Search

Recent studies on pediatric acute myeloid leukemia (pAML) have revealed pediatric-specific driver alterations, many of which are underrepresented in the current classification schemas. To comprehensively define the genomic landscape of pAML, we systematically categorized 895 pAML into 23 molecular categories that are mutually distinct from one another, including new entities such as UBTF or BCL11B, covering 91.4% of the cohort. These molecular categories were associated with unique expression profiles and mutational patterns. For instance, molecular categories characterized by specific HOXA or HOXB expression signatures showed distinct mutation patterns of RAS pathway genes, FLT3, or WT1, suggesting shared biological mechanisms. We show that molecular categories were strongly associated with clinical outcomes using two independent cohorts, leading to the establishment of a prognostic framework for pAML based on molecular categories and minimal residual disease. Together, this comprehensive diagnostic and prognostic framework forms the basis for future classification of pAML and treatment strategies.

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML.Significance:We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes.See related commentary by Hasserjian and Nardi, p. 173.This article is highlighted in the In This Issue feature, p. 171.

Supplementary Data from Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia

Supplementary Figure from Integrated Genomic Analysis Identifies UBTF Tandem Duplications as a Recurrent Lesion in Pediatric Acute Myeloid Leukemia

The genetics of relapsed pediatric acute myeloid leukemia (AML) has yet to be comprehensively defined. Here, we present the spectrum of genomic alterations in 136 relapsed pediatric AMLs. We identified recurrent exon 13 tandem duplications (TD) in upstream binding transcription factor (UBTF) in 9% of relapsed AML cases. UBTF-TD AMLs commonly have normal karyotype or trisomy 8 with cooccurring WT1 mutations or FLT3-ITD but not other known oncogenic fusions. These UBTF-TD events are stable during disease progression and are present in the founding clone. In addition, we observed that UBTF-TD AMLs account for approximately 4% of all de novo pediatric AMLs, are less common in adults, and are associated with poor outcomes and MRD positivity. Expression of UBTF-TD in primary hematopoietic cells is sufficient to enhance serial clonogenic activity and to drive a similar transcriptional program to UBTF-TD AMLs. Collectively, these clinical, genomic, and functional data establish UBTF-TD as a new recurrent mutation in AML. Significance: We defined the spectrum of mutations in relapsed pediatric AML and identified UBTF-TDs as a new recurrent genetic alteration. These duplications are more common in children and define a group of AMLs with intermediate-risk cytogenetic abnormalities, FLT3-ITD and WT1 alterations, and are associated with poor outcomes. See related commentary by Hasserjian and Nardi, p. 173. This article is highlighted in the In This Issue feature, p. 171.

Pediatric acute myeloid leukemia (AML) is often refractory to chemotherapy and remains a devastating disease with poor outcomes. Consequently, there is an urgent need to characterize molecular drivers of pediatric AML with the goal of developing more effective molecular therapies. Our group recently identified somatic tandem duplications (TD) in exon 13 of upstream binding transcription factor (UBTF) as a subtype-defining genomic alteration in pediatric AML that is associated with poor outcomes, high relapse rate, and measurable residual disease (MRD) positivity. However, how these alterations drive leukemogenesis is yet to be investigated. Wild-type UBTF regulates rRNA transcription, ribosome biogenesis, and nucleolar formation. Our recent work suggests that UBTF-TD retains these functions. However, we found that expression of UBTF-TD in cord blood CD34+ cells, but not wild-type UBTF, leads to their increased proliferation, self-renewal, and CD117/CD33 surface marker expression, suggesting a potential gain of function as well. Furthermore, we also observed increased expression of HOXB locus genes, PRDM16, MEIS1, and NKX2-3 in the CD34+ UBTF-TD overexpression model relative to our WT control. These expression patterns are similar to what we see in patients, collectively implicating UBTF-TD driving leukemic phenotypes. Based on our localization studies of UBTF-TD to DNA, we hypothesized that UBTF-TD protein may gain the ability to interact with genomic loci that characterize UBTF-TD AML, like the HOXB locus. We therefore wanted to test if UBTF-TD localized to the HOXB cluster and if this lead direct transcriptional upregulation. To test this, we expressed HA-tagged UBTF-TD in cord-blood CD34+ cells and mapped UBTF-TD genomic occupancy using CUT&RUN. We found that UBTF-TD localized to the promoters and gene bodies of key genes, including PRDM16, MEIS1, NKX2-3, and the HOXB cluster. These sites were accompanied by H3K4me3 and H3K27ac active marks without the H3K27me3 repressive mark. To validate these as bone-fide UBTF-TD-occupied regions, we utilized a expressed FKBP12F36V-HA-UBTF-TD fusion in CD34+ cells. This degradation tag (dTAG) system allows for rapid degradation of UBTF-TD protein upon dTAG-13 treatment. We treated our FKBP12F36V-HA-UBTF-TD expressing cord blood CD34+ cells with 1uM of dTAG-13 or DMSO. We found that a 3-day treatment with dTAG-13 led to downregulation of HOXB gene locus expression, but loss of CD117 expression or cell growth required prolonged treatment. Furthermore, our CUT&RUN data showed that dTAG-13 treatment resulted in loss of UBTF-TD protein at the HOXB gene cluster. Collectively, these data show that UBTF-TD interacts with genomic loci at the HOXB gene cluster to directly upregulate their expression. This loss of HOX gene expression leads then leads to loss of a leukemic phenotype. Our data highlights a potential mechanism for UBTF-TD leukemogenesis and sets a foundation where inhibiting the UBTF-TD molecular signature may be a viable therapeutic option. Citation Format: Juan M Barajas, Masayuki Umeda, Ryan Hiltenbrand, Reethu Krishnan, Tamara Westover, Michael P Walsh, Jing Ma, Sherif Abdelhamed, Jeffery M Klco. UBTF-TD molecular alterations drive leukemogenesis in pediatric AML by directly interacting and regulating the HOXB gene locus [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A45.

Developing CAR T cells for acute myeloid leukemia (AML) has been hampered by a paucity of targets that are expressed on AML blasts and not on hematopoietic progenitor cells (HPCs). Here we demonstrate that GRP78 is expressed on the cell surface of primary AML blasts but not HPCs. To target GRP78, we generate T cell expressing a GRP78-specific peptide-based CAR, which show evidence of minimal fratricide post activation/transduction and antigen-dependent T cell differentiation. GRP78-CAR T cells recognize and kill GRP78-positive AML cells without toxicity to HPCs. In vivo, GRP78-CAR T cells have significant anti-AML activity. To prevent antigen-dependent T cell differentiation, we block CAR signaling and GRP78 cell surface expression post activation by using dasatinib during GRP78-CAR T cell manufacturing. This significantly improves their effector function in vitro and in vivo. Thus, targeting cell surface GRP78-positive AML with CAR T cells is feasible, and warrants further active exploration., There is an unmet need to discover suitable targets for CAR-T therapy in patients with acute myeloid leukemia (AML). Here the authors show that GRP78, a key regulator of the unfolded protein response, is highly expressed on the surface of primary AML blasts, but not on normal lymphocytes and hematopoietic progenitor cells, and that GRP78-CAR T have anti-AML activity in preclinical models.

Children with acute myeloid leukemia (AML) have a dismal prognosis due to a high relapse rate; however, the molecular basis leading to relapsed pediatric AML has not yet been fully characterized. To define the spectrum of alterations common at relapse, we performed integrated profiling of 136 relapsed pediatric AML cases with RNA sequencing (RNA-seq), whole-genome sequencing, and target-capture sequencing. In addition to well-characterized fusion oncoproteins, such as those involving KMT2A (n=36, 26.5%) or NUP98 (n=18, 13.2%), we also identified somatic mutations in UBTF (upstream binding transcription factor) in 12 of 136 cases (8.8%) of this relapsed cohort. Somatic alterations of the UBTF gene, which encodes a nucleolar protein that is a component of the RNA Pol I pre-initiation complex to ribosomal DNA promoters, have rarely been observed in AML. In our cohort, all alterations can be described as heterozygous in-frame exon 13 tandem duplications (UBTF-TD), either at the 3' end of exon 13 of UBTF or of the entire exon 13 (Fig. A). As we noticed limited detection in our pipeline as a result of complex secondary indels alongside the duplications, we established a soft-clipped read-based screening method to detect UBTF-TD more efficiently. Applying the screening to RNA-seq data of 417 additional pediatric AMLs from previous studies and our clinical service, we identified 15 additional UBTF-TDs, many of which have not been previously reported. At the amino acid level, UBTF-TDs caused amino acid insertions of variable sizes (15-181 amino acids), duplicating a portion of high mobility group domain 4 (HMG4), which includes short leucine-rich sequences. UBTF-TD AMLs commonly occurred in early adolescence (median age: 12.6, range: 2.4-19.6), and 19 of the total 27 cases had either normal karyotype (n=12) or trisomy 8 (n=7). UBTF-TD is mutually exclusive from other recurrent fusion oncoproteins, such as NUP98 and KMT2A rearrangements (Fig. B), but frequently occurred with FLT3-ITD (44.4%) or WT1 mutations (40.7%). The median variant allele fraction (VAF) of the UBTF-TD was 48.0% (range: 9.7-66.7%). In four cases with data at multiple disease time points, the identical UBTF-TDs were present at high allele fractions at all time points, suggesting that UBTF-TD is a clonal alteration. tSNE analysis of the transcriptome dataset showed that UBTF-TD AMLs share a similar expression pattern with NPM1 mutant and NUP98-NSD1 AML subtypes, including NKX2-3 and HOXB cluster genes (Fig. C) . Altogether, these findings suggest that UBTF-TD is a unique subtype of pediatric AML. To address the impact of UBTF-TD expression in primary hematopoietic cells, we introduced UBTF-TD and UBTF wildtype expression vectors into cord blood CD34+ cells via lentiviral transduction. UBTF-TD expression promotes colony-forming activity and cell growth, yielding cells with a persistent blast-like morphology (Fig. D). Further, transcriptional profiling of these cells demonstrated expression of HOXB genes and NKX2-3, similar to UBTF-TD AMLs in patients, indicating that UBTF-TD is sufficient to induce the leukemic phenotype. To investigate the prevalence of UBTF-TDs in larger de novo AML cohorts, we applied the above UBTF-TD screening method to the available de novo AML cohorts of TCGA (n=151, adult), BeatAML (n=220, pediatric and adult), and AAML1031 (n=1035, pediatric). We identified UBTF-TDs in 4.3% (45/1035) of the pediatric AAML1031 cohort, while the alteration is less common (0.9%: 3/329, p=0.002) in the adult AML cohorts (Fig. E). In the AAML1031 cohort, UBTF-TDs remain mutually exclusive with known molecular subtypes of AML and commonly occur with FLT3-ITD (66.7%) and WT1 (40.0%) mutations and either normal karyotype or trisomy 8. The presence of UBTF-TDs in the AAML1031 cohort is associated with a poor outcome (Fig. F, median overall survival, 2.3 years) and MRD positivity; multivariate analysis revealed that UBTF-TD and WT1 are independent risk factors for overall survival within FLT3-ITD+ AMLs. In conclusion, we demonstrate UBTF-TD defines a unique subtype of AMLs that previously lacked a clear oncogenic driver. While independent of subtype-defining oncogenic fusions, UBTF-TD AMLs are associated with FLT3-ITD and WT1 mutations, adolescent age, and poor outcomes. These alterations have been under-recognized by standard bioinformatic approaches yet will be critical for future risk-stratification of pediatric AML. Figure 1 Figure 1. Disclosures Iacobucci: Amgen: Honoraria; Mission Bio: Honoraria. Miller: Johnson & Johnson's Janssen: Current Employment. Mullighan: Pfizer: Research Funding; Illumina: Membership on an entity's Board of Directors or advisory committees; AbbVie: Research Funding; Amgen: Current equity holder in publicly-traded company.

Context: The genomic characterization of acute myeloid leukemia (AML) in adults, both at diagnosis and relapse, has been extensively reported. Previous work has shown a clear difference between AML in adults and children, yet the genetic changes associated with relapsed disease in children have yet to be fully described. Objective: To elucidate the genetic background of pediatric AML at relapse and identify gene alterations related to poor prognosis. Design: Transcriptomic and genetic analyses were performed on relapsed AML from multiple clinical studies held in St. Jude Children’s Research Hospital from 2002 to 2020. No blinding was performed in this study. Patients or Other Participants: RNA sequencing was performed from 136 patients (median age of 9.2 years) with relapsed pediatric AML. Ninety-one of these samples were also studied by tumor–normal-paired whole-genome sequencing. Additional transcriptomic data from pediatric AML at diagnosis (n=417) were also obtained from previous reports to define the molecular alterations common in relapsed pediatric AML. The clinical outcome of each molecular category is confirmed using clinical data of the diagnosis cohort of the TARGET pediatric AML study. Results: Genomic profiling of relapsed pediatric AML demonstrated an increase in KMT2A (n=36, 26.5%) and NUP98 rearrangements (n=18, 13.2%) when compared to de novo pediatric AML, as well as an increase in WT1 mutations (n=33, 24.3%). Comprehensive characterization of pediatric AML with extension RNA transcriptome data revealed 18 molecular categories with unique expression patterns and mutually exclusive gene alteration patterns. These molecular categories included rare categories with structural variants involving oncogenes of MECOM (n=3, 2.2%) or BCL11B (n=2, 1.5%), which led to the upregulation and allele-specific expression (ASE) of each gene. The entire molecular categories showed unique distribution in the relapse cohort compared to the TARGET cohort (p Conclusions: Integrated genomic profiling of pediatric AML identified unique molecular categories associated with relapsed disease and poor outcome.

Virus-associated hemorrhagic cystitis (VAHC) is a formidable complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The standard management of severe VAHC after allo-HSCT has not been established. Intravenous administration of cidofovir (CDV), an acyclic nucleoside analogue with broad-spectrum activity against DNA viruses, has been reported to be effective for VAHC, but it can cause severe renal toxicity. Here we report four cases who achieved clinical responses with intravesical instillation of CDV for severe VAHC after allo-HSCT. Median age was 57 years (40-63), and all were male. The underlying diseases were hematological malignancies. Three had received bone marrow transplantation, and one received cord blood transplantation twice. Conditioning regimen was myeloablative for one, and reduced-intensity for three. The viral types were BK virus and/or adenovirus. Two patients had received CDV intravenously prior to the intravesical therapy. A dose of intravesical CDV was 2-5 mg/kg. In all cases, symptoms of cystitis improved dramatically within a few days without showing any systemic adverse effects. The virological response was observed in two cases. This local therapy was effective even in the cases refractory to the intravenous CDV and a case with severe renal failure. Along with the review of literature, we propose that the intravesical instillation of CDV can be a therapeutic option for severe VAHC after allo-HSCT.

SummaryIsolation of specific cell types, including pluripotent stem cell (PSC)-derived populations, is frequently accomplished using cell surface antigens expressed by the cells of interest. However, specific antigens for many cell types have not been identified, making their isolation difficult. Here, we describe an efficient method for purifying cells based on endogenous miRNA activity. We designed synthetic mRNAs encoding a fluorescent protein tagged with sequences targeted by miRNAs expressed by the cells of interest. These miRNA switches control their translation levels by sensing miRNA activities. Several miRNA switches (miR-1-, miR-208a-, and miR-499a-5p-switches) efficiently purified cardiomyocytes differentiated from human PSCs, and switches encoding the apoptosis inducer Bim enriched for cardiomyocytes without cell sorting. This approach is generally applicable, as miR-126-, miR-122-5p-, and miR-375-switches purified endothelial cells, hepatocytes, and insulin-producing cells differentiated from hPSCs, respectively. Thus, miRNA switches can purify cell populations for which other isolation strategies are unavailable.

Key Clinical Message Currently, there is no standard treatment for neurolymphomatosis because of the scarcity of clinical studies. Here, we report the successful treatment of neurolymphomatosis caused by follicular lymphoma with bendamustine, which could be an effective treatment option for this condition.

The Japan Atomic Energy Agency has been establishing a database of operation and maintenance experience for the High Temperature Engineering Test Reactor. The objective of this database is to share...

MicroRNA (miRNA) activity differs with cell type, suggesting it can be used as a cell marker. In this study, we developed novel miRNA-responsive non-viral reporter vectors to continuously monitor and visualize miRNA dynamics during differentiation and to efficiently purify target living cells. Each vector codes miRNA-responsive and reference reporter genes in a single mRNA. These two genes are independent modules but transcribed by a single promoter, which enables us to distinguish miRNA-mediated post-transcriptional repression from transcriptional repression. We generated stable, miRNA-responsive vector-containing human induced pluripotent stem cells (hiPSCs) using the piggyBac transposon or episomal vectors. We could continuously monitor the differentiation status of living hiPSCs by detecting the activity of hiPSC-specific miRNA (miR-302a*). In addition, we could selectively sort hiPSC-derived cardiomyocytes using cardiomyocyte-specific miRNA (miR-208a or miR-1)-reporter vectors. Our miRNA reporter system provides a simple way to quantitatively and continuously monitor and visualize changes in the cellular state and should facilitate a broad range of studies that depend on cellular changes including drug discovery and cell-fate conversion.

Object Modified cervical laminoplasty techniques have been developed to reduce postoperative axial neck pain and preserve function in patients with cervical spondylotic myelopathy (CSM). However, the previous studies demonstrating satisfactory surgical outcomes had a retrospective design. Here, the authors aimed to prospectively evaluate the 2-year outcomes of a modified cervical laminoplasty technique for CSM that preserves the paravertebral muscles. Methods Outcomes were analyzed for 40 patients (22 men and 18 women; mean age, 66.6 years; age range 44–92 years) with CSM who underwent C4–6 laminoplasty with C-3 and C-7 partial laminectomies or C-3 total and C-7 partial laminectomies and received hydroxyapatite spacers. Neurological, pain severity, and spinal radiographic evaluations were performed preoperatively and at 3, 6, 12, 18, and 24 months postoperatively. Plain radiography and MRI of the cervical spine were performed to evaluate the range of motion (ROM), sagittal alignment, and cross-sectional areas of the deep extensor muscles. The extent of bone–spacer bonding and bony union at the gutter was assessed by CT. Results The mean preoperative Japanese Orthopaedic Association CSM score was 10.2, but it increased to 14.4 by 24 months after surgery. Eleven patients had axial neck pain preoperatively, but only 3 reported mild pain at 24 months, and in all 3 cases the pain was mild. The mean angle of lordosis was 11.7° preoperatively and 12.0° 2 years postoperatively. Although the ROM at the C2–7 levels was significantly reduced 3 months postoperatively, an increasing trend was observed up to 12 months, and 86% of the preoperative ROM was achieved by 2 years postoperatively. The mean paravertebral muscle cross-sectional areas were 833 ± 215 mm2 preoperatively and 763 ± 197 mm2 24 months postoperatively, but the difference was not statistically significant. The rates of bone–spacer bonding and bony union at the gutter were low during the early stages but increased to 90% and 93%, respectively, by 2 years after surgery. Conclusions The modified laminoplasty technique used in this study ensured very good neurological status and ROM after 2 years and was associated with low incidences of axial neck pain and serious complications. This simple and easy operative method could benefit future laminoplasty protocols.

Background: Bone marrow cells possess multipotentiality and have been used for several treatments. We hypothesized that bone marrow cells might differentiate into regenerated tendon and that several cytokines within bone marrow cells might accelerate tendon healing. Therefore, we treated Achilles tendon ruptures in a rat model with transplantation of whole bone marrow cells. Methods: Nine F344/Nslc (Fisher) rats were the source of bone marrow cells and mesenchymal stem cells as well as normal Achilles tendons. Eighty-seven Fisher rats were used for the experiments. The rats were divided into three groups: the BMC group (bone marrow cells injected around the tendon), the MSC group (mesenchymal stem cells injected around the tendon), and the non-treated control group (incision only). Outcome measures included mechanical testing, collagen immunohistochemistry, histological analysis, and reverse transcription-polymerase chain reaction to detect expression of transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF). Results: The ultimate failure load in the BMC group was significantly greater than that in the non-treated or the MSC group at seven days after incision (3.8 N vs. 0.9 N or 2.1 N, p < 0.016) and at fourteen days after incision (10.2 N vs. 6.1 N or 8.2 N, p < 0.016). The ultimate failure load in the BMC group at twenty-eight days after incision (33.8 N) was the same as that of normal tendon (34.8 N). The BMC group demonstrated stronger staining for type-III collagen at seven days after incision and stronger staining for type-I collagen at twenty-eight days than did the MSC group. Expression of TGF-β and VEGF in the BMC group was significantly increased compared with that in the other groups at four days after incision (TGF-β: 1.6 vs. 1.3 or 0.6, p < 0.01; VEGF: 1.7 vs. 1.1 or 0.9, p < 0.01). Conclusions: Transplantation of whole bone marrow cells may be a better and more readily available treatment for Achilles tendon rupture than cultured mesenchymal stem cells. Clinical Relevance: The present study provides evidence that transplantation of bone marrow cells may be a promising therapy for the treatment of Achilles tendon rupture.

Sideroblastic anemias consist of a heterogeneous group of inherited and acquired disorders. The most common hereditary type is X-linked sideroblastic anemia (XLSA), which is associated with mutations in the erythroid-specific δ-aminolevulinic acid synthase (ALAS2) gene. Heme synthesis starts with the polymerization of glycine and succinyl CoA polymerization and synthesis of δ-aminolevulinic acid (ALA) in the mitochondria. ALAS2 encodes the enzyme that catalyzes these first steps in the heme synthetic pathway in erythroid cells, steps that require pyridoxal 5'-phosphate (PLP) as a cofactor. It has been found that treatment with PLP is effective for a small fraction of XLSA patients, but there are no effective treatments for the other fraction. The aim of this study is to explore the molecular mechanisms of XLSA and to develop new effective therapies. We used episomal methods to generate induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) of three late-onset XLSA female patients in one family. The cells harbored the heterozygous mutation (R227C) in the ALAS2 gene. Because ALAS2 is located in the X-chromosome, either wild-type or mutant ALAS2 gene is inactivated in the erythroid cells of female heterozygotes. All three patients showed severe anemia and their PBMCs showed skewed X-chromosome inactivation with preferential inactivation of the X chromosome carrying wild-type ALAS2, indicating a condition associated with unbalanced lyonization. From each patient, we successfully established iPSC lines with the active mutant ALAS2 allele and with the active wild-type ALAS2 allele. We assessed the hematopoietic differentiation potential of these two types of iPSC lines derived from the same patient. Differentiation into hematopoietic progenitor cells (HPCs) using embryoid body formation was comparable in the two groups. However, further differentiation in erythroid culture was significantly impaired in iPSC lines harboring the active mutant ALAS2 allele compared with those harboring the active wild-type ALAS2 allele (CD235a+ cells: 59.20±12.16% with the active wild-type ALAS2 allele vs. 3.95±4.71% with the active mutant ALAS2 allele, p We examined the effect of ALA on the erythroid differentiation of the HPCs. The CD235a-positive erythroid cell ratio of HPCs with the active wild-type ALAS2 allele did not increase following administration of ALA. By contrast, the ratio reached normal levels for HPCs with the active mutant ALAS2 allele (CD235a+ cells: 6.10± 5.61% vs. 85.34± 11.05%, p Our data suggest that our iPSC-based system could be useful for studying the precise molecular mechanisms of XLSA and drug testing. Figure Figure. Disclosures Morimoto: Grant-in-Aid for JSPS Research Fellow: Research Funding. Takaori-Kondo: celgene: Honoraria, Research Funding; Bristol-Myers Squibb, Novartis, Janssen pharma, Pfizer: Honoraria.

The treatment of autoimmune diseases by allogeneic bone marrow transplantation remains a promising therapeutic avenue. However, more intensive studies on murine models are essential before application to a large number of human patients. In particular, the use of bone marrow transplantation to treat rheumatoid arthritis has been problematic. We have taken advantage of the SKG/Jcl mouse that develops a chronic T cell-mediated autoimmune disease that mimics rheumatoid arthritis which attempted to prevent the development of immunopathology in these mice by allogeneic bone marrow transplantation (BMT). In particular, we utilized our unique technology in which bone marrow cells (BMCs) of control C57BL/6J mice are directly injected into the bone marrow cavity in the tibia of SKG mice (intra-bone marrow [IBM]-BMT). As controls, SKG/Jcl mice were transplanted with whole BMCs from syngeneic SKG mice. Importantly, 12 months after IBM-BMT [B6-->SKG] demonstrated no evidence of arthritis, whereas the control [SKG-->SKG] mice demonstrated, the expected immunopathology of a rheumatoid arthritis-like condition. Further, hematolymphoid cells in [B6-->SKG] mice were reconstituted by donor-derived cells and the percentages of Treg (Foxp3+/CD4+) cells, the percentages of receptor activator of nuclear factor-kappaB ligand (RANKL)+ cells on the CD4+ T cells and the serum levels of tumor necrosis factor-alpha, interleukin-1 and interleukin-6 were normalized in the [B6-->SKG] mice. These data suggest that IBM-BMT is a viable method of immunological manipulation that suppresses the severe joint destruction and bone absorption in SKG/Jcl mice and lends further credence to the use of this methodology in humans with intractable rheumatoid arthritis.

Cell proliferation and matrix synthesis were compared for rat nucleus pulposus cells cocultured with mesenchymal stem cells (MSCs) or fresh whole bone marrow cells (BMCs), harvested by the perfusion or aspiration methods. Nucleus pulposus cells were isolated from tail intervertebral discs of F344/slc rats, and BMCs were obtained from femora. Proteoglycan synthesis, DNA synthesis, and aggrecan mRNA expression were measured. The level of transforming growth factor-beta in supernatants from the culture system was also measured. Cell number, aggrecan mRNA expression, and uptake of [(35)S]-sulfate and [(3)H]-thymidine by nucleus pulposus cells cocultured with fresh whole BMCs all increased significantly compared with nucleus pulposus cells cocultured with MSCs. TGF-beta secreted by nucleus pulposus cells cocultured with fresh whole BMCs also significantly increased when compared with cocultures with MSCs. The perfusion method was superior to the aspiration method for preventing contamination of BMCs with peripheral red blood cells and lymphocytes, which may cause an autoimmune response in the disc. In conclusion, we suggest that fresh whole BMCs harvested by the perfusion method are more effective for increasing the proliferative and matrix synthesis capacity of nucleus pulposus cells.

Object Surgical outcome and radiographic changes after microsurgical bilateral decompression via a unilateral approach (MBDU) for lumbar spinal canal stenosis during midterm follow-up periods (> 2 years) have not been reported. The authors retrospectively investigated surgical outcomes after MBDU in patients with lumbar degenerative spondylolisthesis with stenosis in comparison with patients with degenerative stenosis during a minimum follow-up period of 2 years. Radiographic changes at the affected intervertebral level were analyzed during that follow-up period. Methods Forty-eight patients (23 in the spondylolisthesis group, 25 in the degenerative stenosis group) were included in the study. The average follow-up period was 46 months (range 24–71 months). Surgical outcome was evaluated using the Neurogenic Claudication Outcome Score (NCOS) and the Oswestry Disability Index (ODI). Additionally, the back pain score within the NCOS was also compared. There were no statistically significant differences between the spondylolisthesis group and the degenerative stenosis group with regard to sex, age, follow-up period, operating time, blood loss, surgical sites, approach side, preoperative NCOS, preoperative back pain score, and preoperative ODI. Comparisons were also made between groups using 2 satisfaction measurements at the last follow-up visit. Radiographically, intervertebral angles of 80 sites and slip percentages of 24 sites were measured preoperatively and at the last follow-up. Results No patient in either group had additional surgery in the lumbar spine, including fusion procedures. The NCOS, back pain score, and ODI had significantly improved at the last follow-up in both groups. There were no significant differences between the 2 groups in these 3 parameters and the 2 satisfaction measurements at the last follow-up, although those for the spondylolisthesis group indicated a somewhat worse outcome. Intervertebral angles, dynamic intervertebral angles, and dynamic slip percentage did not significantly change after surgery, whereas only slip percentage significantly increased postoperatively (p = 0.0319). Conclusions A satisfactory outcome of MBDU persisted for a period longer than 2 years for patients with degenerative spondylolisthesis with stenosis as well as for those with degenerative stenosis. Radiographically in both groups this less invasive procedure was not likely to result in postoperative dynamic instability at the affected level, although the slippage progressed in the spondylolisthesis group.

Osteonecrosis (ON) of the femoral head is one of the most serious complications associated with steroid administration. Here, we treated corticosteroid-induced ON in the rabbit by transplanting mesenchymal cells (MCs). Rabbits were injected once with 20 mg/kg of methylprednisolone (MPSL) and divided into three groups as follows: (1) MPSL alone (no further treatment); (2) MPSL+MCs (7 days after MPSL, MCs [1 x 10(7)/2 ml] were injected into the bone marrow cavity of the femurs); (3) MPSL+saline (7 days after MPSL, saline [2 ml] was injected into the bone marrow cavity of the femurs). Subsequently, the incidence of ON in the femurs 4 weeks after MPSL alone and MPSL+saline was 80 and 68.4%, respectively. In contrast, no ON was recorded in rabbits treated with MPSL+MCs. Vascular endothelial growth factor (VEGF) staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end-labeling (TUNEL) staining was more marked in the MPSL alone and MPSL+saline groups than in the MPSL+MCs rabbits. The percentages of cells in the G1 phase in the MPSL+MCs group were significantly lower than in the other two groups. These findings suggest that the injection of autologous MCs into the femur could prevent corticosteroid-induced ON in patients treated with high-dose short-term steroid medication.

The future post-irradiation program for the first-loading fuel of the HTTR is scheduled using the HTTR fuel handling facilities and the Hot Laboratory in the Japan Materials Testing Reactor (JMTR) to confirm its irradiation resistance and to obtain data on its irradiation characteristics in the core. This report describes the preliminary test results and the future plan for a post-irradiation examination for the HTTR fuel. In the preliminary test, fuel compacts made with the same SiC-coated fuel particle as the first loading fuel were used. In the preliminary test, dimension, weight, fuel failure fraction, and burnup were measured, and X-ray radiograph, SEM, and EPMA observations were carried out. Finally, it was confirmed that the first-loading fuel of the HTTR showed good quality under an irradiation condition. The future plan for the post-irradiation tests was described to confirm its irradiation performance and to obtain data on its irradiation characteristics in the HTTR core.

Object The authors retrospectively investigated the surgical outcomes and radiographically documented changes after microsurgical posterior foraminotomy with en bloc laminoplasty in patients with cervical spondylotic radiculo-myelopathy (CSRM), including cervical spondylotic amyotrophy (CSA), during a period greater than 2 years. Methods Thirty-four consecutive patients (24 men and 10 women) were included in this study. Twenty patients had preoperative radicular pain, and CSA was diagnosed in 14 patients. The mean age at the time of surgery was 61 years (range 43–77 years). The follow-up period ranged from 2 to 6.5 years (mean 3.4 years). Foraminotomy was performed at 49 sites. Neurological improvement was evaluated using the Japanese Orthopaedic Association (JOA) scoring system; radicular pain and deltoid muscle strength were also evaluated clinically. Cervical lordosis, flexion–extension angles, range of motion (ROM), and the angulation and the extent of vertebral slippage at the affected nerve root levels were measured preoperatively and at last follow-up examination. The mean rate of JOA score improvement was 67.2% (range 22.2–100%). In all 20 patients, preoperative radicular pain completely resolved after surgery. In all 14 patients with CSA, deltoid muscle strength improved; in approximately 80% of these patients, there was either no muscle weakness or only slight weakness. The flexion angles and ROM significantly decreased at the time of the last follow-up examination (p = 0.0402 and 0.0196, respectively). No other items changed significantly. Conclusions The aforementioned surgical procedure was safely completed and the surgical outcomes were satisfactory for CSRM including CSA. The instability (the angulation and the vertebral slippage) did not significantly change after surgery. This procedure yielded outstanding results and should be considered an option for cervical laminoplasty in the future.

The high temperature engineering test reactor (HTTR) is a graphite-moderated and helium gas-cooled reactor with a thermal output of 30 MW and a reactor outlet coolant temperature of 950 °C during the high temperature test operation. Fuel elements, control rod guide blocks and replaceable reflector blocks are main core components of the HTTR. The core components are mainly made of ceramics, such as graphite, silicon carbide, etc. which are excellent thermal resistant materials. Design requirements for the core components were laid down and extensive design efforts and R&D were performed in order to maintain their structural integrity. The structural integrity of the core components was ascertained by performing evaluations on the basis of the R&D and stress analysis results. This paper describes the design requirements and design details of the core components, and the structural integrity evaluation results for the fuel and graphite blocks.

正射影速度場の時系列から剛体の立体構造と3D速度とを復元するために, まず時間的に隣接する速度場間での対応決定を行い, ついで剛体条件, 瞬時剛体条件を用いて復元を行う手法が提案されている.本報告は, 変位の総和の最小化と, 速度変化の連続性と, 剛体性とに注目した, 前者についての効率的な手法の提案とコンピュータシミュレーションによる検証とからなる.

Modified cervical laminoplasty techniques have been developed to reduce postoperative axial neck pain and preserve function in patients with cervical spondylotic myelopathy (CSM). However, the previous studies demonstrating satisfactory surgical outcomes had a retrospective design. Here, the authors aimed to prospectively evaluate the 2-year outcomes of a modified cervical laminoplasty technique for CSM that preserves the paravertebral muscles.Outcomes were analyzed for 40 patients (22 men and 18 women; mean age, 66.6 years; age range 44-92 years) with CSM who underwent C4-6 laminoplasty with C-3 and C-7 partial laminectomies or C-3 total and C-7 partial laminectomies and received hydroxyapatite spacers. Neurological, pain severity, and spinal radiographic evaluations were performed preoperatively and at 3, 6, 12, 18, and 24 months postoperatively. Plain radiography and MRI of the cervical spine were performed to evaluate the range of motion (ROM), sagittal alignment, and cross-sectional areas of the deep extensor muscles. The extent of bone-spacer bonding and bony union at the gutter was assessed by CT.The mean preoperative Japanese Orthopaedic Association CSM score was 10.2, but it increased to 14.4 by 24 months after surgery. Eleven patients had axial neck pain preoperatively, but only 3 reported mild pain at 24 months, and in all 3 cases the pain was mild. The mean angle of lordosis was 11.7° preoperatively and 12.0° 2 years postoperatively. Although the ROM at the C2-7 levels was significantly reduced 3 months postoperatively, an increasing trend was observed up to 12 months, and 86% of the preoperative ROM was achieved by 2 years postoperatively. The mean paravertebral muscle cross-sectional areas were 833 ± 215 mm(2) preoperatively and 763 ± 197 mm(2) 24 months postoperatively, but the difference was not statistically significant. The rates of bone-spacer bonding and bony union at the gutter were low during the early stages but increased to 90% and 93%, respectively, by 2 years after surgery.The modified laminoplasty technique used in this study ensured very good neurological status and ROM after 2 years and was associated with low incidences of axial neck pain and serious complications. This simple and easy operative method could benefit future laminoplasty protocols.

The purpose of this study was to compare anterior cruciate ligament (ACL) regeneration between animal groups subjected to intra-articular injection of fresh whole bone marrow cells (BMCs), cultured mesenchymal stem cells (MSCs), or saline. Partially transected ACLs in Fischer 344/Nslc rats were prepared, followed by injection of BMCs, MSCs, or saline into the articular cavity at 1 week after transection. Donor cells expressing green fluorescent protein were detected in the recipient's transected ACLs at 4 weeks in the BMC and MSC groups, and their ACLs appeared almost normal histologically. Further, there were significantly more mature spindle cells in the BMC group than in the saline group at 4 weeks. Biomechanically, the tensile strength in the BMC group reached near normal levels at 4 weeks after injection. The levels of transforming growth factor-β1 in the ACL tissue and knee joint fluid in the BMC group were increased significantly compared with that of the saline group at 4 weeks as detected by immunohistochemical analysis. In conclusion, intra-articular bone marrow transplantation using fresh whole BMCs is an effective treatment for ACL partial rupture. This therapy is easy to apply in a clinical setting because no culture system is required for collecting MSCs.

Surgical outcome and radiographic changes after microsurgical bilateral decompression via a unilateral approach (MBDU) for lumbar spinal canal stenosis during midterm follow-up periods (2 years) have not been reported. The authors retrospectively investigated surgical outcomes after MBDU in patients with lumbar degenerative spondylolisthesis with stenosis in comparison with patients with degenerative stenosis during a minimum follow-up period of 2 years. Radiographic changes at the affected intervertebral level were analyzed during that follow-up period.Forty-eight patients (23 in the spondylolisthesis group, 25 in the degenerative stenosis group) were included in the study. The average follow-up period was 46 months (range 24-71 months). Surgical outcome was evaluated using the Neurogenic Claudication Outcome Score (NCOS) and the Oswestry Disability Index (ODI). Additionally, the back pain score within the NCOS was also compared. There were no statistically significant differences between the spondylolisthesis group and the degenerative stenosis group with regard to sex, age, follow-up period, operating time, blood loss, surgical sites, approach side, preoperative NCOS, preoperative back pain score, and preoperative ODI. Comparisons were also made between groups using 2 satisfaction measurements at the last follow-up visit. Radiographically, intervertebral angles of 80 sites and slip percentages of 24 sites were measured preoperatively and at the last follow-up.No patient in either group had additional surgery in the lumbar spine, including fusion procedures. The NCOS, back pain score, and ODI had significantly improved at the last follow-up in both groups. There were no significant differences between the 2 groups in these 3 parameters and the 2 satisfaction measurements at the last follow-up, although those for the spondylolisthesis group indicated a somewhat worse outcome. Intervertebral angles, dynamic intervertebral angles, and dynamic slip percentage did not significantly change after surgery, whereas only slip percentage significantly increased postoperatively (p = 0.0319).A satisfactory outcome of MBDU persisted for a period longer than 2 years for patients with degenerative spondylolisthesis with stenosis as well as for those with degenerative stenosis. Radiographically in both groups this less invasive procedure was not likely to result in postoperative dynamic instability at the affected level, although the slippage progressed in the spondylolisthesis group.

Objectives. Femoral head osteonecrosis (ON) is a serious complication of steroid administration. We evaluated bone marrow transplantation (BMT) for preventing corticosteroid-induced ON. Methods. Rabbits, injected with methylprednisolone (MPSL; 20mg/kg), were divided into four groups: (i) MPSL alone; MPSL injection only, (ii) MPSL+needling; 2 days after MPSL injection, a hole (1.2 mm diameter) was drilled from the outer cortex 2.5cm distal to the proximal end of the greater trochanter, (iii) MPSL+saline; 2 days after MPSL injection, 2ml saline was injected directly into the bone marrow cavity, and (iv) MPSL+BMT; 2 days after MPSL injection, 1 x 10 7 /2 ml bone marrow cells (BMCs) were injected directly into the bone marrow cavity. Platelets, fibrinogen, prothrombin time and total cholesterol in peripheral blood were measured before and after treatment. Tissues were stained with haematoxylin and eosion and terminal deoxynucleotidyl-mediated deoxyuridine triphosphate nick-end labelling stain and immunostained for VEGF, while cell proliferation and viability of whole BMCs in the femur were analysed by cell cycle analysis and [ 3 H]-thymidine uptake. Results. The ON incidence in rabbits treated with MPSL alone, MPSL+needling and MPSL+saline was 72.7, 70.0 and 66.7%, respectively, while in the MPSL+BMT group, the incidence was 0%. Serological findings in the MPSL+BMT group were almost normalized. VEGF and TUNEL staining were reduced in the MPSL+BMT group compared with all other groups. There were significantly fewer BMCs in G1 phase from the MPSL+BMT group than the other groups, while uptake of [ 3 H]-thymidine was significantly increased. Conclusion. Direct injection of autologous BMCs into femurs prevents corticosteroid-induced ON following treatment with high-dose, short-term steroids.

Previous research on inventory distributions between local warehouses or retailers (bases) has focused separately on either of two types of stock transshipment policies: preventive lateral transshipments or emergency lateral transshipments. Each of these has its advantages and disadvantages, and combining these policies may well enable merchandisers to achieve higher service levels. Thus, the combined use of these policies is the focus of the present study. A stochastic programming problem is formulated with demand as a stochastic variable, and the policy of using both preventive and emergency lateral transshipment is examined for its effectiveness while solution methods are examined for their efficiency.