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Background The reliable diagnosis of human T-cell leukemia virus type 1 (HTLV-1) infection is important, particularly as it can be vertically transmitted by breast feeding mothers to their infants. However, current diagnosis in Japan requires a confirmatory western blot (WB) test after screening/primary testing for HTLV-1 antibodies, but this test often gives indeterminate results. Thus, this collaborative study evaluated the reliability of diagnostic assays for HTLV-1 infection, including a WB-based one, along with line immunoassay (LIA) as an alternative to WB for confirmatory testing. Results Using peripheral blood samples from blood donors and pregnant women previously serologically screened and subjected to WB analysis, we analyzed the performances of 10 HTLV-1 antibody assay kits commercially available in Japan. No marked differences in the performances of eight of the screening kits were apparent. However, LIA determined most of the WB-indeterminate samples to be conclusively positive or negative (an 88.0% detection rate). When we also compared the sensitivity to HTLV-1 envelope gp21 with that of other antigens by LIA, the sensitivity to gp21 was the strongest. When we also compared the sensitivity to envelope gp46 by LIA with that of WB, LIA showed stronger sensitivity to gp46 than WB did. These findings indicate that LIA is an alternative confirmatory test to WB analysis without gp21. Therefore, we established a novel diagnostic test algorithm for HTLV-1 infection in Japan, including both the performance of a confirmatory test where LIA replaced WB on primary test-reactive samples and an additional decision based on a standardized nucleic acid detection step (polymerase chain reaction, PCR) on the confirmatory test-indeterminate samples. The final assessment of the clinical usefulness of this algorithm involved performing WB analysis, LIA, and/or PCR in parallel for confirmatory testing of known reactive samples serologically screened at clinical laboratories. Consequently, LIA followed by PCR (LIA/PCR), but neither WB/PCR nor PCR/LIA, was found to be the most reliable diagnostic algorithm. Conclusions Because the above results show that our novel algorithm is clinically useful, we propose that it is recommended for solving the aforementioned WB-associated reliability issues and for providing a more rapid and precise diagnosis of HTLV-1 infection.

BackgroundSimian T-cell leukemia virus type 1 (STLV-1) is disseminated among various non-human primate species and is closely related to human T-cell leukemia virus type 1 (HTLV-1), the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. Notably, the prevalence of STLV-1 infection in Japanese macaques (JMs) is estimated to be > 60%, much greater than that in other non-human primates; however, the mechanism and mode of STLV-1 transmission remain unknown. The aim of this study is to examine the epidemiological background by which STLV-1 infection is highly prevalent in JMs.ResultsThe prevalence of STLV-1 in the JMs rearing in our free-range facility reached up to 64% (180/280 JMs) with variation from 55 to 77% among five independent troops. Anti-STLV-1 antibody titers (ABTs) and STLV-1 proviral loads (PVLs) were normally distributed with mean values of 4076 and 0.62%, respectively, which were mostly comparable to those of HTLV-1-infected humans. Our initial hypothesis that some of the macaques might contribute to frequent horizontal STLV-1 transmission as viral super-spreaders was unlikely because of the absence of the macaques exhibiting abnormally high PVLs but poor ABTs. Rather, ABTs and PVLs were statistically correlated (p ConclusionsTogether with the fact that almost all of the full-adult JMs older than 9 years old were infected with STLV-1, our results of this study demonstrated for the first time that frequent horizontal and mother-to-child transmission may contribute to high prevalence of STLV-1 infection in JMs.

Both natural viral infections and therapeutic interventions using viral vectors pose significant risks of malignant transformation. Monitoring for clonal expansion of infected cells is important for detecting cancer. Here we developed a novel method of tracking transgene integration sites. RAISING (Rapid Amplification of Integration Sites without Interference by Genomic DNA contamination) is a sensitive, inexpensive alternative to established methods. Its compatibility with Sanger sequencing combined with our CLOVA (Clonality Value) software is critical for those without access to expensive next-generation sequencing. To model our method, we analyzed samples from 698 patients infected with the retrovirus HTLV-1, which causes adult T-cell leukemia/lymphoma (ATL). We defined a clonality value identifying ATL patients with 100% sensitivity and 95.3% specificity, and our preliminary longitudinal analysis suggests this may also be useful for ATL risk assessment. We anticipate future studies will confirm the broad applicability of our technology, especially in the emerging gene therapy sector.

The diagnosis of human T -cell leukemia virus type 1 (HTLV-1) infection in Japan is usually performed by serological testing, but the high rate of indeterminate results from western blotting makes it difficult to assess the infection accurately. Nucleic acid tests for HTLV-1 and/or HTLV-2 are used to confirm infection with HTLV-1 and/or HTLV-2 and are also used for the follow-up of HTLV-1 related diseases. To prepare a highly sensitive method that can discern infection with HTLV-1 and HTLV-2, a multiplex quantitative polymerase chain reaction (qPCR) by large-scale primer screening was developed. Sensitivity and specificity were evaluated by serial dilution of cell lines and by testing with known clinical samples. The resulting multiplex qPCR can detect about four copies of HTLV-1 provirus per 105 cells. Moreover, HTLV-1 provirus could be detected in 97.2% (205 of 211) of HTLV-1 seropositive clinical samples. These sensitivities were sufficiently high compared with the methods reported previously. Also, all the HTLV-2 seropositive clinical samples tested were found to be positive by this method (three of three). In conclusion, this method can successfully and simultaneously detect both types of HTLV-1 and HTLV-2 provirus with extremely high sensitivity.

Human T-cell leukemia virus type 2 (HTLV-2) is non-endemic in Japan unlike the related HTLV type 1. Previously, although HTLV-2-seropositivity was identified via western blotting in one male blood donor in Japan, there have been no reports of HTLV-2 provirus detection by nucleic acid testing. In this report, one Japanese pregnant woman was clinically diagnosed as being HTLV-2-infected with a line immunoassay for specific antibodies after primary testing through prenatal screening in Japan. In genomic DNA of her peripheral blood mononuclear cells, HTLV-2 proviral genome was detected by nucleic acid testing (three methods) with quantitative polymerase chain reaction. The full-genome sequence of this strain was successfully determined. The identified virus was interestingly characterized as a presumed progenitor of subtypes a and c by recombination region and phylogenetic tree analyses. In conclusion, the present infection is, to our knowledge, the first case of molecularly identified and genetically characterized HTLV-2 infection found via prenatal screening in non-endemic Japan.

Approximately 10–20 million of Human T-cell leukemia virus type-1 (HTLV-1)-infected carriers have been previously reported, and approximately 5% of these carriers develop adult T-cell leukemia/lymphoma (ATL) with a characteristic poor prognosis. In Japan, Southern blotting has long been routinely performed for detection of clonally expanded ATL cells in vivo, and as a confirmatory diagnostic test for ATL. However, alternative methods to Southern blotting, such as sensitive, quantitative, and rapid analytical methods, are currently required in clinical practice. In this study, we developed a high-throughput method called rapid amplification of integration site (RAIS) that could amplify HTLV-1-integrated fragments within 4 h and detect the integration sites in > 0.16% of infected cells. Furthermore, we established a novel quantification method for HTLV-1 clonality using Sanger sequencing with RAIS products, and the validity of the quantification method was confirmed by comparing it with next-generation sequencing in terms of the clonality. Thus, we believe that RAIS has a high potential for use as an alternative routine molecular confirmatory test for the clonality analysis of HTLV-1-infected cells.

Quantitative PCR (qPCR) of human T-cell leukemia virus type 1 (HTLV-1) provirus is used for HTLV-1 testing and for assessment of risk of HTLV-1-related diseases. In this study, a reference material was developed for standardizing HTLV-1 qPCR. Freeze-dried TL-Om1 cells diluted with Jurkat cells were prepared and an assigned value for proviral load (PVL) of 2.71 copies/100 cells was determined by digital PCR. Nine Japanese laboratories using their own methods evaluated the PVLs of this reference material as 1.08-3.49 copies/100 cells. The maximum difference between laboratories was 3.2-fold. Correcting measured PVLs by using a formula incorporating the assigned value of this reference material should minimize such discrepancies.

Human T cell leukemia virus type 1 (HTLV-1) is mainly transmitted vertically through breast milk. The rate of mother-to-child transmission (MTCT) through formula feeding, although significantly lower than through breastfeeding, is approximately 2.4%-3.6%, suggesting the possibility of alternative transmission routes. MTCT of HTLV-1 might occur through the uterus, birth canal, or placental tissues; the latter is known as transplacental transmission. Here, we found that HTLV-1 proviral DNA was present in the placental villous tissues of the fetuses of nearly half of pregnant carriers and in a small number of cord blood samples. An RNA ISH assay showed that HTLV-1-expressing cells were present in nearly all subjects with HTLV-1-positive placental villous tissues, and their frequency was significantly higher in subjects with HTLV-1-positive cord blood samples. Furthermore, placental villous trophoblasts expressed HTLV-1 receptors and showed increased susceptibility to HTLV-1 infection. In addition, HTLV-1-infected trophoblasts expressed high levels of viral antigens and promoted the de novo infection of target T cells in a humanized mouse model. In summary, during pregnancy of HTLV-1 carriers, HTLV-1 was highly expressed in placental villous tissues, and villous trophoblasts showed high HTLV-1 sensitivity, suggesting that MTCT of HTLV-1 occurs through the placenta.

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens.

Patients with adult T-cell leukemia (ATL) exhibit a poor prognosis and overall survival rate when treated with standard chemotherapy, highlighting the continued requirement for the development of novel safe and effective therapies for human T-cell leukemia virus type 1 (HTLV-1)-related diseases. In this study, we demonstrated that MK-2048, a second-generation HIV-1 integrase (IN) inhibitor, potently and selectively kills HTLV-1–infected cells. Differential transcriptome profiling revealed significantly elevated levels of gene expression of the unfolded protein response (UPR) PKR-like ER kinase (PERK) signaling pathway in ATL cell lines following MK-2048 treatment. We also identified a significant downregulation in glucose regulated protein 78 (GRP78), a master regulator of the UPR in the CD4+CADM1+ HTLV-1–infected cell population of primary HTLV-1 carrier peripheral blood mononuclear cells (PBMCs) (n = 9), suggesting that HTLV-1–infected cells are hypersensitive to endoplasmic reticulum (ER) stress-mediated apoptosis. MK-2048 efficiently reduced proviral loads in primary HTLV-1 carrier PBMCs (n = 4), but had no effect on the total numbers of these cells, indicating that MK-2048 does not affect the proliferation of HTLV-1–uninfected PBMCs. MK-2048 specifically activated the ER stress–related proapoptotic gene, DNA damage-inducible transcript 3 protein (DDIT3), also known as C/EBP homologous protein (CHOP), in HTLV-1–infected but not uninfected cells of HTLV-1–carrier PBMCs. Our findings demonstrated that MK-2048 selectively induces HTLV-1–infected cell apoptosis via the activation of the UPR. This novel regulatory mechanism of the HIV IN inhibitor MK-2048 in HTLV-1–infected cells provides a promising prophylactic and therapeutic target for HTLV-1–related diseases including ATL.

BACKGROUND Dengue fever is caused by four related RNA viruses of the genus Flavivirus, dengue virus (DENV)-1, -2, -3, and -4, which are transmitted to humans by mosquitoes. Although DENV is not endemic in Japan, an autochthonous dengue outbreak occurred in 2014. Several transfusion-transmitted cases have also been reported after the use of blood and plasma products in DENV-endemic countries. The aim of this study was to develop a novel multiplex reverse transcription–polymerase chain reaction (RT-PCR) assay for DENV blood screening. STUDY DESIGN AND METHODS Large-scale oligonucleotide screening was performed to obtain DENV-specific primers and probes using a variety of DENV clinical isolates. A multiplex RT-PCR assay was then developed using the identified oligonucleotides and the ability of this assay to detect DENV RNA was evaluated. RESULTS A number of oligonucleotides suitable for DENV RNA detection were identified and a novel DENV serotype-specific multiplex RT-PCR assay was successfully established. Comparative analysis revealed that the multiplex assay could detect levels of viral contamination as low as 100 viral copies/mL. CONCLUSION This established serotype-specific multiplex RT-PCR assay provides a simple, sensitive, and quantitative detection method for DENV, which could be applied in the screening of blood samples to prevent transfusion-transmitted DENV infection.

We report two complete proviral genome sequences of human T-cell leukemia virus type 1 (HTLV-1) isolated from the peripheral blood specimens of acute type adult T-cell leukemia (ATL) patients in Oita Prefecture, Japan.

Influenza becomes epidemic worldwide every year, and many individuals receive vaccination annually. Quality control relating to safety and potency of influenza vaccines is important to maintain public confidence. The safety of influenza vaccines has been assessed by clinical trials, and animal safety tests are performed to monitor the consistent quality between vaccines used for clinical trials and marketing; the biological responses in vaccinated animals are evaluated, including changes in body weight and white blood cell count. Animal safety tests have been contributing to the quality relating to the safety of influenza vaccines for decades, but improvements are needed. Although precise mechanisms involving biological changes in animal safety tests have not been fully elucidated, the application of cDNA microarray technology make it possible to reliably identify genes related to biological responses in vaccinated animals. From analysis of the expression profile of >10,000 genes of lung in animals treated with an inactivated whole virion influenza vaccine, we identified 17 marker genes whose expression patterns correlated well to changes in body weight and leukocyte count in vaccinated animals. In influenza HA vaccine-treated animals exhibiting subtle changes in biological responses, a robust expression pattern of marker genes was found. Furthermore, these marker genes could also be used in the evaluation of adjuvanted influenza vaccines. The expression profile of marker genes is expected to be an alternative indicator for safety control of various influenza vaccines conferring high sensitivity and short turnaround time. Thus, gene expression profiling may be a powerful tool for safety control of vaccines in the future.

Inherited bone-marrow-failure syndromes (IBMFSs) include heterogeneous genetic disorders characterized by bone-marrow failure, congenital anomalies, and an increased risk of malignancy. Many lines of evidence have suggested that p53 activation might be central to the pathogenesis of IBMFSs, including Diamond-Blackfan anemia (DBA) and dyskeratosis congenita (DC). However, the exact role of p53 activation in each clinical feature remains unknown. Here, we report unique de novo TP53 germline variants found in two individuals with an IBMFS accompanied by hypogammaglobulinemia, growth retardation, and microcephaly mimicking DBA and DC. TP53 is a tumor-suppressor gene most frequently mutated in human cancers, and occasional germline variants occur in Li-Fraumeni cancer-predisposition syndrome. Most of these mutations affect the core DNA-binding domain, leading to compromised transcriptional activities. In contrast, the variants found in the two individuals studied here caused the same truncation of the protein, resulting in the loss of 32 residues from the C-terminal domain (CTD). Unexpectedly, the p53 mutant had augmented transcriptional activities, an observation not previously described in humans. When we expressed this mutant in zebrafish and human-induced pluripotent stem cells, we observed impaired erythrocyte production. These findings together with close similarities to published knock-in mouse models of TP53 lacking the CTD demonstrate that the CTD-truncation mutations of TP53 cause IBMFS, providing important insights into the previously postulated connection between p53 and IBMFSs.

Human T-cell leukemia virus type 1 (HTLV-1) infection causes adult T-cell leukemia (ATL), which is frequently resistant to currently available therapies and has a very poor prognosis. To prevent the development of ATL among carriers, it is important to control HTLV-1-infected cells in infected individuals. Therefore, the establishment of novel therapies with drugs specifically targeting infected cells is urgently required. This study aimed to develop a potential therapy by generating recombinant vesicular stomatitis viruses (rVSVs) that lack an envelope glycoprotein G and instead encode an HTLV-1 receptor with human glucose transporter 1 (GLUT1), neuropilin 1 (NRP1), or heparan sulfate proteoglycans (HSPGs), including syndecan 1 (SDC1), designated VSVΔG-GL, VSVΔG-NP, or VSVΔG-SD, respectively. In an attempt to enhance the infectivity of rVSV against HTLV-1-infected cells, we also constructed rVSVs with a combination of two or three receptor genes, designated VSVΔG-GLN and VSVΔG-GLNS, respectively. The present study demonstrates VSVΔG-GL, VSVΔG-NP, VSVΔG-GLN, and VSVΔG-GLNS have tropism for HTLV-1 envelope (Env)-expressing cells. Notably, the inoculation of VSVΔG-GL or VSVΔG-NP significantly eliminated HTLV-1-infected cells under the culture conditions. Furthermore, in an HTLV-1-infected humanized mouse model, VSVΔG-NP was capable of efficiently preventing HTLV-1-induced leukocytosis in the periphery and eliminating HTLV-1-infected Env-expressing cells in the lymphoid tissues. In summary, an rVSV engineered to express HTLV-1 primary receptor, especially human NRP1, may represent a drug candidate that has potential for the development of unique virotherapy against HTLV-1 de novo infection. IMPORTANCE Although several anti-ATL therapies are currently available, ATL is still frequently resistant to therapeutic approaches, and its prognosis remains poor. Control of HTLV-1 de novo infection or expansion of HTLV-1-infected cells in the carrier holds considerable promise for the prevention of ATL development. In this study, we developed rVSVs that specifically target and kill HTLV-1 Env-expressing cells (not ATL cells, which generally do not express Env in vivo ) through replacement of the G gene with HTLV-1 receptor gene(s) in the VSV genome. Notably, an rVSV engineered to express human NRP1 controlled the number of HTLV-1-infected Env-expressing cells in vitro and in vivo , suggesting the present approach may be a promising candidate for novel anti-HTLV-1 virotherapy in HTLV-1 carriers, including as a prophylactic treatment against the development of ATL.

Background The risk of transferring blood-borne infections during transfusion is continually increasing because of newly emerging and reemerging viruses. Development of a rapid screening method for emerging viruses that might be transmitted by transfusion is required to eliminate such pathogens during blood donor screening. Owing to increased use of human materials in organ transplants and cell therapy, the risk of donor-transmitted viral infections is also increasing. Although nucleic acid amplification technology (NAT) is dedicated to blood screening, a small, convenient detection system is needed at the laboratory and hospital level. Study Design and Methods We developed a new pathogen detection system that can detect multiple viruses simultaneously, using originally designed degenerate polymerase chain reaction primers to amplify a wide range of viral genotypes. Amplified samples were identified using a DNA microarray of pathogen-specific probes. Results We detected very low copy numbers of multiple subtypes of viruses, such as human hepatitis C virus (HCV), human hepatitis B virus (HBV), human parvovirus B19 (PVB19), and West Nile virus (WNV), using a single plate. We also detected all genotypes of human immunodeficiency virus (HIV) but sensitivity was less than for the other viruses. Conclusion We developed a microarray assay using novel primers for detection of a wide range of multiple pathogens and subtypes. Our NAT system was accurate and reliable for detection of HIV, HBV, HCV, PVB19, and WNV, with respect to specificity, sensitivity, and genotype inclusivity. Our system could be customized and extended for emerging pathogens and is suitable as a future NAT system.

Background Bacterial infections cause an increase in the population of hematopoietic stem cells (HSCs). To investigate the downstream factors associated with hematopoietic stem cells, mice are infected with Mycobacterium avium (M. avium). Results Mycobacterium avium (M. avium) infection induces the enlargement of the spleen and changes in histopathology, including changes to the lineage populations. A dramatic expansion of Lin−c-kit+Sca-1+ (KSL) cells in mouse bone marrow cells and spleen cells was detected 4 weeks after infection with M. avium; however, there was no difference in the engraft activity between infected and un-infected mouse bone marrow cells. We tested the cytokine and cytokine-related gene expression after M. avium infection and found that IFN-γ expression increased and peaked at 4 weeks in both bone marrow and spleen cells. The expression of Sca-1 gene peaked at 4 weeks in the bone marrow but peaked at 2 weeks in spleen cells, although the Sca-1 surface marker peaked at 4 weeks after infection in both bone marrow and spleen cells. Interferon regulatory factor-2 (IRF-2) expression did not change in the bone marrow cells, whereas it decreased in spleen cells at 4 weeks and IRF-1 expression was up-regulated in both bone marrow and spleen cells after infection. However, the up-regulation of IRF-1 was not correlated with IFN-γ expression in the M. avium-infected mouse spleen cells. Conclusions This finding suggests that the IFN-γ production mediated by M. avium infection alters the population of KSL cells during host defense, and the down-regulation of the IFN-γ response in spleen cells occurs at the late stage after M. avium infection.

Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

Human immunodeficiency virus type 1 (HIV-1) utilizes a highly complex splice site regulation system, taking advantage of host proteins, to express its own viral protein in an orderly way. We show here that one of the host proteins, high mobility group A protein 1a (HMGA1a), is involved in splice site regulation of 3′ splice site 2 (A2) and 5′splice site 3 (D3) of HIV-1 genomic RNA. shRNA knockdown of HMGA1 in HeLa cells resulting in a decrease of HMGA1 showed a significant decrease of Vpr mRNA. RNA electophoretic mobility shift assays showed HMGA1a specifically binds to a sequence adjacently upstream D3. In vitro splicing using heterologous pre-mRNA with A2 and D3, showed HMGA1a induced a splicing intermediate which decreased when an RNA decoy of the HMGA1a binding site was added. RT-PCR of in vitro splicing products revealed that HMGA1a induced an incomplete splicing product resulting from usage of A2 but inhibition of D3, which is reminiscent of the splicing pattern necessary for Vpr mRNA formation. HMGA1a interacted with hnRNPA1 shown by coimmunoprecipitation and supershifted U1 snRNP in an RNA electophoretic mobility shift assay. We conclude that HMGA1a anchors U1 snRNP to inhibit D3 function, and that HMGA1a inhibits hnRNPA1 function on exon splicing silencer of Vpr (ESSV) to activate A2 function. We show here for the first time that HMGA1a is involved in specific splice site regulation of HIV-1.

Vpr, an accessory gene product of human immunodeficiency virus type 1 (HIV-1), affects both viral and cellular proliferation by mediating long terminal repeat activation, cell cycle arrest at the G2 phase, and apoptosis. We previously found that Vpr plays a novel role as a regulator of pre-mRNA splicing both in vivo and in vitro. However, the cellular target of Vpr, as well as the mechanism of cellular pre-mRNA splicing inhibition by Vpr, is unknown. Here, we show clearly that Vpr inhibits the splicing of cellular pre-mRNA, such as beta-globin pre-mRNA and immunoglobulin (Ig) M pre-mRNA and that the third alpha-helical domain and arginine-rich region are important its ability to inhibit splicing. Additionally, using mutants with specific substitutions in two domains of Vpr, we demonstrated that the interaction between Vpr and SAP145, an essential splicing factor, was indispensable for splicing inhibition. Finally, co-immunoprecipitation and in vitro competitive binding assays indicated that Vpr associates with SAP145 and interferes with SAP145-SAP49 complex formation. Thus, these results suggest that cellular expression of Vpr may block spliceosome assembly by interfering with the function of the SAP145-SAP49 complex in host cells.

Conventional animal tests such as leukocytosis promoting tests have been used for decades to evaluate toxicity of pertussis vaccine. Here, we examined gene expression in relation to the vaccine toxicity using a DNA microarray. Comparison of conventional animal test data with the DNA microarray-based gene expression data revealed a gene expression pattern highly correlated with leukocytosis in animals. Of 10,490 rat genes analyzed, two genes, alpha1-acid-glycoprotein (Agp) and hemopexin (Hpx), were found up-regulated by the toxin administration in a dose-dependent manner (assayed by a quantitative PCR based on the microarray). Variation of the gene expression was very small amongst the test animals, and the results were highly reproducible. These findings suggest that gene expression analysis of vaccine-treated animals can be used as an accurate and simple method of pertussis vaccine safety assessment.

Quantitative PCR (qPCR) analysis of human T-cell leukemia virus type 1 (HTLV-1) was used to assess the amount of HTLV-1 provirus DNA integrated into the genomic DNA of host blood cells. Accumulating evidence indicates that a high proviral load is one of the risk factors for the development of adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis. However, interlaboratory variability in qPCR results makes it difficult to assess the differences in reported proviral loads between laboratories. To remedy this situation, we attempted to minimize discrepancies between laboratories through standardization of HTLV-1 qPCR in a collaborative study. TL-Om1 cells that harbor the HTLV-1 provirus were serially diluted with peripheral blood mononuclear cells to prepare a candidate standard. By statistically evaluating the proviral loads of the standard and those determined using in-house qPCR methods at each laboratory, we determined the relative ratios of the measured values in the laboratories to the theoretical values of the TL-Om1 standard. The relative ratios of the laboratories ranged from 0.84 to 4.45. Next, we corrected the proviral loads of the clinical samples from HTLV-1 carriers using the relative ratio. As expected, the overall differences between the laboratories were reduced by half, from 7.4-fold to 3.8-fold on average, after applying the correction. HTLV-1 qPCR can be standardized using TL-Om1 cells as a standard and by determining the relative ratio of the measured to the theoretical standard values in each laboratory.

Background Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) infection. However, there are no therapies to prevent ATL development in high-risk asymptomatic carriers. To develop a therapy targeting HTLV-1-infected cells that are known to express CCR4 frequently, we tested whether truncated Pseudomonas exotoxin (PE38) fused to a CCR4 ligand, CCL17/thymus and activation-regulated chemokine (TARC), selectively eliminates such cells. Results Our data show that TARC–PE38 efficiently killed HTLV-1-infected cell lines. It also shrank HTLV-1-associated solid tumors in an infected-cell-engrafted mouse model. In HTLV-1-positive humanized mice, TARC–PE38 markedly inhibited the proliferation of HTLV-1-infected human CD4+CD25+ or CD4+CD25+CCR4+ cells and reduced the proviral loads (PVLs) in peripheral blood mononuclear cells (PBMCs). Importantly, TARC–PE38 significantly reduced the PVLs in PBMCs obtained from asymptomatic carriers. We show that the cytotoxicity of TARC–PE38 is mediated by the expression of the proprotein convertase, furin. The expression of furin was enhanced in HTLV-1-infected cells and correlated positively with PVLs in HTLV-1-infected individuals, suggesting that infected cells are more susceptible to TARC–PE38 than normal cells. Conclusions TARC–PE38 robustly controls HTLV-1 infection by eliminating infected cells in both a CCR4- and furin-dependent manner, indicating the excellent therapeutic potential of TARC–PE38. Electronic supplementary material The online version of this article (doi:10.1186/s12977-015-0199-8) contains supplementary material, which is available to authorized users.

Quantitative PCR (qPCR) for human T-lymphotropic virus 1 (HTLV-1) is useful for measuring the amount of integrated HTLV-1 proviral DNA in peripheral blood mononuclear cells. Many laboratories in Japan have developed different HTLV-1 qPCR methods. However, when six independent laboratories analyzed the proviral load of the same samples, there was a 5-fold difference in their results. To standardize HTLV-1 qPCR, preparation of a well-defined reference material is needed. We analyzed the integrated HTLV-1 genome and the internal control (IC) genes of TL-Om1, a cell line derived from adult T-cell leukemia, to confirm its suitability as a reference material for HTLV-1 qPCR. Fluorescent in situ hybridization (FISH) showed that HTLV-1 provirus was monoclonally integrated in chromosome 1 at the site of 1p13 in the TL-Om1 genome. HTLV-1 proviral genome was not transferred from TL-Om1 to an uninfected T-cell line, suggesting that the HTLV-1 proviral copy number in TL-Om1 cells is stable. To determine the copy number of HTLV-1 provirus and IC genes in TL-Om1 cells, we used FISH, digital PCR, and qPCR. HTLV-1 copy numbers obtained by these three methods were similar, suggesting that their results were accurate. Also, the ratio of the copy number of HTLV-1 provirus to one of the IC genes, RNase P, was consistent for all three methods. These findings indicate that TL-Om1 cells are an appropriate reference material for HTLV-1 qPCR.

Vpr, one of the accessory gene products of human immunodeficiency virus type 1 (HIV-1), affects aspects of both viral and cellular proliferation, being involved in long terminal repeat (LTR) activation, arrest of the cell cycle at the G2 phase, and apoptosis. We have discovered a novel role for Vpr as a regulator of the splicing of pre-mRNA both in vivo and in vitro. We found, by RT-PCR and RNase protection analysis, that Vpr caused the accumulation of incompletely spliced forms of alpha-globin 2 and beta-globin pre-mRNAs in cells that had been transiently transfected with a Vpr expression vector. We postulated that this novel effect of Vpr might occur via a pathway that is distinct from arrest of the cell cycle at G2. By analyzing splicing reactions in vitro, we showed that Vpr inhibited the splicing of beta-globin pre-mRNA in vitro. The splicing of intron 1 of alpha-globin 2 pre-mRNA was modestly inhibited by Vpr but the splicing of intron 2 was unaffected. Interestingly, an experimental infection system which utilizes high-titered HIV-1/vesticular stomatitis virus G protein showed that Vpr expressed from an HIV-1 provirus was sufficient to accumulate endogenous alpha-globin 2 pre-mRNA. Thus, it is likely that Vpr contributes to selective inhibition of the splicing of cellular pre-mRNA.

Diamond-Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond-Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond-Blackfan anaemia, we performed whole-exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond-Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond-Blackfan anaemia. In vitro knockdown of gene expression disturbed pre-ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond-Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond-Blackfan anaemia.

Vaccines are beneficial and universal tools to prevent infectious disease. Thus, safety of vaccines is strictly evaluated in the preclinical phase of trials and every vaccine batch must be tested by the National Control Laboratories according to the guidelines published by each country. Despite many vaccine production platforms and methods, animal testing for safety evaluation is unchanged thus far. We recently developed a systems biological approach to vaccine safety evaluation where identification of specific biomarkers in a rat pre-clinical study evaluated the safety of vaccines for pandemic H5N1 influenza including Irf7, Lgals9, Lgalsbp3, Cxcl11, Timp1, Tap2, Psmb9, Psme1, Tapbp, C2, Csf1, Mx2, Zbp1, Ifrd1, Trafd1, Cxcl9, β2m, Npc1, Ngfr and Ifi47. The current study evaluated whether these 20 biomarkers could evaluate the safety, batch-to-batch and manufacturer-to-manufacturer consistency of seasonal trivalent influenza vaccine using a multiplex gene detection system. When we evaluated the influenza HA vaccine (HAv) from four different manufactures, the biomarker analysis correlated to findings from conventional animal use tests, such as abnormal toxicity test. In addition, sensitivity of toxicity detection and differences in HAvs were higher and more accurate than with conventional methods. Despite a slight decrease in body weight caused by HAv from manufacturer B that was not statistically significant, our results suggest that HAv from manufacturer B is significantly different than the other HAvs tested with regard to Lgals3bp, Tapbp, Lgals9, Irf7 and C2 gene expression in rat lungs. Using the biomarkers confirmed in this study, we predicted batch-to-batch consistency and safety of influenza vaccines within 2 days compared with the conventional safety test, which takes longer. These biomarkers will facilitate the future development of new influenza vaccines and provide an opportunity to develop in vitro methods of evaluating batch-to-batch consistency and vaccine safety as an alternative to animal testing.

Megakaryopoiesis is associated with inflammatory reactions. To investigate the role of interferon regulatory factors (IRFs) in inflammation-associated megakaryopoiesis, mouse bone marrow hematopoietic stem cells (HSCs) were analyzed. IFN-γ treatment induced IRF-2 expression as well as the expression of CD41 and IRF-1 in HSCs. An in vitro clonogenic assay showed that IRF-2- but not IRF-1-overexpressing cells increased the number of megakaryocytic colonies. IRF-2 transfection up-regulated CD41 promoter activity in hematopoietic cell lines. The number of CD41-positive bone marrow cells increased in mice injected with IRF-2-expressing bone marrow cells. These findings suggest that IRF-2 plays an important role in megakaryopoiesis in inflammatory states.

Germ-line stem cells (GSCs) constitute a stem cell population with remarkable stability and proliferative potential in vitro and are a useful model for studying the mechanism of self-renewal and "stemness" function of committed tissue stem cells. To identify GSC-specific genes, we performed subtractive hybridization using cDNA from GSCs, testis, and embryonic stem (ES) cells, and successfully identified 11 genes highly expressed in GSCs. Histological analysis confirmed expression of Cry alpha b, Mcpt8, Cxcl5, Fth1, Ctla2 alpha, and Spp1 in undifferentiated spermatogonia on the basement membrane area of the seminiferous epithelium of the testis, where the GSC niche is thought to be located. Among GSC-specific genes, quantitative PCR analysis showed seven genes-Fth1, Cry alpha b, Spp1, Bcap31, Arhgap1, Ctla2 alpha, and Serpina3g-to be common transcripts highly expressed in hematopoietic stem cells (HSCs). Histological analysis confirmed that Ctla2 alpha-, Serpina3g-, and Spp1-expressing cells were observed in the trabecular bone region of the bone marrow, where the HSC niche is located. Furthermore, histological analysis revealed that only Spp1 was expressed in the hair follicle bulge in the area of the hair follicle stem cell niche. Thus, identifying stemness genes by comparative analysis to GSCs is a powerful tool with which to explore the fundamental commonalities of HSCs and other stem cell types.

The gene encoding ribosomal protein S19 (RPS19) is one of the responsible genes for Diamond-Blackfan anaemia (DBA), a congenital erythroblastopenia. Although haplo-insufficiency of RPS19 has been suggested to be the onset mechanism underlying the pathogenesis of DBA, the sequential mechanism has not been elucidated. In order to analyse the consequences of the missense mutation of RPS19 specific for DBA patients, we made mutated RPS19 expression vectors. Twelve C-terminally Flag-tagged missense mutants were exogenously expressed from retroviral vectors and analysed by Western blot analysis and flow cytometry. When these 12 mutants were expressed in the erythro-leukaemic cell lines K562 and human bone marrow CD34(+) cells, almost all of the mutant proteins (except for G120R) were unstable, and the levels of mutated RPS19 protein were significantly low. To address the effect of deficient RPS19 expression on cell proliferation, RPS19 was downregulated by siRNA. Repressive expression of RPS19 in human CD34(+) cells produced an elevated number of cells at G0 and induced erythroid progenitor-specific defects in BM cells. These results suggest that abnormal ribosomal biogenesis causes inadequate cell cycle arrest in haematopoietic progenitors, and that, subsequently, erythroid progenitors are specifically hampered. These in vitro phenotypes of genetically manipulated CD34(+) cells mimic DBA pathogenesis.

We have previously identified 17 biomarker genes which were upregulated by whole virion influenza vaccines, and reported that gene expression profiles of these biomarker genes had a good correlation with conventional animal safety tests checking body weight and leukocyte counts. In this study, we have shown that conventional animal tests showed varied and no dose-dependent results in serially diluted bulk materials of influenza HA vaccines. In contrast, dose dependency was clearly shown in the expression profiles of biomarker genes, demonstrating higher sensitivity of gene expression analysis than the current animal safety tests of influenza vaccines. The introduction of branched DNA based-concurrent expression analysis could simplify the complexity of multiple gene expression approach, and could shorten the test period from 7 days to 3 days. Furthermore, upregulation of 10 genes, Zbp1, Mx2, Irf7, Lgals9, Ifi47, Tapbp, Timp1, Trafd1, Psmb9, and Tap2, was seen upon virosomal-adjuvanted vaccine treatment, indicating that these biomarkers could be useful for the safety control of virosomal-adjuvanted vaccines. In summary, profiling biomarker gene expression could be a useful, rapid, and highly sensitive method of animal safety testing compared with conventional methods, and could be used to evaluate the safety of various types of influenza vaccines, including adjuvanted vaccine.

Tie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell–derived Tie2+Flk1+ fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-1 and found that LYVE-1+ cells derived from Tie2+Flk1+ cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2-/- ES cells. Although the initial numbers of LYVE-1+ and PECAM-1+ cells derived from Tie2-/- cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2-/- ES cells did not show measurable defects in development of the hematopoietic system, suggesting that Tie2 is not essential for hematopoietic cell development.

Adult T cell leukemia (ATL) is a lymphoproliferative disorder caused by infection with HTLV-I. Although various chemotherapies have shown significant complete remission rates, most of the treated patients relapse. These data indicate the existence of leukemic stem cells (LSCs) and a specific niche that regulates stemness and protects LSCs from various chemotherapies. We have reported in previous studies that the ATL-LSCs isolated from a Tax-transgenic (Tax-Tg) mouse are enriched in the CD117+/CD38–/CD71– fraction of the lymphoma, and LSCs have the potential to reproduce the original tumor when transplanted into a NOD/SCID mouse (Yamazaki et al., Blood, 2009). However, the niche of ATL-LSCs is still unclear. To identify the ATL-LSC niche in vivo, we performed a homing assay. Splenic lymphoma cells isolated from a Tax-Tg mouse were GFP transduced by a lentivirus, and then sorted GFP+ cells were transplanted intra-peritoneally into a non-irradiated NOD/SCID mouse. The homing of GFP+ cells to tissues was assessed by flow cytometry (FCM) at 16 hours and 3, 7, 14 and 21 days after transplantation. As a result, GFP+ lymphoma cells were first detected in the spleen and BM at 16 hours after transplantation. No GFP+ lymphoma cells were detected in the thymus and LN. Interestingly, more than 60% of first colonized cells in the spleen and BM at 16 hours after transplantation were AT-LSCs (GFP+/CD117+ cells). From day 3 to 7, more than 40% of colonizing cells in the BM and spleen were ATL-LSCs. To identify the specific niche of ATL-LSCs in the BM, we performed imaging analysis of ATL-LSCs. ATL-LSCs (GFP+/CD117+ and CD38–/CD71–/CD117+ cells) were mainly localized near the endosteal region of trabecular bone in the BM. We found that ATL-LSCs were also attached to the reticular cells in the trabecular bone. In addition, we found the number of osteoclast was significantly increased at the trabecular region. Increasing number of osteoclasts correlates the increased the serum calcium concentration and decreased the mass of trabecular bone. FCM analysis and in vitro differentiation assay confirmed that the number of osteoclast precursors was increased in the ATL BM. To clarify the role of osteoclast in the ATL BM, we treated osteoclast inhibitor Zoledronic acid (ZOL) to the ATL mouse model. As a result, ZOL itself significantly reduced the number of GFP+ ATL cells in the BM. When we treated ZOL with anti cancer drug, GFP+ ATL cells were dramatically reduced in the BM and extend the mouse survival rate significantly despite anti cancer drug does not reduced the number of ATL cells itself. In addition, abnormal trabecular bone morphology was completely recovered in the treated mouse. These data suggest that osteoclast may have a function to support leukemic stem cell niche. To clarify the key signals to induce osteoclast in ATL BM, we checked the expression of RANKL and PTHrP. We found that RANKL was up-regulated both in the lymphoma cell and stromal cells in the bone marrow. In this study, we found that ATL-LSC niche is located at the trabecular bone region in the BM and osteoclasts have a role to support ATL cell and develop LSCs niche in a mouse model of ATL. We conclude that osteoclast have a potential therapeutic target in the mouse model of ATL. Disclosures: No relevant conflicts of interest to declare.

Abstract 1877 Adult T cell leukemia (ATL) is a lymphoproliferative disorder caused by infection with HTLV-I. Although various chemotherapies have shown significant complete remission rates, most of the treated patients relapse. These data indicate the existence of leukemic stem cells (LSCs) and a specific niche that regulates stemness and protects these cells from chemotherapy. We have reported in previous studies that the ATL-LSCs isolated from a Tax-transgenic (Tax-Tg) mouse are enriched in the CD117+/CD38–/CD71– fraction of the lymphoma, and LSCs have the potential to reproduce the original tumor when transplanted into a NOD/SCID mouse (Yamazaki et al., Blood, 2009). However, the niche of ATL-LSCs in the spleen, bone marrow (BM), thymus and lymph node (LN) is still unclear. To identify the ATL-LSC niche in vivo, we performed a homing assay. Lymphoma cells isolated from a Tax-Tg mouse were GFP transduced by a lentivirus, and then sorted GFP+ cells (2×106) were transplanted intraperitoneally into a non-irradiated NOD/SCID mouse. The homing of GFP+ cells to tissues was traced by flow cytometry (FCM) at 16 hours and 3, 7, 14 and 21 days after transplantation. At 16 hours after transplantation, GFP+ lymphoma cells were detected in the spleen and BM. No GFP+ lymphoma cells were detected in the thymus and LN. Interestingly, more than 60% of first colonized cells in the spleen and BM at 16 hours were AT-LSCs (GFP+/CD117+ cells). From day 3 to 7, more than 40% of proliferating cells in the BM and spleen were ATL-LSCs. At day 3, only a few non-ATL-LSCs (GFP+/CD117–cells) were detected in the thymus, LN and peripheral blood. The number of GFP+ cells was drastically increased at day 14 in the spleen. These data indicate that ATL-LSCs prefer to colonize and proliferate in the spleen and BM. To identify the specific niche of ATL-LSCs in the spleen and BM, we performed imaging analysis of ATL-LSCs. ATL-LSCs (GFP+/CD117+ and CD38–/CD71–/CD117+cells) were mainly localized near the vascular region in the spleen and endosteal region of trabecular bone in the BM. We found that some ATL-LSCs were attached to reticular cells (RC) in the spleen. In the BM, ATL-LSCs cells were localized at the endosteal region of the trabecular bone. Interestingly, similar to the spleen, RCs were observed at the endosteal region and contacted ATL-LSCs in the BM. FCM analysis confirmed that the number of reticular cells and mesenchymal stem cells (MSCs), were increased in the ATL BM and spleen. These data suggest that RCs are a possible candidate for the ATL-LSC niche and may be a new target of therapy. Finally, to characterize the ATL-LSC niche, we isolated osteoblastic cells, blood endothelial cells, lymphatic endothelial cells and reticular cells from normal and ATL BM to compare the gene expression profiles of each niche cell type. Here, together with DNA microarray analysis of ATL-LSCs both in the BM and spleen, we have characterized ATL-LSC niche cells both in the spleen and BM. Disclosures: No relevant conflicts of interest to declare.

Abstract 984 Diamond-Blackfan anemia (DBA) is a congenital bone marrow failure syndrome, characterized by red blood cell aplasia, macrocytic anemia, and increased risk of malignancy. Approximately 90% of patients present during the first year of life or in early childhood. About 40–50% of DBA cases are familial with autosomal dominant, while the remainder is sporadic cases whose mode of inheritance is largely unknown. Although anemia is the most prominent feature of DBA, up to 40% of patients also accompany other symptoms including growth retardation and/or a variety of congenital malformations. Recent studies have shown that the disease could be associated with heterozygous mutations in ribosomal protein (RP) genes, including six small subunit RP genes RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26 as well as four large subunit RP genes RPL5, RPL11, RPL26, and RPL35A, which collectively account for about 50% of patients with DBA. In addition, germline mutations in the GATA1 gene encoding a hematopoietic transcription factor, have been also reported in two DBA families. However, it is clear that the molecular etiology of many DBA cases remains to be covered. To identify new mutations that are responsible for DBA, we performed whole-exome sequencing on 40 DBA patients with no documented mutations/deletions involving known DBA genes. After excluding all variants registered in the 1000 Genomes Project, or dbSNP131, or found in our inhouse SNP database, we searched for non-synonymous mutations involving RP genes as possible candidate for novel DBA genes. In this study, we identified probable pathogenic mutations in two novel RP genes, RPS27 and RPL27 in two patients. The first case was a 1-year-old girl who harbored a single nucleotide substitution at the splice acceptor site in intron 1 of RPL27 (c.-2–1G>A), which results in splicing error. She had atrial septal defect and pulmonary stenosis, and responded to steroid treatment. The second case was a 2-year-old girl carrying a frameshift deletion of RPS27 (c.90delC, p.Tyr31ThrfsX5), leading to a premature truncation. This patient had no abnormalities and responded to steroid treatment. An additional five missense SNVs affecting single cases was identified in five genes, including RPL3L, RPL8, RPL13, RPL18A, and RPL31, together with two in-frame deletions of RPL6 and RPL14 in two patients, which cause deletion of a single amino-acid. However, the pathological significance in these 7 cases is uncertain. In the remaining 31 patients, no mutations were detected in RP genes. In conclusion, we identified novel germline mutations of RP genes that could be responsible for DBA, further confirming the concept that the RP genes are common targets of germline mutations in DBA patients and also suggested the presence of non-RP gene targets for DNA. Disclosures: No relevant conflicts of interest to declare.

Introduction Interferon regulatory factor-2 (IRF-2) is a transcriptional repressor in the interferon system and is thought to function by competing with IRF-1. Its physiological role in lymphoid and hematopoietic development has been investigated in IRF-2−/− mice, in which a general bone marrow suppression of hematopoiesis and B lymphopoiesis has been reported. Methods We investigate the role of IRF-2 in mouse bone marrow hematopoietic stem cells using IRF-2−/− mice. Hematopoietic stem cells (as KSL) are isolated from IRF-2−/− mice bone marrow and transplanted to lethally ittadiated mice for chimerismanalysis. Results IRF-2 is strongly expressed in hematopoietic stem cells (HSC) in mouse bone marrow. The population of bone marrow Lin-c-Kit+Sca-1+(KSL) cells is increased in IRF-2−/− mice due to the general enhancement of Sca-1-positive cells. SCs are enriched in KSL CD150+CD48-cells. A very small population of CD150-positive cells was detected in bone marrow from IRF-2−/− mice. Real-time polymerase chain reaction showed reduced CD150 gene expression in bone marrow cells from these mice compared to wild type mice. Although the population of bone marrow KSL cells in IRF-2−/− mice increased, a reduction of HSC was observed due to a decrease of the CD150-positive cell population. In IRF-2−/− interferon-a receptor−/− double-knockout mice, the CD150 expression levels were comparable to wild-type mice, indicating that this gene is regulated by the type [1] interferon (IFN) response via transcriptional regulation. Transplantation of KSL cells from IRF-2−/− mice into wild-type mice together with a competitor resulted in failed to engraft. Even the transplantation of a >20-fold excess of KSL cells from IRF-2−/− mice resulted in engraftment that was still poorer than wild-type cells evaluated at 3–4 months after transplantation. However, transplantation of a higher dose (2 × 10 6 cells) of whole bone marrow mononuclear cells from IRF-2−/− mice rescued recipients from lethal irradiation, although the engraftment was poorer than wild-type cells. This documents the presence of some active long-term repopulating hematopoietic stem cells (LT-HSCs) in IRF-2−/− mice. Conclusion Our results reveal unknown HSC markers in IRF-2−/− mouse bone marrow and demonstrate that IRF-2 acts on LT-HSCs not only through protective type I IFN responses, but also by directly regulating HSC cell surface molecules.

Abstract 4231 Introduction: Fifty percent of Diamond–Blackfan anemia (DBA) patients possess mutations in ribosomal protein genes. Although several ribosomal protein genes, RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, have been reported to be mutated in some DBA patients, including point mutations, nonsense mutations, deletions, splice site mutations, and translocations, other DBA patients appear to have intact ribosomal protein genes. To identify new mutations in ribosomal protein genes from a different aspect, we focused on extensive deletions in these genes, such as mutations involving loss of a whole allele. In this study, we applied quantitative genomic PCR, and successfully developed a convenient method for detecting extensive deletions designated the “DBA gene copy number assay”. Methods: DBA patients should have an intact allele and a mutated allele for the responsible ribosomal protein gene, meaning that they will have an abnormal karyotype (gene copy number of N) if they have an extensive deletion. We attempted to clarify the copy numbers of ribosomal protein genes by the difference in a 1-cycle delay of threshold in a quantitative PCR (q-PCR) assay. To detect extensive deletions, at least 2 sets of gene-specific primers for each DBA responsible gene (RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26) were prepared. Appropriate primers to fit the setting that the threshold cycle (Ct) of the q-PCR should occur within 1 cycle of the Ct scores of other primer sets were selected. After validation, we identified 6, 3, 4, 3, 3, 6, 9, 3, and 2 specific primer sets for RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26, respectively. By simply looking at the q-PCR amplification curves by eye, we were easily able to judge the copy numbers of 2N (normal) or N (abnormal) for the ribosomal protein genes. Results: We performed the DBA gene copy number assay for 14 randomly selected undiagnosed patients from the Japanese DBA genomic resource at the University of Hirosaki, who had no mutations by genomic sequencing analyses. For each case, all the DBA responsible genes were confirmed using the diagnostic primers. The results of the DBA gene copy number assays revealed that 5 of the 14 probands (36%) had an extensive deletion in one of the DBA responsible genes. As an interesting case among the 5 positive cases, we confirmed an extensive deletion in the RPS19 gene. The Ct scores for 4 of the 9 primer sets for RPS19 demonstrated a 1-cycle delay, while the scores for the other 5 primer sets were normal. By genomic PCR amplification analyses, we identified a deletion from nt. -1400 to +5757 (7157 nucleotides) in the RPS19 gene. The deleted region included the promoter region, and exons 1, 2, and 3 of the RPS19 gene. The remaining 4 cases were 1 proband with an RPL5 deletion, 1 with an RPL35A deletion and 2 with RPS17 deletions. In particular, the extensive deletions in the RPL5 and RPS17 alleles are the first such cases reported. Discussion: Since it has been difficult to address the loss of a whole allele in DBA, such mutations have not been precisely examined within the DBA responsible genes. Our data suggest that extensive deletions in ribosomal protein genes comprise a significant proportion of DBA cases in Japan. Our novel method could become a useful tool for screening the gene copy numbers of ribosomal protein genes, and for identifying new pathological mutations. Disclosures: No relevant conflicts of interest to declare.

In normal hematopoiesis, hematopoietic stem cells (HSCs) in bone marrow mainly produce various types of blood cells. HSCs have a function of self-renewal and are located in the osteoblastic and vascular niche, which regulates the stemness function as a stem cell microenvironment. Recent studies clearly show that several factors (N-Cadherin, b-Catenin, SDF-1a and Spp1) are involved in the maintenance of the stemness function in HSCs. However, it has also been shown that embryonic HSCs can proliferate and differentiate in extrameduller tissues like liver, spleen and placenta. In adults, extrameduller hematopoiesis (EMH) can be induced by a hematological malignancy and some infectious diseases. However, the mechanism of HSCs regulation and niche cells at the EMH has not been well defined. To reveal the mechanism of HSCs in the EMH, we utilized the c-fos knockout mouse (c-fos −/−) as an EMH model mouse. In c-fos −/−, hematopoiesis in bone marrow was absent as a result of marrow spaces being occupied by increasing the number of bone forming osteoblasts; EMH then began in the spleen. First, to identify the main site of the HSC-niche in the EMH spleen, we performed HSC localization analysis using in situ hybridization of various HSC markers. Surprisingly, some CD34, Sca-1, c-kit, SCL/tal-1, and Tie-2 expressing cells were located near megakaryocyte like cells (MLCs). These MLCs were mainly located in the red pulp region in the spleen, and 3–5 cells form a syncytium. Interestingly, these MLCs express various types of osteoblastic niche related molecules, (N-Cadherin, b-Catenin, Spp1 and SDF-1a) in addition to megakaryocytic markers (CD41, CD61, and b3-integrin), suggesting that MLC is a HSC niche candidate in the EMH. To confirm our hypothesis, we next performed CFU-S (colony forming unit-spleen) assay as a naturally induced EMH model. Total 1×105 bone marrow mononuclear cells isolated from Ly5.2 or GFP-transgenic mouse were transplanted into lethally irradiated Ly5.2 mouse. In this system, MLCs were first seen in the spleen at day 1. At day 8, the number of MLCs had increased and units of 10–15 MLCs aggregated and formed syncytium. About 80% of MLCs were derived from donor (Ly5.2) cells. More interestingly, and contrary to our expectations, these aggregated MLCs with Sca-1+ or c-kit+ hematopoietic progenitor cells (HPCs) were mainly located, not inside the colony, but in the interstitial region between the developing colonies. They also expressed osteoblastic niche molecules. It has been suggested that each colonies in spleen are derived from HPCs and that HPCs exist inside the colony. Our data indicate the possibility that HPCs were transiently- located outside the colony. To undertake a detailed characterization of MLCs, we sorted donor derived MLCs as a Lin-/CD41+ cells and performed Q-PCR analysis. In agreement with our histological analysis, the sorted MLCs expressed various types of niche molecules and cytokines, compared to megakaryocytes. As well, when we co-cultured HSCs with isolated MLCs, the numbers of HSC were significantly increased compare to with a liquid culture system. Taking these data together, we suggested that MLCs have the potential to support HSC proliferation. In this study, we first identified a candidate for EMH-niche cells and postulated a developmental mechanism in the spleen. Our findings provide a new functional insight into HSCs outside the bone marrow, and extend a new tool that supports ex vivo expansion of HSCs.

Hematopoietic stem cells (HSCs) have a potential to differentiate into variety types of blood cells, and have an ability of self-renewing in the bone marrow (BM). All these fates are regulated by intrinsic genetic pathways in the micro-enviroment (niche) where HSCs are located. Osteoblasts are thought to be the hematopoietic niche which regulates HSC function in the BM. To clarify the molecular basis of hematopoietic niche system, we compared the two somatic stem cells (Germ line stem cell (GS) and HSC) using cDNA suppression subtractive hybridization, and identified 12 stem cell-specific genes which are expressed both in the HSCs and GS. Among these genes, we analyzed Spp1, since it is a major glycoprotein produced by osteoblast and it has been reported to be a negative regulator of hematopoietic niche size in the BM. In situ Hybridization (ISH) with Spp1-specific probes demonstrated that Spp1-expressing osteobalsts were located at the endosteal region of trabecular bone (TB). In the TB of adult mouce, Spp1-expressing hematopoietic cells attached with Spp1-expressing osteobalsts. Double ISH analysis revealed that Spp1 and Tie2 were partially co-expressed in the TB. These data suggests that Spp1 is an important molecule of hematopoietic niche system which were produced and interacted both HSCs and niche cell. When we analyzed spp1 knock out mouse (Spp1−/−), adult Spp1−/− mice (8 weeks) appeared to be normal and fertile. However, the abnormal TB formation was observed in the younger Spp1−/− mice (2 weeks). A number of osteobalsts were increased and formed a multi-layer of osteoblasts at the endosteal region of TB. Along with the morphological change of the TB, distribution pattern of CD34, Tie2 and Bmi-1 expressing HSC was unusual. Tie2 expressing cells of Spp1−/− mouse were mainly located at vascular-enriched zone in the bone marrow, but few Tie2 expressing cells at osteoblastic zone. These data suggest that HSC and osteobalst do not form the correct niche in the Spp1−/− TB, and Spp1 is thought to be essential for the initial niche formation. To confirm the function of Spp1 at the initiation phase of niche formation, we performed the bone marrow transplantation. Mononuclear cells (1×105 cells) from Ly5.1 mice were transplanted into lethally irradiated Ly5.2 mice. At 12 days after transplantation, donor hematopoietic cells were observed in the recipient TB. At this day 12, the expression of Spp1 was up-regulated in the endosteal region of TB, however, Spp1 expression was decreased in the course of reconstitution. Thus, the findings regarding the hematopoietic niche formation in Spp1−/− mice and in the irradiated mice allow to understand the Spp1 function in the initial phase of niche formation in the bone marrow.

Mutations in ribosomal protein S19 (RPS19) gene is closely related to Diamond-Blackfan anemia (DBA). We have found point mutation, deletion and insertion in RPS19 gene in DBA patients (Blood, 100: 2724–31, 2002). In our analysis of DBA patients, gene expression of RPS19 from these mutated genomes were down regulated. Retroviral transduction of RPS19 gene to patient bone marrow cells restored the ability to differentiate into erythroid in vitro (Mol Ther., 7: 613–22, 2003). However, the mechanisms of how these mutations in RPS19 associate with anemia remains unknown. To analyze the mutated genome, we have made constructs of mutated RPS19 gene-expression vectors. From reported 56 different RPS19 mutations, we focused on the missense mutants and tried to characterize them. Flag-tagged twelve missense mutants (V15F, L18R, P47L, W52R, R56Q, S59F, A61Q, R62W, R101H, G120R, G127Q, and G131R) were exogenously expressed in several cell lines by retroviral vectors and were analysed by western blotting or FACS. When these 12 mutants were expressed in erythro-leukemic cell lines K562 and HEL, almost all the mutant proteins (except for G120R) were expressed at significantly low level compared to that of wild type. However, some of these mutants were expressed in human kidney epithelial cell line, 293T cells. These findings suggest that the expression level of RPS19 protein derived from mutated RPS19 genome is dependent on cell types, whether erythroid-lineage cells or others. In order to analyze the function of RPS19, doxycycline (Dox)-inducible expression of siRNA against RPS19 was induced in K562 cells and observed growth arrest at day 4 after induction of siRNA by Dox. Expression level of RPS19 decreased to one tenth of the normal level. Cell cycle analysis and quantitative-PCR analysis revealed that the growth arrest of K562 cells expressing siRNA against RPS19 was due to the induction of cell cycle arrest at G1 phase (G0/G1 54 % vs 36% ) coincide with the up-regulation of p21 and p57 mRNA level. From these results, we could possibly suggested that, in DBA patients, RPS19 protein expression is not stable and the decreased level of RPS19 protein prolongs G1 phase of cell cycle especially in the course of differentiation on erythroid cells, which may contribute to the retarded production of red blood cells. Our findings allow to understand the entire feature of DBA-associated RPS19 mutations especially on the erythorid differentiation and may help defining the therapeutic target to improve DBA.