineage differentiation in the early hematopoietic hierarchy. Shortly after conditional inactivation of Mi-2β, there is an increase in cycling and a decrease in quiescence in an HSC (hematopoietic stem cell)-enriched bone marrow population. These cycling mutant cells readily differentiate into the erythroid lineage but not into the myeloid and lymphoid lineages. Together, these effects result in an initial expansion of mutant HSC and erythroid progenitors that are later depleted as more differentiated proerythroblasts accumulate at hematopoietic sites exhibiting features of erythroid leukemia. Examination of gene expression in the mutant HSC reveals changes in the expression of genes associated with self-renewal and lineage priming and a pivotal role of Mi-2β in their regulation. Thus, Mi-2β provides the hematopoietic system with immune cell capabilities as well as with an extensive regenerative capacity.

Ikaros DNA binding factor plays critical roles in lymphocyte development. Changes in Ikaros expression levels during lymphopoiesis are controlled by redundant but also unique regulatory elements of its locus that are critical for this developmental process. We have recently shown that Ikaros binds its own locus in thymocytes in vivo. Here, we evaluated the role of an Ikaros binding site within its major lympho-myeloid promoter. We identified an Ikaros/Ets binding site within a promoter sub-region that was highly conserved in mouse and human. Deletion of this binding site increased the percentage of the reporter-expressing mouse lines, indicating that its loss provided a more permissive chromatin environment. However, once transcription was established, the lack of this site decreased transcriptional activity. These findings implicate a dual role for Ikaros/Ets1 binding on Ikzf1 expression that is exerted at least through its promoter.

Regulated expression of Ikaros is critical for normal hemopoiesis and lymphocyte development. To elucidate the mechanisms underlying transcription of Ikaros, tissue-specific DNase I-hypersensitive sites (DHS) were mapped throughout the Ikaros locus, and several promoters were identified. The activity of these regulatory regions was elucidated using an enhanced green fluorescent protein (EGFP) reporter in transgenic mice. Two genomic fragments, each containing a distinct promoter and its associated DHS cluster, were found to be active in the myeloid (DHS-C2 and DHS-C3) and B-cell (DHS-C3) lineages. Although neither of these regulatory regions was active within the majority of differentiating thymocytes and mature T cells, the DHS-C3 region was active at the earliest stages (DN1–DN3) of T-cell differentiation. However, when the DHS-C3 region was combined with the downstream intronic DHS-C6 cluster, its activity was maintained and raised to higher levels at subsequent stages of T-cell differentiation. This combination of regulatory elements provided reporter expression that closely resembles that of endogenous Ikaros during hemo-lymphopoiesis, and it decreased (but did not alleviate) position effect variegation within the expressing cell types.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of precursor B-lymphocytes affecting both children and adults. Deletions and dominant-negative mutations in IKZF1, the gene encoding the Ikaros transcription factor, are found in ~85% of Ph+ B-ALL and in some cases of Ph– B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph– or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and imatinib have been added to chemotherapy regimens for Ph+ B-ALL, over half of these patients will still relapse, which correlates with residual disease burden in the bone marrow (BM) following induction therapy. Hence, new therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph– B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) where the recombined allele is similar to the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated recently that Ikaros DNA-binding function is required in the B-lymphoid lineage for transition from the large to small pre-B cell stage of differentiation, and that arrest at this stage of development can give rise to B-ALL (Joshi et al., Nat. Immunol. 2014;15:294). The survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK, downstream of integrins and growth factor receptors, which plays important roles in cancer stem cell biology, the tumor microenvironment and tumorigenesis. VS-4718 and VS-6063 (defactinib) are potent, orally bioavailable FAK inhibitors that inhibit tumor growth and metastasis in preclinical models, and are currently under evaluation in clinical trials in patients with various solid tumors. VS-6063 has demonstrated tolerability and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in phase I trials (ASCO, 2014). Here, we show that BCR-ABL1 cooperates with Ikzf1 mutation to accelerate B-leukemogenesis in mice. BCR-ABL1+ Ikaros-mutant B-ALLs exhibit stroma-mediated resistance to ABL1 TKIs, while the FAK inhibitors VS-4718 and VS-6063 are effective in blocking stromal adhesion and inducing apoptosis in both mouse and human Ikaros-mutant B-ALL samples. To test whether dysregulation of TK signaling cooperates with Ikzf1 mutation in the pathogenesis of high-risk B-ALL, we isolated BM B-lymphoid progenitor cells from wild-type (WT), IkE5fl/+ CD2-Cre, and IkE5fl/fl CD2-Cre donors, transduced them with BCR-ABL1 retrovirus and transplanted the cells into recipient mice. We observed a dramatic acceleration of precursor B-lymphoid leukemia induced by BCR-ABL1 in IkE5Δ/+ and particularly in IkE5Δ/Δ donor cells that correlated with a striking (~30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Relative to Ikzf1 WT BCR-ABL1+ leukemic cells, Ikzf1-mutant BCR-ABL1+ blasts showed significant resistance to imatinib and dasatinib that was dependent on the presence of OP9 stroma. The effect of FAK inhibition, using the FAK inhibitors VS-4718, VS-6062, and VS-6063 (Verastem), was first tested on murine B-ALL cells (genotypes Ikzf1 mutant, Ikzf1 mutant BCR-ABL1+, and Ikzf1 WT BCR-ABL1+) grown on OP9 stroma. FAK inhibitor treatment abolished stromal adhesion of Ikzf1-mutant B-ALL and induced apoptosis in non-adherent cells, but had little effect on Ikzf1 WT B-ALL cells. VS-4718 and VS-6063 were each synergistic with dasatinib in reducing the viability of Ikzf1-mutant BCR-ABL1+ B-ALL cells cultured on OP9 stroma. For primary human B-ALL samples grown on OP9 stroma, IKZF1-mutant cells were also more sensitive to FAK inhibitor treatment than WT IKZF1 WT B-ALL, with or without BCR-ABL1 expression. Collectively, these observations suggest a new model to explain the pathogenesis of high-risk B-ALL and its resistance to therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM LSCs. Inhibition of FAK signaling in Ph+ or Ph­–IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. Therefore, FAK inhibitors warrant further investigation for the treatment of high-risk IKZF1-mutant B-ALL patients. Disclosures Joshi: Verastem: Research Funding. Yoshida:Verastem, Inc.: Research Funding. Paraskevopoulou:Verastem, Inc.: Research Funding. Zhang:Verastem, Inc.: Research Funding. Krause:Glycomimetics. Inc.: Research Funding. Shapiro:Verastem: Employment, Equity Ownership. Weaver:Verastem: Employment, Equity Ownership. Pachter:Verastem Inc.: Employment, Equity Ownership. Georgopoulos:Verastem, Inc.: Research Funding.

Precise control of gene expression plays fundamental roles in brain development, but the roles of chromatin regulators in neuronal connectivity have remained poorly understood. We report that depletion of the NuRD complex by invivo RNAi and conditional knockout of the core NuRD subunit Chd4 profoundly impairs the establishment of granule neuron parallel fiber/Purkinje cell synapses in the rodent cerebellar cortex invivo. By interfacing genome-wide sequencing of transcripts and ChIP-seq analyses, we uncover a network of repressed genes and distinct histone modifications at target gene promoters that are developmentally regulated by the NuRD complex in the cerebellum invivo. Finally, in a targeted invivo RNAi screen of NuRD target genes, we identify a program of NuRD-repressed genes that operate as critical regulators of presynaptic differentiation in the cerebellar cortex. Our findings define NuRD-dependent promoter decommissioning as a developmentally regulated programming mechanism that drives synaptic connectivity in the mammalian brain.

Survivin is expressed in the nucleus and/or cytoplasm of various types of malignant tumor cells. Nuclear survivin is indispensable for complete mitosis, while cytoplasmic survivin functions as an apoptosis inhibitor. We examined the difference in the survivin expression among stromal cells of fibroadenoma, and benign and malignant phyllodes tumors.Tumor sections were immunohistochemically stained with an anti-human survivin antibody and the labeling index of survivin was calculated.In stromal cells of all tumors, survivin was expressed in the nuclei but not in the cytoplasm. The labeling indices of the stromal cells in five malignant phyllodes tumors (20.5±3.0) were significantly greater than those observed in eight fibroadenomas (1.9±0.6) or nine benign phyllodes tumors (3.0±0.9).In the present study it was shown that stromal cells in malignant phyllodes tumors express nuclear survivin more extensively than stromal cells in benign phyllodes tumors or fibroadenomas.

Deletion of the DNA-binding domain of the transcription factor Ikaros generates dominant-negative isoforms that interfere with its activity and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemia (B-ALL). Here we found that conditional inactivation of the Ikaros DNA-binding domain in early pre-B cells arrested their differentiation at a stage at which integrin-dependent adhesion to niches augmented signaling via mitogen-activated protein kinases, proliferation and self-renewal and attenuated signaling via the pre-B cell signaling complex (pre-BCR) and the differentiation of pre-B cells. Transplantation of polyclonal Ikaros-mutant pre-B cells resulted in long-latency oligoclonal pre-B-ALL, which demonstrates that loss of Ikaros contributes to multistep B cell leukemogenesis. Our results explain how normal pre-B cells transit from a highly proliferative and stroma-dependent phase to a stroma-independent phase during which differentiation is enabled, and suggest potential therapeutic strategies for Ikaros-mutant B-ALL.

electric impulse単独の正常肝への影響および肝癌への治療効果についてラットを用い検討した. 正常肝と癌周囲に対し2または4本の針状電極を電極間距離10mmで肝葉に刺入し, 100μsecの矩形パルスを8パルス通電した. 通電電圧を500, 1, 000, 1, 500V/cmとし, 通電形態を電極2本通電, 4本4辺通電, 4本4辺と対角線を加えた6辺通電に分け検討した. 正常肝での肝機能は, 通電電圧に相関して上昇し, 壊死範囲を反映していた. 正常肝および肝癌の通電では, いずれも通電電圧に相関して完全壊死頻度が上昇し, 4辺1, 500V/cm, 6辺1, 000V/cmにより完全壊死が得られた. 7日後の組織所見は, 通電部のみ肉芽組織に置換されていた. 副作用は, electric impulseに同調した筋収縮のみで, 他には認められなかった. electric impulse単独療法は, 電圧により目的とする部位の完全壊死が副作用なく得られ, 肝癌の新しい治療法となる可能性が示唆された.

Ikaros is a critical regulator of lymphocyte development and homeostasis; thus, understanding its transcriptional regulation is important from both developmental and clinical perspectives. Using a mouse transgenic reporter approach, we functionally characterized a network of highly conserved cis-acting elements at the Ikzf1 locus. We attribute B-cell and myeloid but not T-cell specificity to the main Ikzf1 promoter. Although this promoter was unable to counter local chromatin silencing effects, each of the 6 highly conserved Ikzf1 intronic enhancers alleviated silencing. Working together, the Ikzf1 enhancers provided locus control region activity, allowing reporter expression in a position and copy-independent manner. Only 1 of the Ikzf1 enhancers was responsible for the progressive upregulation of Ikaros expression from hematopoietic stem cells to lymphoid-primed multipotent progenitors to T-cell precursors, which are stages of differentiation dependent on Ikaros for normal outcome. Thus, Ikzf1 is regulated by both epigenetic and transcriptional factors that target its enhancers in both redundant and specific fashions to provide an expression profile supportive of normal lymphoid lineage progression and homeostasis. Mutations in the Ikzf1 regulatory elements and their interacting factors are likely to have adverse effects on lymphopoiesis and contribute to leukemogenesis., link_to_OA_fulltext

Cell fate depends on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2β nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid lineage-determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros DNA-binding activity caused a local increase in chromatin remodeling and histone deacetylation and suppression of lymphoid cell-specific gene expression. Without Ikaros, the NuRD complex also redistributed to transcriptionally poised genes that were not targets of Ikaros (encoding molecules involved in proliferation and metabolism), which induced their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks.

In radiation heat transfer evaluation, the hemispherical emittance spectrum is the most important characteristic quantity. This emittance is directly related to the hemispherical reflectance RHH for perfect-diffuse hemispherical irradiation. However, there has been no proper technique for measuring RHH of real surfaces which reflect radiation imperfect-diffusely. In this paper, we present a new reflectometer system for measuring a spectrum of RHH in a wavelength region of 0.35-1.10μm. This system has two ellipsoidal mirrors and a beam splitter to realize the perfect-diffuse irradiation and the hemispherical reflection detection. This system also measures the spectra of normal- and obliqueincident hemispherical reflectances. We apply this technique to the investigation of the spectral and angular characteristics of reflection from rough surfaces of mild steel, and demonstrate the capability of the system in a fundamental study of radiation characteristics of materials.

We established a new method of plasma treatment for the removal of interfering factors in the plasma to allow detection of endotoxin by limulus test. The limulus test used was an endotoxin-specific chromogenic test, the Endospecy test. Perchloric acid (PCA) treatment and centrifugation (PCA method) is usually used to remove interfering factors from plasma, with the precipitate being discarded and the supernatant used to detect endotoxin. As the solubilized precipitates of endotoxin-spiked plasma and some patient plasma were found to contain the Endospecy activity, we have devised a new method assaying endotoxin in both the supernatant and precipitate. This study confirmed that the solubilized precipitate of endotoxin-spiked plasma had Endospecy activity and found that the precipitate had other endotoxin activities, such as lethality in galactosamine-sensitized mice and pyrogenicity in rabbits. We also confirmed that interfering factors were completely removed from plasma samples by this new method. The endotoxin level after the new PCA method was found to be about 8 times higher than that determined after PCA treatment and the new PCA method surpasses the conventional PCA method with regard to the positive rate of endotoxin contents in clinical samples. These results indicate that the new PCA method is superior to the PCA method as a plasma pretreatment method for limulus test.

A new reflectometer system is developed for measuring the radiative characteristics of solid materials from a thermal engineering point of view. This system employs an ellipsoidal mirror in a simple optical alignment, and measures the spectrum of the normal-incident hemispherical reflectance in a wavelength region from 0.35 to 1.10μm. A single spectral scanning offers the hemispherical reflectance spectrum which completely covers both of the specular and diffuse components of reflection. The reflectance is directly interpreted into the absorptance for the normal incidence. The system also measures the angular dependence of reflection readily. The system is applied to a systematic study on the reflection characteristics of rough metallic surfaces. The predominant contribution of the specular component in the angular characteristics is demonstrated.

漢方薬が原因と推定された劇症肝炎の1例を経験した.症例は62歳男性.痔核治療のため漢方薬(金鵄丸)の服用を開始したところ,6週間後に倦怠感と尿濃染が出現した.服用中止により一旦症状の消失をみたが,服薬再開後5週間で上記症状が再出現,黄疸の出現をみ入院となった.凝固能低下が著明で(PT 28%, HPT 19%),種々の治療にもかかわらず,4週間後多臓器不全の状態で死亡した.剖検肝は495gと萎縮著明で,肝組織像は広範性肝壊死を示した.金鵄丸による薬剤性肝炎は本例を含め9例が報告されている.その特徴は,金鵄丸が原因との認識が遅れたため反復服用により肝炎の繰り返しをみる例が多いこと,発疹,好酸球増多がみられないことであった.本例は,漢方薬により劇症肝炎を来した初めての報告である.漢方薬の使用が増加しているおり,漢方薬によっても本例のごとき重症肝障害が惹起されうることに注意すべきである.

Deletions and dominant-negative mutations in IKZF1, the gene encoding Ikaros transcription factor, are found in ∼85% of Ph+ B-ALL and in some cases of Ph− B-ALL, and are associated with poor prognosis. Genomic studies of high-risk Ph− or “Ph-like” B-ALLs have revealed frequent mutation and activation of TK genes and signaling pathways. While ABL1 tyrosine kinase inhibitors (TKIs) such as dasatinib and ponatinib have been incorporated into chemotherapy regimens for Ph+ B-ALL, the majority of patients still relapse, which correlates with residual bone marrow disease following induction therapy. New therapeutic strategies are needed for patients with Ikaros-mutant, high-risk Ph+ and Ph− B-ALL. Using mice with a conditional Ikzf1 mutation (Ike5fl) that mimics the dominant-negative Ik6 mutant found in human B-ALL, we demonstrated that loss of Ikaros DNA-binding function arrests B-lymphoid development at a large pre-B cell stage that can give rise to B-ALL. Survival and proliferation of Ikaros mutant pre-B cells is dependent on increased integrin-mediated stromal adhesion and activation of focal adhesion kinase (FAK). FAK is a non-receptor TK downstream of integrins and growth factor receptors that plays important roles in cancer stem cell biology and the tumor microenvironment. Here, we show that dysregulated tyrosine kinases, including BCR-ABL1, cooperate with Ikaros mutation to accelerate the development of B-lymphoblastic leukemia in mice, correlated with a striking (∼30-fold) increase in the frequency of engrafting leukemia-initiating or leukemic stem cells (LSCs). Ikzf1-mutant BCR-ABL1+ lymphoblasts exhibit relative resistance to ABL1 TKIs including dasatinib that is dependent on the presence of stroma. VS-4718 is a potent, orally bioavailable FAK inhibitor that inhibits tumor growth and metastasis in preclinical models, and is currently under evaluation in clinical trials in patients with various solid tumors. VS-4718 treatment abolished stromal adhesion and induced apoptosis of Ikzf1-mutant B-ALL and synergized with dasatinib, but had little effect on Ikzf1 WT B-ALL cells. Primary human Ph+ B-ALL samples showed a correlation between IKZF1 mutation status, stromal adherence, and sensitivity to FAK and ABL inhibitors in vitro, and BM tropism in vivo in xenografted NSG mice. Results from ongoing in vivo therapy experiments will be presented. Together, these results suggest a new model for the pathogenesis of high-risk B-ALL and its resistance to conventional therapy. B-ALLs with IKZF1 mutations may be resistant to TKIs and to chemotherapy by virtue of their stromal adhesion phenotype, resulting in failure to eliminate BM leukemic stem cells. Inhibition of FAK signaling in Ph+ or Ph− IKZF1-mutant B-ALL may reverse the stromal-mediated resistance to ABL1 TKIs and/or chemotherapy. FAK inhibitors represent a promising new approach for the treatment of high-risk IKZF1-mutant B-ALL patients. Citation Format: Nilamani Jena, Ila Joshi, Toshimi Yoshida, Zhihong Zhang, Zhong-Ying Liu, Prasanthi Tata, Irina M. Shapiro, Jonathan A. Pachter, David T. Weaver, Katia Georgopoulos, Richard A. Van Etten. Targeting focal adhesion kinase is a novel approach to therapy of high-risk, Ikaros-mutant acute B-cell lymphoblastic leukemia. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2015-4202

Ikaros is expressed in early hematopoietic progenitors and is required for lymphoid differentiation. Analysis of Ikaros null populations revealed a lack of defining markers for early fate-restricted progenitors, but it was difficult to discern whether Ikaros was required for formation of these populations, or for expression of these markers. Here we use a GFP reporter based on Ikaros regulatory elements to identify the HSC and separate early progenitors in both wild-type and Ikaros-null mice. The presence of lympho-myeloid progenitors is revealed in Ikaros-null mice, which lack the defining factor Flt3 and are capable of myeloid, but not lymphoid differentiation. In contrast, lack of Ikaros in the common myeloid progenitor results in increased formation of erythro-megakaryocyte at the expense of myeloid progenitors and influences their subsequent differentiation. By this approach, pivotal roles for Ikaros in distinct fate decisions in the early hematopoietic hierarchy are revealed.

Deletion of the IKAROS DNA-binding domain generates dominant-negative isoforms that interfere with the transcriptional activity of the IKAROS family and correlate with poor prognosis in human precursor B cell acute lymphoblastic leukemias (B-ALL). In this study, we defined the role of the Ikaros family during pre-B cell differentiation, the stage from which human B-ALLs arise, by conditionally inactivating IKAROS DNA binding in the immediate precursors of pre-B cells in mice. We demonstrate a novel niche-dependent phase in early pre-B cell differentiation that supports self-renewal and proliferative expansion. Expression of dominant-negative IKAROS arrests cells in this state by augmenting integrin and MAPK signaling and attenuating pre-B cell receptor signaling and differentiation. Up-regulated genes in Ikaros mutant pre-B cells were highly enriched in pathways involved in focal adhesion and remodeling of the actin cytoskeleton. The mutant pre-B cells had increased β1 integrin-mediated adhesion and elevated levels of activated focal adhesion kinase (FAK), whereas treatment with a small molecule FAK inhibitor greatly reduced pre-B cell stromal adhesion and selectively induced apoptosis in Ikaros mutant but not WT pre-B cells. Transplantation of polyclonal Ikaros mutant pre-B cells into recipient mice resulted in long-latency oligoclonal pre-B-ALL, demonstrating that loss of IKAROS contributes to multistep B-leukemogenesis. The highly proliferative and aberrantly self-renewing phenotype of Ikaros-deficient pre-B cells illuminates mechanisms underlying human IKAROS mutant B-ALL and suggests new therapeutic strategies for treatment of this aggressive leukemia. Disclosures: Van Etten: Bristol Myers Squibb: Consultancy; Deciphera Pharmaceuticals: Consultancy; TEVA Pharmaceuticals: Consultancy, Research Funding.

We generated interleukin-5 receptor alpha chain (IL-5R alpha)-deficient (IL-5R alpha-/-) mice by gene targeting. The IL-5R alpha-/- mice showed decreased numbers of B-1 cells concomitant with low serum concentrations of IgM and IgG3. They showed no IL-5-induced enhancement of B cell responses to T-independent antigens. The number of alpha beta T cell receptor-positive thymocytes tended to decrease in 3-week-old IL-5R alpha-/- mice, returning to normal by 6 weeks of age. The IL-5R alpha-/- mice produced basal levels of eosinophils, while their bone marrow cells failed to form eosinophilic colonies in response to IL-5. Impaired eosinophilopoiesis in IL-5R alpha-/-mice enhanced the survival of Angiostrongylus cantonensis. These results indicate that IL-5-induced eosinophils serve as potent effector cells in the killing of Angiostrongylus cantonensis in mice.

Serum FDP was found to be elevated in patients with liver cirrhosis, but we are still unable to clearly differentiate between primary fibrinolysis and fibrinolysis secondary to disseminated intravascular coagulation (DIC)Whether elevated FDP shows secondary fibrinolysis or not was investigated in 31 patients with decompensated liver cirrhosis.The following tests were performed: hepaplastin test, prothrombin time, anti-thrombin III, plasminogen activator, fibrinogen, plasminogen, α2 plasmin inhibitor and FDP.The following results were obtained.1) Decompensated liver cirrhosis is associated with hypercoagulable and hyper-fibrinolytic states.2) The patients with 20μg/ml or less of FDP (25 of 31 cases (84%) of decompensated liver cirrhosis), were not in hypercoagulable state but were in hyper-fibrinolytic state . Therefore, we thought that these cases do not have a tendency of developing DIC.3) On the other hand, in the patients with 40μg/ml or more of FDP (5 of 31 cases (16%) of decompensated liver cirrhosis), hypercoagulable and hypofibrinolytic states were in front. Therefore, these cases have secondary fibrinolysis.

Hepaplastintest, thrombotest, prothrombin time and blood coagulation factors II, V, VII, VIII, IX and X were measured in 9 cases with fulminant hepatitis, in 34 cases with acute hepatitis and in mice with MHV-2 hepatitis. Also carbon index as pagocytic activity was measured in mice with MHV-2 hepatitis. The following results were obtained.1) The activities of the blood coagulation except for factor VIII showed a decrease in fulminant hepatitis and in acute stage of acute hepatitis as compared to healthy subjects, especially in fulminant hepatitis.2) In acute hepatitis including fulminant hepatitis, there was a correlation between the decrease of the activities of blood coagulation except for factor VIII and severity of the disease.3) In mice with MHV-2 hepatitis, the activities of the blood coagulation except for factor VIII decreased with the progression of hepatic necrosis. But the activities of factor VIII increased in early stage, and decreased in terminal stage of MHV-2 hepatitis with parallel to the pagocytic activity.Therefore, it is believed that the determination of blood coagulation tests in hepatitis is useful to determine the diagnosis and prognosis of the diseases, and that the changes of factor VIII in MHV-2 hepatitis are closely related to the function of RES.

Previously, Lee & Walsh reported that the FDP was cleared by the RES. And we reported previously that the change of blood coagulation factor VIII are closely related to the activity of the RES.Hepaplastintest (HPT), Thrombotest (TT), Prothrombin time (PT), blood coagulation factors I, V, VII, VIII, plasminogen, plasmin inhibitor plasminogen activator and FDP were measured in 5 patients with fulminant hepatitis and in mice with MHV-2 hepatitis.The following results about FDP in blood were obtained.1) In mice with MHV-2 hepatitis, the FDP in blood increased with the progression of hepatic necrosis. And furthermore, hepatic necrosis progressed, the FDP in blood decreased.2) Increased FDP and increased factor VIII suggested primary fibrolysis.3) Increased FDP and decreased factor VIII suggested secondary fibrinolysis.4) Decreased FDP and increased factor VIII suggested hyperclearance of FDP by the RES.5) Decreased FDP and normal factor VIII suggested slight hepatic necrosis.6) Decreased FDP and factor VIII suggested final stage.Therefore, FDP was determined by two important factors producing FDP by hepatic necrosis and clearing FDP by the RES.

円形および縦フイン付円管放熱器に関して,真空容器内でふく射伝熱実験を行い,相互ふく射を考慮したフイン列のふく射伝導伝熱の解析結果と有効度,フイン温度分布について比較した.円形フインの場合は両端フインの放熱量が大きいために系全体の有効度では計算結果より20～30%高いが系の中央部では有効度温度分布ともに良好な一致をみた.縦フインの場合は系全体の有効度で高温領域の4～5%の偏差を最大とする一致を得た.

自然対流ふく射同時熱伝達下のフィン付平面放熱器に関して自然対流ふく射複合伝熱解析をもとに,まず,基板温度が一様な場合についてフィン体積さらにはフィン占有体積をも評価した目的量に関して最適計算を行ない,最適形状(フィン数,フィン間隔,フィン高さ,フィン厚み)を明らかにした.つぎに,基板の一部のみが等温である場合についての伝熱解析手法を提案し,実験との比較によりその有用性を明らかにした.

HBe抗体陽性キャリア妊婦から垂直感染し出生50日目に出生児が急性B型肝炎を発症し,さらに患児の発症から92日目に父親がB型劇症肝炎を発症し救命しえた家族内感染例を経験した.患児は発熱・哺乳力の低下で発症.GOT 11,380U, GPT 5,060Uと上昇しプロトンビン時間(PT)10%と著明な低下を示した.HBs-Ag陽性,anti HBc (200倍)74%であった.総ビリルビン(T. Bil)が漸増し32mg/dlとなり凝固能の改善がないため,プレドニゾロンを使用したところ改善し,急性肝炎重症型と考えられた.父親は嘔気・全身倦怠感で発症.T. Bil 14.7mg/dl, GOT 1,675U, GPT 4,690U, PT12%, HBsAg陽性,IgMHBc3.29と陽性.昏睡II度となりプレドニゾロン,グルカゴン-インスリン療法,特殊組成アミノ酸などの治療にて改善した.母親から出生児に垂直感染し,患児から父親に水平感染したと思われる.HBe抗体陽性キャリア妊婦からの出生児に対してもHBIGなどの対策が必要と考えられる症例である.

Sjogren症候群(SjS)を合併した原発性胆汁性肝硬変症(PBC)において,肝,唾液腺とともに,膵を生検材料にて検索した.症例は55歳女性,黄疸と掻痒感を主訴とし,トランスアミナーゼの軽度上昇と,Al-P,γ-GTP, IgMの著明高値を認めた.抗ミトコンドリア抗体陽性であり,肝は,ckronic non-suppurative destructive cholangitisの組織像であった.乾燥症状を有し,唾液腺は,典型的SjSの像を呈した.経過中,膵型アイソザイム優位のアミラーゼ高値持続をみたため,外科的肝生検と同時に膵生検を行なった.膵には,び漫性に,小円形細胞の浸潤巣が認められ,同部の腺房細胞の退行性変化は著明であった.これらの像は,いずれも非特異的な変化とも考えられたが,同胞女性に無症候性PBCや高アミラーゼ血症が認められたことから,膵障害がPBCやSjSと病因の共通性を有する可能性も否定はできなかった.本例はPBCの膵を生検材料にて検索し得た初めての報告である.

Here we investigate the mechanisms that underlie the induction of developmental potential and establishment of cell fate during early hematopoiesis. A cascade of lineage-affiliated gene expression signatures, primed in hematopoietic stem cells (HSC) and differentially propagated in lineage-restricted progenitors, is identified. First evidence is provided for a stochastic sampling of lymphoid, erythroid and myeloid transcripts in HSC and multipotent progenitors (MPP). Multi-lineage priming is subsequently resolved upon lineage restrictions. Nonetheless, an unexpected association of lymphoid and myeloid signatures is detected past a nominal myeloid restriction point and a previously unappreciated lymphoid potential is revealed for this stage in development. New insight is provided into Ikaros' role as a bivalent regulator of multi-lineage priming during early hematopoiesis. Whereas Ikaros is responsible for activation of a cascade of lymphoid signatures in the HSC, at subsequent restriction points it is also involved in the repression of lineage-inappropriate signatures including stem cell-specific genes.

Rare individuals with inactivating mutations in the Huntington’s disease gene (HTT) exhibit variable abnormalities that imply essential HTT roles during organ development. Here we report phenotypes produced when increasingly severe hypomorphic mutations in the murine HTT orthologue Htt, (HdhneoQ20, HdhneoQ50, HdhneoQ111), were placed over a null allele (Hdhex4/5). The most severe hypomorphic allele failed to rescue null lethality at gastrulation, while the intermediate, though still severe, alleles yielded recessive perinatal lethality and a variety of fetal abnormalities affecting body size, skin, skeletal and ear formation, and transient defects in hematopoiesis. Comparative molecular analysis of wild-type and Htt-null retinoic acid-differentiated cells revealed gene network dysregulation associated with organ development that nominate polycomb repressive complexes and miRNAs as molecular mediators. Together these findings demonstrate that Htt is required both pre- and post-gastrulation to support normal development., Author summary The HTT gene mutated in Huntington’s Disease (HD) has essential roles during normal development. However, still not fully understood are the functional consequences of its partial inactivation. Our genetic study provides a comprehensive description of the effects of progressively more severe decreases in expression of Htt, the murine HTT counterpart. The most severe Htt decrease leads to lethality of early embryos, while intermediate but still severely reduced Htt dosages yield a variety of recessively inherited developmental abnormalities affecting body size, skin, skeletal and ear formation, and hematopoiesis. Comparative molecular analysis of differentiating wild-type cells and cells lacking Htt function further elucidate genes networks dysregulated during organ development. These nominate chromatin regulators and short non-coding RNAs as key molecular mediators. Together these findings demonstrate that Htt is required from conception to support normal embryonic and fetal development.