Influence of differences in the hardness and calcium content of diets on the growth of craniofacial bone in rats

Objective:To examine the effects of a soft diet and a low-calcium diet on the craniofacial growth and bone architectures of the maxilla and mandible.Materials and Methods:Male rats (n  =  20, 3 weeks old) were divided into four groups. Ten rats were given a normal-calcium diet, and the other rats were given a low-calcium diet. Each group was then divided into two subgroups, which were fed a hard or a soft diet. After 4 weeks, craniofacial growth and architecture in maxillary and mandibular bone were analyzed using cephalometry, micro-computed tomography, and histopathology.Results:The low-calcium diet had no effect on serum calcium levels. The low-calcium diet had the greatest effect on craniofacial bone growth, while the soft diet affected the growth of several bone sites that are attached to the masseter muscle. A low-calcium diet resulted in the deterioration of the connectivity of the trabeculae in the furcation region of the maxillary and mandibular first molar, while a soft diet resulted in the diffuse disappearance of trabeculae in the central part of the furcation regions. In the midpalatal suture, a low-calcium diet resulted in inhibition of cartilaginous ossification, although the midpalatal suture had a normal cartilaginous structure. A soft diet resulted in narrower cartilage cell layers in the midpalatal suture.Conclusions:We demonstrated that a low-calcium diet and a soft diet resulted in a deterioration of bone structures in both the maxilla and in the mandible; however, the mechanisms underlying these effects differed between diets.     

Clinical outcomes of a novel therapeutic vaccine with Tax peptide‐pulsed dendritic cells for adult T cell leukaemia/lymphoma in a pilot study

Adult T cell leukaemia/lymphoma (ATL) is a human T cell leukaemia virus type‐I (HTLV‐I)‐infected T cell malignancy with poor prognosis. We herein developed a novel therapeutic vaccine designed to augment an HTLV‐I Tax‐specific cytotoxic T lymphocyte (CTL) response that has been implicated in anti‐ATL effects, and conducted a pilot study to investigate its safety and efficacy. Three previously treated ATL patients, classified as intermediate‐ to high‐risk, were subcutaneously administered with the vaccine, consisting of autologous dendritic cells (DCs) pulsed with Tax peptides corresponding to the CTL epitopes. In all patients, the performance status improved after vaccination without severe adverse events, and Tax‐specific CTL responses were observed with peaks at 16–20 weeks. Two patients achieved partial remission in the first 8 weeks, one of whom later achieved complete remission, maintaining their remission status without any additional chemotherapy 24 and 19 months after vaccination, respectively. The third patient, whose tumour cells lacked the ability to express Tax at biopsy, obtained stable disease in the first 8 weeks and later developed slowly progressive disease although additional therapy was not required for 14 months. The clinical outcomes of this pilot study indicate that the Tax peptide‐pulsed DC vaccine is a safe and promising immunotherapy for ATL.     

Coexpression of CD40 and CD40 ligand in Epstein-Barr virus-infected T and NK cells and their role in cell survival

We investigated the role that CD40-CD40 ligand (CD40L) signaling plays in survival of Epstein-Barr virus (EBV)-infected T and NK cells. EBV-infected T and NK cell lines derived from patients with either chronic active EBV infection (CAEBV) or nasal T/NK cell lymphoma, as well as virus-infected peripheral T cells freshly isolated from a patient with CAEBV, were shown to express both CD40 and CD40L on their surface. Apoptosis of these cells was enhanced by blockade of CD40-CD40L signaling by a fusion protein of CD40 and immunoglobulin G (CD40Ig). Expression of CD40 was induced in human CD40L-positive Jurkat T cells after experimental EBV infection, and apoptosis of infected cells was enhanced by CD40Ig. These results suggest that CD40-CD40L signaling promotes survival of EBV-infected T and NK cells and, thus, plays an important role in the pathogenesis of T/NK lymphoproliferative disorders associated with the virus.     

Identification and functional characterization of novel telomerase variant alleles in Japanese patients with bone-marrow failure syndromes

As the incidence of bone-marrow failure syndromes (BMFS) is 2-3x higher in East Asia than in the West, we examined peripheral blood or marrow cells of 100 Japanese patients for possible pathogenic mutations in the two main components of the telomere-synthesizing enzyme telomerase (hTERC RNA and hTERT protein) that have recently been implicated in the disease pathogenesis. We analyzed samples collected from 34 patients with acquired aplastic anemia (AA), 66 patients with myelodysplastic syndromes (MDS) and 120 healthy controls. In addition to two polymorphic germ-line sequence changes (n-771A/G and n-714 C insertion) in the promoter region of hTERC and eleven hTERT polymorphisms that were identified in both patients and healthy individuals, we found a novel germ-line C323T mutation in the hTERC RNA in an MDS patient only. This heterozygous C323T mutation abolished telomerase enzymatic activity and functioned in a haploinsufficiency manner to modulate telomerase activity in cells. In summary, this study reports a novel telomerase natural variant that abolishes telomerase function, which may lead to telomere shortening and marrow hypocellularity in patients with BMFS. This study also highlights the rarity of genetic alterations in BMFS patients in Japan, which suggests that other factors may play a more prominent role in the disease pathogenesis in East Asia.     

Helicobacter pylori induces antiapoptosis through buclear factor-kappaB activation

Although Helicobacter pylori is classified as a definite carcinogen, the mechanism underlying gastric carcinogenesis is not yet clear. We previously have shown that H. pylori activates an antiapoptotic gene, the cellular inhibitor of apoptosis protein 2 (c-IAP2), the underlying mechanism of which was investigated in the present study. cDNA array and real-time PCR analyses indicated that H. pylori showed a stimulatory effect on the expression of c-IAP2. Isogenic mutant strains with impaired cag pathogenicity island (cagPAI) expression showed weaker induction. Analyses that used the in situ terminal deoxynucleotide transferase-mediated dUTP nick end-labeling method indicated suppression of antiapoptosis by the antisense c-IAP2 oligonucleotide. Reporter assays with deletion and mutation constructs for the c-IAP2 promoter showed that nuclear factor-kappaB (NF-kappaB) binding sites are indispensable for transactivation. Super-repressor IkappaBalpha or NF-kappaB inhibitor reduced c-IAP2 transactivation by H. pylori, and exogenous expression of c-IAP2 inhibited apoptosis seen with H. pylori. In conclusion, H. pylori induces antiapoptosis through c-IAP2 transactivation following cagPAI-dependent NF-kappaB activation. The interaction of these stimuli may play a role in gastric carcinogenesis.     

Genomic imprinting of XX spermatogonia and XX oocytes recovered from XX<-->XY chimeric testes

We produced XX<-->XY chimeras by using embryos whose X chromosomes were tagged with EGFP (X*), making the fluorescent green female (XX*) germ cells easily distinguishable from their nonfluorescent male (XY) counterparts. Taking advantage of tagging with EGFP, the XX* "prospermatogonia" were isolated from the testes, and the status of their genomic imprinting was examined. It was shown that these XX cells underwent a paternal imprinting, despite their chromosomal constitution. As previously indicated in sex-reversal XXsxr testes, we also found a few green XX* germ cells developed as "eggs" within the seminiferous tubules of XX*<-->XY chimeric testes. These cells were indistinguishable from XX* prospermatogonia at birth but resumed oogenesis in a testicular environment. The biological nature of the "testicular eggs" was examined by recovering the eggs from chimeric testes. The testicular eggs not only formed an egg-specific structure, the zona pellucida, but also were able to fuse with sperm. The collected testicular eggs were indicated to undergo maternal imprinting, despite the testicular environment. The genomic imprinting did not always follow the environmental conditions of where the germ cells resided; rather, it was defined by the sex that was chosen by the germ cells at early embryonic stage.     

Low incidence of MYC/BCL2 double‐hit in Burkitt lymphoma

Translocations involving MYC are highly characteristic for Burkitt lymphoma (BL). BCL2 expression has also been found previously in about 10 to 20% of BL cases, and BCL2 translocation is a major mechanism for the deregulation of BCL2 expression in non‐Hodgkin lymphomas. However, we know little about the incidence of MYC/BCL2 double‐hit (DH) in BL. We examined BL cases to determine how frequently they contained BCL2 translocations in combination with MYC translocations using fluorescence in situ hybridization. We also determined the effect of BCL2 expression on clinical outcomes of BL. BCL2 translocations were detected in 3.5% (2/57 cases) of the cases, and BCL2 expression was detected in 33%. Two cases with BCL2 translocation also showed BCL2 expression. The incidence of BCL2 expression was significantly higher in patients 16 years of age and older (46%) than in patients under 16 years of age (6%). Among patients 16 years of age and older, we did not detect significant differences in overall survival with respect to BCL2 expression status. In conclusion, BCL2 translocation is a rare cytogenetic abnormality in BL, and BL probably accounts for only a small fraction of MYC/BCL2 DH lymphomas. BCL2 expression in BL is probably not associated with BCL2 translocations.     

CLEC-2 in megakaryocytes is critical for maintenance of hematopoietic stem cells in the bone marrow

Hematopoietic stem cells (HSCs) depend on the bone marrow (BM) niche for their maintenance, proliferation, and differentiation. The BM niche is composed of nonhematopoietic and mature hematopoietic cells, including megakaryocytes (Mks). Thrombopoietin (Thpo) is a crucial cytokine produced by BM niche cells. However, the cellular source of Thpo, upon which HSCs primarily depend, is unclear. Moreover, no specific molecular pathway for the regulation of Thpo production in the BM has been identified. Here, we demonstrate that the membrane protein C-type lectin-like receptor-2 (CLEC-2) mediates the production of Thpo and other factors in Mks. Mice conditionally deleted for CLEC-2 in Mks (Clec2^MkΔ/Δ) produced lower levels of Thpo in Mks. CLEC-2–deficient Mks showed down-regulation of CLEC-2–related signaling molecules Syk, Lcp2, and Plcg2. Knockdown of these molecules in cultured Mks decreased expression of Thpo. Clec2^MkΔ/Δ mice exhibited reduced BM HSC quiescence and repopulation potential, along with extramedullary hematopoiesis. The low level of Thpo production may account for the decline in HSC potential in Clec2^MkΔ/Δ mice, as administration of recombinant Thpo to Clec2^MkΔ/Δ mice restored stem cell potential. Our study identifies CLEC-2 signaling as a novel molecular mechanism mediating the production of Thpo and other factors for the maintenance of HSCs.Maintenance of hematopoietic stem cells (HSCs) within the adult BM is crucial for the healthy production of hematopoietic cells (Orkin and Zon, 2008). HSCs reside in a specialized microenvironment in the BM called the niche (Schofield, 1978). Along with cell-intrinsic programs, the niche influences the cell fate of HSCs, which in turn govern the homeostasis of the hematopoietic system (Nakamura-Ishizu et al., 2014a). The HSC niche is chiefly composed of nonhematopoietic cells, including immature osteoblasts (OBLs; Arai and Suda, 2007), endothelial cells (ECs; Butler et al., 2010; Ding et al., 2012), perivascular cells (Sugiyama et al., 2006; Ding et al., 2012), mesenchymal stem cells (MSCs; Méndez-Ferrer et al., 2010), sympathetic nervous cells (Katayama et al., 2006), adipocytes (Naveiras et al., 2009), and nonmyelinating Schwann cells (Yamazaki et al., 2011). Nonetheless, mature hematopoietic cells such as macrophages/monocytes (Chow et al., 2011), osteoclasts (Kollet et al., 2006), and regulatory T cells (Fujisaki et al., 2011) also regulate HSCs, albeit mainly in an indirect manner, through the modulation of nonhematopoietic niche cells. Recently, mature megakaryocytes (Mks) were described as hematopoietic progeny that directly regulate HSC quiescence (Heazlewood et al., 2013; Bruns et al., 2014; Zhao et al., 2014; Nakamura-Ishizu et al., 2014b); one of the mechanisms underlying Mk niche function is the production of the cytokine thrombopoietin (Thpo) by Mks themselves (Nakamura-Ishizu et al., 2014b). However, among the Mk-related niche factors reported to date, no molecular mechanism that is specific to Mks has been identified.Thpo is a crucial cytokine for both the maturation of Mks and the maintenance of quiescent HSCs (Zucker-Franklin and Kaushansky, 1996; Qian et al., 2007; Yoshihara et al., 2007). Thpo is produced in multiple organs, including the liver, kidney, spleen, and muscle (Nomura et al., 1997). Baseline production of serum Thpo is thought to be maintained by the liver and regulated in response to inflammatory stress or changes in glycosylation of aged platelets (Kaser et al., 2001; Stone et al., 2012; Grozovsky et al., 2015). Serum Thpo levels also fluctuate according to circulating platelet number: platelets sequester Thpo via the myeloproliferative leukemia virus oncogene (c-Mpl), the receptor for Thpo (Kuter and Rosenberg, 1995; de Graaf et al., 2010), thereby lowering Thpo levels. Thus, platelet number is not as tightly regulated by Thpo production as erythrocyte number is by erythropoietin production (Fandrey and Bunn, 1993). It is likely that BM HSCs depend on Thpo, which is produced in the BM by niche cells. Depletion of circulating platelets by neuraminidase does not affect HSCs (Bruns et al., 2014), indicating that serum Thpo up-regulation through thrombocytopenia does not affect HSC maintenance. Moreover, HSCs reside near bone-lining OBLs and mature Mks, which both support HSCs by producing Thpo (Yoshihara et al., 2007; Nakamura-Ishizu et al., 2014b). However, the main cellular source of Thpo, upon which BM HSCs depend, and the molecular signaling pathway that mediates BM Thpo production remain elusive.Recent studies showed that signals mediated through C-type lectin-like domain-containing receptors (CLEC-4H1 and CLEC-4H2; also known as Ashwell–Morell receptor) stimulate Thpo production in hepatocytes through recognition of desialylated platelets (Grozovsky et al., 2015). Platelets and Mks express CLEC-2 (Suzuki-Inoue et al., 2006, 2007), which is among the top 25 genes specifically expressed on Mks (Senis et al., 2007). Activation of platelet CLEC-2 through binding to sialylated podoplanin is essential for the segregation of lymphatic and blood vessels during development (Bertozzi et al., 2010; Suzuki-Inoue et al., 2010). CLEC-2–podoplanin signaling also functions in maintenance of lymphocyte- and dendritic cell–related responses in the stroma of lymph nodes (Acton et al., 2012, 2014; Herzog et al., 2013).The significance of CLEC-2 expression on Mks in BM hematopoiesis, and whether it is involved in Thpo production in Mks, has not been previously explored. Here, we demonstrate that Mk-specific deficiency of CLEC-2 disrupts HSC quiescence and alters HSC potential as a result of defective Mk niche function. Moreover, we demonstrate that CLEC-2 signaling is involved in various molecular pathways for production of niche factors, including Thpo in Mks. Through the identification of CLEC-2, a novel Mk-specific factor, our data elucidate the organ-dependent production and function of Thpo and reinforce the idea that Mks contribute to a niche that regulates HSC quiescence.