Clonal hematopoiesis was prevalent in aplastic anemia. Some mutations were related to clinical outcomes. A highly biased set of mutations is evidence of Darwinian selection in the failed bone marrow environment. The pattern of somatic clones in individual patients over time was variable and frequently unpredictable. (Funded by Grant-in-Aid for Scientific Research and others.).

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Memory stem T cells (TSCMs) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. Hallmarks of autoimmune disease pathogenesis are abnormal CD4+ and CD8+ T cell activation. We investigated the TSCM subset in 55, 34, 43, and 5 patients with acquired aplastic anemia (AA), autoimmune uveitis, systemic lupus erythematosus, and sickle cell disease, respectively, as well as in 41 age-matched healthy controls. CD8+ TSCM frequency was significantly increased in AA compared with healthy controls. An increased CD8+ TSCM frequency at diagnosis was associated with responsiveness to immunosuppressive therapy, and an elevated CD8+ TSCM population after immunosuppressive therapy correlated with treatment failure or relapse in AA patients. IFN-γ and IL-2 production was significantly increased in various CD8+ and CD4+ T cell subsets in AA patients, including CD8+ and CD4+ TSCMs. CD8+ TSCM frequency was also increased in patients with autoimmune uveitis or sickle cell disease. A positive correlation between CD4+ and CD8+ TSCM frequencies was found in AA, autoimmune uveitis, and systemic lupus erythematosus. Evaluation of PD-1, CD160, and CD244 expression revealed that TSCMs were less exhausted compared with other types of memory T cells. Our results suggest that the CD8+ TSCM subset is a novel biomarker and a potential therapeutic target for AA.

The online version of this article has a Supplementary Appendix. Acknowledgments: the authors would like to thank Rie Ohmi and Kenichi Takemoto for excellent technical assistance, and Yasuhiko Yamamoto for excellent cell sorting technical support. We also thank the doctors who contributed patients to this study : Mitsufumi Nishio, Ryosuke Yamamura, Yoshiko Okikawa, Takeaki Tomoyose, Takuya Machida, Hiroshi Kanashima, Masahiro Manabe, Yukiyoshi Moriuchi, Takashi Nakaike, Yutaka Imamura, Kenji Shinohara, Taro Masunari, Akio Maeda, Hirokazu Okumura, Kazuyuki Shigeno, Masayuki Kikukawa, Hidemi Ogura, Tadashi Nagai, Hidetaka Niitsu and Senji Kasahara. Funding: this study was supported by grants awarded to SN. Manuscript received on December 23, 2011. Revised version arrived on May 6, 2012. Manuscript accepted on June 7, 2012 To characterize bone marrow failure with del(13q), we reviewed clinical records of 22 bone marrow failure patients possessing del(13q) alone or del(13q) plus other abnormalities. All del(13q) patients were diagnosed with myelodysplastic syndrome-unclassified due to the absence of apparent dysplasia. Elevated glycosylphosphatidylinositol-anchored protein-deficient blood cell percentages were detected in all 16 with del(13q) alone and 3 of 6 (50%) patients with del(13q) plus other abnormalities. All 14 patients with del(13q) alone and 2 of 5 (40%) patients with del(13q) plus other abnormalities responded to immunosuppressive therapy with 10-year overall survival rates of 83% and 67%, respectively. Only 2 patients who had abnormalities in addition to the del(13q) abnormality developed acute myeloid leukemia. Given that myelodysplastic syndrome-unclassified with del(13q) is a benign bone marrow failure subset characterized by good response to immunosuppressive therapy and a high prevalence of increased glycosylphosphatidylinositol-anchored protein-deficient cells, del (13q WHO 'MDS-U' designation. Haematologica 2012;97(12):1845-1849. doi:10.3324/haematol.2011 This is an open-access paper. ABSTRACT© F e r r a t a S t o r t i F o u n d a t i o n refractory cytopenia with multilineage dysplasia (RCMD) and 60 with MDS-U, whose blood samples were sent to our laboratory between May 1999 and July 2010 for screening of GPI-AP -granulocytes and erythrocytes. BM smear slides were reviewed by 2 independent hematologists. BM cellularity was defined as the percentage of BM volume occupied by hematopoietic cells in the trephine biopsy specimens. Hypocellular marrow was defined as less than 30% cellularity in patients under the age of 70 years, or less than 20% cellularity in patients 70 years and over. 6 Chromosomal analysis was performed and described according to the International System for Human Cytogenetic Nomenclature (ISCN). 7 Responses to IST were defined according to the established criteria. 8 The ethics committee of Kanazawa University Graduate School of Medical Science approved the study protocol, and all patients provided their informed consent prior to sampling. Monoclonal antibodiesMonoclona...

Abstract. The allyl sulfides, including diallyl sulfide (DAS), diallyl disulfide (DAD), and diallyl trisulfide (DAT), contained in garlic and members of the Allium family, have a variety of pharmacological activities. Therefore, allyl sulfides have been evaluated as potential novel chemotherapeutic agents. Here, we found that DAT inhibited nuclear factor-κB (NF-κB) signaling and induced apoptosis in primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV). We examined the cytotoxic effects of DAS, DAD and DAT on PEL cells. DAT significantly reduced the viability of PEL cells compared with uninfected B-lymphoma cells, and induced the apoptosis of PEL cells by activating caspase-9. DAT induced stabilization of IκBα, and suppressed NF-κB transcriptional activity in PEL cells. We examined the mechanism underlying DAT-mediated IκBα stabilization. The results indicated that DAT stabilized IκBα by inhibiting the phosphorylation of IκBα by the IκB kinase (IKK) complex. Furthermore, DAT induced proteasomal degradation of TRAF6, and DAT suppressed IKKβ-phosphorylation through downregulation of TRAF6. It is known that activation of NF-κB is essential for survival of PEL cells. In fact, the NF-κB inhibitor BAY11-7082 induced apoptosis in PEL cells. In addition, DAT suppressed the production of progeny virus from PEL cells. The administration of DAT suppressed the development of PEL cells and ascites in SCID mice xenografted with PEL cells. These findings provide evidence that DAT has antitumor activity against PEL cells in vitro and in vivo, suggesting it to be a novel therapeutic agent for the treatment of PEL. IntroductionPrimary effusion lymphoma (PEL, also termed body-cavitybased lymphoma) is a malignant B-cell lymphoma caused by Kaposi's sarcoma-associated herpesvirus (KSHV, also named HHV-8) in immunosuppressed individuals, such as AIDS patients or those that have undergone organ transplantation (1,2). PEL is a subtype of non-Hodgkin's lymphoma and is characterized by lymphomatous effusions of pleural and abdominal cavities. KSHV is a rhadinovirus of the γ-herpesvirus subfamily and is closely related to herpesvirus Saimiri and Epstein-Barr virus (EBV). KSHV is the causative agent of Kaposi's sarcoma and AIDS-related lymphoproliferative disorders, such as PEL and multicentric Castleman's disease (3). Similar to other herpesviruses, KSHV has two life cycles (latency and lytic replication). The KSHV genome circularizes and forms a double-stranded DNA, the episome, in the nucleus of PEL cells during latent infection. To establish a latent infection, KSHV expresses several viral genes, including latency-associated nuclear antigen (LANA), v-FLIP, v-cyclin, kaposin and microRNAs, in PEL cells. LANA is required for the replication and maintenance of viral DNA, and contributes to KSHV-associated oncogenesis through interaction with cellular molecules, such as p53, Rb and GSK-3. These viral proteins and RNA manipulate cellular signaling pathways, includ...

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Kaposi’s sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV-encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late (L) gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the viral preinitiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, -24, -30, -31, -34, and -66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a bacterial artificial chromosome (BAC) system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased virus production, viral DNA replication was unaffected. Chromatin immunoprecipitation analysis showed that ORF66 bound to the promoters of a late gene (K8.1) but did not bind to those of a latent gene (ORF72), an immediate early gene (ORF16), or an early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine repeat in the C-terminal region of ORF66 were essential for the interaction with ORF34, the transcription of K8.1, and virus production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model in which ORF66 serves as a critical vPIC component to promote late viral gene expression and virus production. IMPORTANCE KSHV ORF66 is expressed during the early stages of lytic infection, and ORF66 and vPIC are thought to contribute significantly to late gene expression. However, the physiological importance of ORF66 in terms of vPIC formation remains poorly understood. Therefore, we generated an ORF66-deficient BAC clone and evaluated its viral replication. The results showed that ORF66 plays a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing the function of ORF66 in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacts with the transcription start site of the K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among beta- and gammaherpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

ABSTRACTstrate that down-regulation of miR-126-3p and miR-145-5p promotes CD4+ and CD8 + T-cell activation by increasing MYC and PIK3R2 expression levels and T-cell proliferation, of potential importance in the pathogenesis of AA. Our results provide a pharmacological rationale for the potential use of synthetic miRNA mimics to limit disease.