Polycomb repressive complex 1.1 coordinates homeostatic and emergency myelopoiesis.

Polycomb repressive complex (PRC) 1 regulates stem cell fate by mediating mono-ubiquitination of histone H2A at lysine 119. While canonical PRC1 is critical for hematopoietic stem and progenitor cell (HSPC) maintenance, the role of non-canonical PRC1 in hematopoiesis remains elusive. PRC1.1, a non-canonical PRC1, consists of PCGF1, RING1B, KDM2B, and BCOR. We recently showed that PRC1.1 insufficiency induced by the loss of PCGF1 or BCOR causes myeloid-biased hematopoiesis and promotes transformation of hematopoietic cells in mice. Here we show that PRC1.1 serves as an epigenetic switch that coordinates homeostatic and emergency hematopoiesis. PRC1.1 maintains balanced output of steady-state hematopoiesis by restricting C/EBPα-dependent precocious myeloid differentiation of HSPCs and the HOXA9- and ß-catenin-driven self-renewing network in myeloid progenitors. Upon regeneration, PRC1.1 is transiently inhibited to facilitate formation of granulocyte-macrophage progenitor (GMP) clusters, thereby promoting emergency myelopoiesis. Moreover, constitutive inactivation of PRC1.1 results in unchecked expansion of GMPs and eventual transformation. Collectively, our results define PRC1.1 as a novel critical regulator of emergency myelopoiesis, dysregulation of which leads to myeloid transformation.

PCGF1-PRC1 links chromatin repression with DNA replication during hematopoietic cell lineage commitment.

Polycomb group proteins (PcG), polycomb repressive complexes 1 and 2 (PRC1 and 2), repress lineage inappropriate genes during development to maintain proper cellular identities. It has been recognized that PRC1 localizes at the replication fork, however, the precise functions of PRC1 during DNA replication are elusive. Here, we reveal that a variant PRC1 containing PCGF1 (PCGF1-PRC1) prevents overloading of activators and chromatin remodeling factors on nascent DNA and thereby mediates proper deposition of nucleosomes and correct downstream chromatin configurations in hematopoietic stem and progenitor cells (HSPCs). This function of PCGF1-PRC1 in turn facilitates PRC2-mediated repression of target genes such as Hmga2 and restricts premature myeloid differentiation. PCGF1-PRC1, therefore, maintains the differentiation potential of HSPCs by linking proper nucleosome configuration at the replication fork with PcG-mediated gene silencing to ensure life-long hematopoiesis.

Genetic, Genomic, and Imaging Approaches to Dissect the Role of Polycomb Group Epigenetic Regulators in Mice.

Among the most important histone methyltransferases for metazoan development are EZH1/2 and their homologs, which methylate histone H3 lysine 27 and act as part of a highly conserved set of chromatin regulators called Polycomb Group (PcG) proteins. Reaching a precise understanding of the roles of PcG proteins in the orchestration of differentiation and the maintenance of cell identity requires a variety of genetic and molecular approaches. Here, we present a full suite of methods for the study of PcG proteins in early murine development, including mutant strain generation, embryonic stem cell derivation, epigenomic profiling, and immunofluorescence and in situ hybridization.

Vaccination with Intradermal Bacillus Calmette-Guérin Provides Robust Protection against Extrapulmonary Tuberculosis but Not Pulmonary Infection in Cynomolgus Macaques.

Recently, the efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccination is being reassessed in accordance with the achievements of clinical tuberculosis (TB) vaccine research. However, the mechanisms ultimately determining the success or failure of BCG vaccination to prevent pulmonary TB remain poorly understood. In this study, we analyzed the protective effects of intradermal BCG vaccination by using specific pathogen-free cynomolgus macaques of Asian origin that were intradermally vaccinated with BCG (Tokyo strain) followed by Mycobacterium tuberculosis (Erdman strain) infection. Intradermal BCG administration generated TB Ag-specific multifunctional CD4 T cell responses in peripheral blood and bronchoalveolar lavage and almost completely protected against the development of TB pathogenesis with aggravation of clinical parameters and high levels of bacterial burdens in extrapulmonary organs. However, interestingly, there were no differences in bacterial quantitation and pathology of extensive granulomas in the lungs between BCG-vaccinated monkeys and control animals. These results indicated that the changes in clinical parameters, immunological responses, and quantitative gross pathology that are used routinely to determine the efficacy of TB vaccines in nonhuman primate models might not correlate with the bacterial burden and histopathological score in the lung as measured in this study.

Development of an effective alternative model for in vivo hypnozoite-induced relapse infection: A Japanese macaque (Macaca fuscata) model experimentally infected with Plasmodium cynomolgi.

In the present study, we demonstrate that the Japanese macaque (Macaca fuscata) can be used as an effective alternative in vivo model for investigating hypnozoite-induced relapsing infection caused by Plasmodium cynomolgi B strain, and that this model is comparable to the rhesus macaque model. Two female Japanese macaques (JM-1 and JM-2; aged 5 years; weighing about 4.0 kg) were used for the experiment. To produce sporozoites in mosquitoes, blood infected with P. cynomolgi B strain was collected from the donor monkey JM-1 and fed to approximately 200 mosquitoes using the standard artificial membrane feeding method. The isolated sporozoites (2 × 105) were intravenously inoculated into the JM-2 monkey, and the blood stage of the parasite was detected on day 8 after the infection. Chloroquine sulfate (CQ) was intramuscularly administered at a dosage of 6.0 mg/kg into the JM-2 monkey for 6 consecutive days from day 12 onward, after which the parasites disappeared from the peripheral blood. The first relapse occurred on day 26, which was treated again with CQ. Then, the second relapse occurred on day 44, which was cured by CQ treatment followed by the administration of primaquine phosphate (PQ) at a dosage of 1.0 mg/kg/day for 15 days. The JM-2 monkey was observed until 69 days after PQ administration, and there was no relapse during the entire follow-up period. We propose that the Japanese macaque model could contribute not only to drug screening for anti-hypnozoite activity, but could also be used as a powerful tool for investigating hypnozoite biology.

Intranasal vaccination with HBs and HBc protein combined with carboxyl vinyl polymer induces strong neutralizing antibody, anti-HBs IgA, and IFNG response.

Hepatitis B virus (HBV) infection causes acute and chronic hepatitis, which is a major public health concern worldwide. Immunization methods incorporating hepatitis B surface-small (HBs-S) antigen and hepatitis B core antigen (HBc) have been proposed as candidate therapeutic vaccines, but the elimination of existing HBV infection remains a challenge. To enhance the efficacy of HBs and HBc vaccination, we investigated HBs-large (HBs-L) as an immunogen, and carboxyl vinyl polymer (CVP) as an excipient. HBs-S or HBs-L, in combination with HBc antigen, was administered subcutaneously (without CVP) or intranasally (with or without CVP) for the evaluation of immune response in the tree shrew, which is considered to be a suitable small animal model of HBV infection. Immunization with HBs-L antigen by either route induced a rapid IgG response. Intranasal immunization with HBs-S or HBs-L and HBc formulated with CVP strongly induced neutralizing antibody activity, IgA response, and HBc-specific expression of the interferon gamma-encoding gene. These data indicated the potential of HBs-L and HBc intranasal immunization with CVP, not only as a therapeutic vaccine, but also as a prophylactic vaccine candidate.

Construction of complete Tupaia belangeri transcriptome database by whole-genome and comprehensive RNA sequencing.

The northern tree shrew (Tupaia belangeri) possesses high potential as an animal model of human diseases and biology, given its genetic similarity to primates. Although genetic information on the tree shrew has already been published, some of the entire coding sequences (CDSs) of tree shrew genes remained incomplete, and the reliability of these CDSs remained difficult to determine. To improve the determination of tree shrew CDSs, we performed sequencing of the whole-genome, mRNA, and total RNA and integrated the resulting data. Additionally, we established criteria for the selection of reliable CDSs and annotated these sequences by comparison to the human transcriptome, resulting in the identification of complete CDSs for 12,612 tree shrew genes and yielding a more accurate tree shrew genome database (TupaiaBase: http://tupaiabase.org ). Transcriptome profiles in hepatitis B virus infected tree shrew livers were analyzed for validation. Gene ontology analysis showed enriched transcriptional regulation at 1 day post-infection, namely in the "type I interferon signaling pathway". Moreover, a negative regulator of type I interferon, SOCS3, was induced. This work, which provides a tree shrew CDS database based on genomic DNA and RNA sequencing, is expected to serve as a powerful tool for further development of the tree shrew model.

Effect of blue light-emitting diode light and antioxidant potential in a somatic cell.

Light is an indispensable part of routine laboratory work in which conventional light is generally used. Light-emitting diodes (LEDs) have come to replace conventional light, and thus could be a potent target in biomedical studies. Since blue light is a major component of visible light wavelength, in this study, using a somatic cell from the African green monkey kidney, we assessed the possible consequences of the blue spectra of LED light in future animal experiments and proposed a potent mitigation against light-induced damage. COS-7 cells were exposed to blue LED light (450 nm) and the growth and deoxyribonucleic acid (DNA) damage were assessed at different exposure times. A higher suppression in cell growth and viability was observed under a longer period of blue LED light exposure. The number of apoptotic cells increased as the light exposure time was prolonged. Reactive oxygen species (ROS) generation was also elevated in accordance to the extension of light exposure time. A comparison with dark-maintained cells revealed that the upregulation of ROS by blue LED light plays a significant role in causing cellular dysfunction in DNA in a time-dependent manner. In turn, antioxidant treatment has been shown to improve cell growth and viability under blue LED light conditions. This indicates that antioxidants have potential against blue LED light-induced somatic cell damage. It is expected that this study will contribute to the understanding of the basic mechanism of somatic cell death under visible light and maximize the beneficial use of LED light in future animal experiments.

Avian H5N1 influenza virus infection causes severe pneumonia in the Northern tree shrew (Tupaia belangeri).

Avian-origin influenza viruses like H5N1 and H7N9 often cause severe symptoms with high mortality in humans. Animal models are useful for clarification of the mechanisms of pathogenicity of these infections. In this study, to expand the potential utility of the Northern tree shrew (Tupaia belangeri) for influenza virus infection, we assessed the pathogenicity of H5N1 and H7N9 avian influenza viruses in tupaia. Infectious virus was detected continuously from nasal, oral, tracheal, and conjunctival swab samples in the animals infected with these viruses. H5N1 influenza virus infection of tupaia caused severe diffuse pneumonia with fever and weight loss. In contrast, H7N9 influenza virus infection caused focal pneumonia. The severity of pneumonia was correlated with proinflammatory cytokine transcript levels. These results indicated that tupaia can be another suitable animal model for avian influenza virus research.

Late-stage myeloma invades kidney without significant effect on renal function: findings from 53 autopsies in a single institute.

Renal manifestations of multiple myeloma (MM) including cast nephropathy, amyloidosis, and renal calcification have been widely recognized. However, the severity of histopathological findings has not been addressed so far, and the clinical significance of these pathological findings is unclear. We sought to clarify the relationship between the severity of renal pathology and clinical characteristics. We analyzed 53 autopsies performed on patients who died from MM. The kidneys were evaluated using light microscopy, and the severity of pathological findings was recorded. The most common renal lesion was cast nephropathy (n = 27). Other findings included amyloidosis (n = 10), renal calcification (n = 5), microbial infection (n = 4), and MM infiltration (n = 17). The incidence of MM infiltration was substantially higher than previously reported. Renal MM infiltration was detected even when bone marrow plasmacytosis was limited. However, a significantly higher degree of renal MM infiltration was observed when MM cells invaded the liver. No correlation was observed between serum creatinine levels and degree of MM infiltration, but these tended to be elevated when cast nephropathy was severe. These findings may provide clues to understand both renal injury and extramedullary diseases in patients with MM.

Three-step transcriptional priming that drives the commitment of multipotent progenitors toward B cells.

Stem cell fate is orchestrated by core transcription factors (TFs) and epigenetic modifications. Although regulatory genes that control cell type specification are identified, the transcriptional circuit and the cross-talk among regulatory factors during cell fate decisions remain poorly understood. To identify the "time-lapse" TF networks during B-lineage commitment, we used multipotent progenitors harboring a tamoxifen-inducible form of Id3, an in vitro system in which virtually all cells became B cells within 6 d by simply withdrawing 4-hydroxytamoxifen (4-OHT). Transcriptome and epigenome analysis at multiple time points revealed that ∼10%-30% of differentially expressed genes were virtually controlled by the core TFs, including E2A, EBF1, and PAX5. Strikingly, we found unexpected transcriptional priming before the onset of the key TF program. Inhibition of the immediate early genes such as Nr4a2, Klf4, and Egr1 severely impaired the generation of B cells. Integration of multiple data sets, including transcriptome, protein interactome, and epigenome profiles, identified three representative transcriptional circuits. Single-cell RNA sequencing (RNA-seq) analysis of lymphoid progenitors in bone marrow strongly supported the three-step TF network model during specification of multipotent progenitors toward B-cell lineage in vivo. Thus, our findings will provide a blueprint for studying the normal and neoplastic development of B lymphocytes.

Large Plasmacytoma Occupying the Upper Limb in a Myeloma Patient.

Extramedullary disease (EMD) is an issue for patients with multiple myeloma (MM), since extramedullary spread of MM is associated with an aggressive course and a poor prognosis. Moreover, the mechanism of EMD development is uncertain. Here, we present extensive extramedullary plasmacytoma occupying the left upper limb of a 66-year-old female patient with MM with an extremely aggressive course and multiple visceral organ involvement without bone marrow infiltration or plasma cell leukemia. EMD of this large size is extremely rare and this case may provide a clue for better understanding of clinical features of EMD in MM.

Interferon-ß response is impaired by hepatitis B virus infection in Tupaia belangeri.

To date, the chimpanzee has been used as the natural infection model for hepatitis B virus (HBV). However, as this model is very costly and difficult to use because of ethical and animal welfare issues, we aimed to establish the tupaia (Tupaia belangeri) as a new model for HBV infection and characterized its intrahepatic innate immune response upon HBV infection. First, we compared the propagation of HBV genotypes A2 and C in vivo in tupaia hepatocytes. At 8-10days post infection (dpi), the level of HBV-A2 propagation in the tupaia liver was found to be higher than that of HBV-C. Abnormal architecture of liver cell cords and mitotic figures were also observed at 8 dpi with HBV-A2. Moreover, we found that HBV-A2 established chronic infection in some tupaias. We then aimed to characterize the intrahepatic innate immune response in this model. First, we infected six tupaias with HBV-A2 (strains JP1 and JP4). At 28 dpi, intrahepatic HBV-DNA and serum hepatitis B surface antigens (HBsAg) were detected in all tupaias. The levels of interferon (IFN)-ß were found to be significantly suppressed in the three tupaias infected with HBV A2_JP4, while no significant change was observed in the three infected with HBV A2_JP1. Expression of toll-like receptor (TLR) 1 was suppressed, while that of TLR3 and TLR9 were induced, in HBV A2_JP1-infected tupaias. Expression of TLR8 was induced in all tupaias. Next, we infected nine tupaias with HBV-A2 (JP1, JP2, and JP4), and characterized the infected animals after 31 weeks. Serum HBsAg levels were detected at 31 weeks post-infection (wpi) and IFN-ß was found to be significantly suppressed in all tupaias. TLR3 was not induced, except in tupaia #93 and #96. Suppression of TLR9 was observed in all tupaias, except tupaia #93. Also, we investigated the expression levels of cyclic GMP-AMP synthase, which was found to be induced in all tupaias at 28 dpi and in four tupaias at 31 wpi. Additionally, we evaluated the expression levels of sodium-taurocholate cotransporting polypeptide, which was found to be suppressed during chronic HBV infection. Thus, the tupaia infection model of HBV clearly indicated the suppression of IFN-ß at 31 wpi, which might have contributed to the establishment of chronic HBV infection.

Conversion of T cells to B cells by inactivation of polycomb-mediated epigenetic suppression of the B-lineage program.

In general, cell fate is determined primarily by transcription factors, followed by epigenetic mechanisms fixing the status. While the importance of transcription factors controlling cell fate has been well characterized, epigenetic regulation of cell fate maintenance remains to be elucidated. Here we provide an obvious fate conversion case, in which the inactivation of polycomb-medicated epigenetic regulation results in conversion of T-lineage progenitors to the B-cell fate. In T-cell-specific Ring1A/B-deficient mice, T-cell development was severely blocked at an immature stage. We found that these developmentally arrested T-cell precursors gave rise to functional B cells upon transfer to immunodeficient mice. We further demonstrated that the arrest was almost completely canceled by additional deletion of Pax5 These results indicate that the maintenance of T-cell fate critically requires epigenetic suppression of the B-lineage gene program.

Safety and efficacy of treatment with liposomal amphotericin B in elderly patients at least 65 years old with hematological diseases.

The safety and efficacy of treatment with liposomal amphotericin B (L-AMB) in elderly patients has not been clarified, especially in Japanese patients. Therefore, we retrospectively analyzed 33 elderly patients with hematological diseases of at least 65 years old who received L-AMB between 2009 and 2012. Their clinical outcomes were compared to those of 21 patients who were younger than 65 years. L-AMB was administered for empirical therapy (n = 2) or target therapy for possible (n = 14) or probable/proven (n = 17) invasive fungal infection. There was no discontinuation of L-AMB due to adverse events. More than 2-fold increases from the baseline Cre, AST, and ALT values were observed in 21.2%, 39.4%, and 45.5% of the older group and 38.1%, 61.9%, and 52.4% of the younger group, respectively. The concurrent use of nephrotoxic antibiotics was the only risk factor for the development of a 2-fold increase in the serum Cre level. The duration of L-AMB was significantly longer in patients who developed grade III-IV hypokalemia. A partial or complete response was observed in 54.8% and 62.5% of the elderly and younger groups, respectively. In conclusion, L-AMB therapy appeared to be acceptably safe as empirical therapy or treatment for invasive fungal infection.

Effective use of the TSPY gene-specific copy number in determining fetal DNA in the maternal blood of cynomolgus monkeys.

Since the available concentration of single-copy fetal genes in maternal blood DNA is sometimes lower than detection limits by PCR methods, the development of specific and quantitative PCR detection methods for fetal DNA in maternal blood is anticipated, which may broaden the methods that can be used to monitor pregnancy. We used the TaqMan qPCR amplification for DYS14 multi-copy sequence and the SRY gene in maternal blood plasma (cell-free DNA) and fractional precipitated blood cells (cellular DNA) from individual cynomolgus monkeys at 22 weeks of pregnancy. The availability of cell-free fetal DNA was higher in maternal blood plasma than that of cellular DNA from fractional precipitated blood cells. There was a significantly higher (P < 0.001) mean copy number of fetal male DYS14 from maternal plasma (4.4 × 10(4) copies/mL) than that of detected fetal cellular DNA from fractional blood cell pellets. The sensitivity of the DYS14 PCR assay was found to be higher than that of the SRY assay for the detection of fetal DNA when its presence was at a minimum. The DYS14 assay is an improved method for quantifying male fetal DNA in circulating maternal blood in the primate model.

Detection and quantification of male-specific fetal DNA in the serum of pregnant cynomolgus monkeys (Macaca fascicularis).

Because of their developmental similarities to humans, nonhuman primates are often used as a model to study fetal development for potential clinical applications in humans. The detection of fetal DNA in maternal plasma or serum offers a source of fetal genetic material for prenatal diagnosis. However, no such data have been reported for cynomolgus monkeys (Macaca fascicularis), an important model in biomedical research. We have developed a specific, highly sensitive PCR system for detecting and quantifying male-specific fetal DNA in pregnant cynomolgus monkeys. We used multiplex quantitative real-time PCR to analyze cell-free DNA in maternal blood serum obtained from 46 pregnant monkeys at gestational weeks 5, 12, and 22. The presence of SRY gene and DYS14 Y chromosomal sequences was determined in 28 monkeys with male-bearing pregnancies. According to confirmation of fetal sex at birth, the probe and primers for detecting the Y chromosomal regions at each time point revealed 100% specificity of the PCR test and no false-positive or false-negative results. Increased levels of the SRY-specific sequences (mean, 4706 copies/mL serum DNA; range, 1731 to 12,625) and DYS14-specific sequences (mean, 54,814 copies/mL serum DNA; range, 4175-131,250 copies) were detected at week 22. The SRY- and DYS14-specific probes appear to be an effective combination of markers in a multiplex PCR system. To our knowledge, this report is the first to describe the detection of cell-free DNA in cynomolgus monkeys.

Emergence of infectious malignant thrombocytopenia in Japanese macaques (Macaca fuscata) by SRV-4 after transmission to a novel host.

We discovered a lethal hemorrhagic syndrome arising from severe thrombocytopenia in Japanese macaques kept at the Primate Research Institute, Kyoto University. Extensive investigation identified that simian retrovirus type 4 (SRV-4) was the causative agent of the disease. SRV-4 had previously been isolated only from cynomolgus macaques in which it is usually asymptomatic. We consider that the SRV-4 crossed the so-called species barrier between cynomolgus and Japanese macaques, leading to extremely severe acute symptoms in the latter. Infectious agents that cross the species barrier occasionally amplify in virulence, which is not observed in the original hosts. In such cases, the new hosts are usually distantly related to the original hosts. However, Japanese macaques are closely related to cynomolgus macaques, and can even hybridize when given the opportunity. This lethal outbreak of a novel pathogen in Japanese macaques highlights the need to modify our expectations about virulence with regards crossing species barriers.

Autopsy Analysis may Contribute to Establish Actual Incidence of Second Primary Malignancies in Myeloma.

Second primary malignancies (SPMs) are issues for patients with multiple myeloma (MM). There may have been some limitations in prior studies, such as difficulties in a longer follow-up and absence of established screening methods. Therefore, we studied autopsied cases to overcome these limitations. This study aimed to examine SPMs using autopsy reports. Ninety-one cases of MM autopsied at our institution from 1979 to 2013 were analyzed. Median age of autopsied patients was 64.1 years, and proportion of male/female was 59/32. Autopsy was performed in 35.3% of patients died of MM. There were five cases of SPMs with a median confirmation time of 38 (12-132) months from the diagnosis of MM. In three of the five patients, the diagnosis of SPMs was established at autopsy. One case was of myelodysplastic syndrome, and the others were of non-hematological malignancies. The annual risk of SPM estimated using the Kaplan-Meier method was approximately 1%. Three of five SPM cases were detected at autopsy. Analysis of autopsy may contribute to estimate the actual risk of SPMs in MM.

Isolation and DNA characterization of a simian retrovirus 5 from a Japanese monkey (Macaca fuscata).

An SRV-like virus was isolated from a colony-born Japanese monkey. To identify this SRV-like virus, we designed universal primers at regions that were conserved among the reported SRV sequences in the 5'-LTR and the short ORF and we obtained plasmid clones containing the complete gag, prt, pol and env genes. The full-length sequences of the isolate were determined from the plasmids and by direct sequencing. Sequence comparisons and phylogenetic analyses indicated that this SRV-like virus had a sequence identical to the reported 626 bp of SRV-5. In this study, we isolated SRV5/JPN/2005/V1 from a Japanese monkey and characterized the full-length SRV-5 sequence.