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Recently, our lab reported that CUBIC, a tissue-clearing technique, could help reveal the initial route of infection, its spread, and the localization of pathogens in infected goldfish Carassius auratus. ​However, this technique, in its original form, failed to clear melanin pigmented parts of the fish. ​We show here that bleaching with H2O2 clears melanin pigments of Black Moor goldfish without causing severe histological damage. ​Furthermore, nuclear staining with PI helps visualize the internal structures of cleared fish in a dark field. ​Since bleaching does not significantly quench the fluorescence of GFP, it may be applied to the in vivo imaging of GFP.

Introduction Late non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation(allo-HCT) is a problem that is yet to be solved. Moreover, no efficient markers exist to predict late NRM. Vascular endothelial damage is known to be a cause of late NRM after allo-HCT. The transplant endothelial activation and stress index (EASIX) was initially established for the diagnosis of thrombotic microangiopathy. Pre-transplant EASIX (EASIX-pre) quartiles were reported to be predictive markers for early NRM after allo-HCT, but EASIX could also potentially help in the evaluation of late NRM. Since late NRM may be affected by time dependent factors that cannot be evaluated before allo-HCT, the timing of EASIX evaluation may be important. Therefore, we focused on EASIX 1 year after allo-HCT (EASIX-1year). Aims This study aimed to clarify the usefulness of EASIX-pre and EASIX-1year as predictive markers of late NRM and overall survival (OS) after allo-HCT. Methods Among 210 patients with hematological disease who underwent a first allo-HCT between 2006 and 2019at our facility, we evaluated EASIX-1year in 102(53 males and 49 females) patients who were alive after 1 year without relapse and/or withdrawal. EASIX was calculated as LDH level (U/I) × Cre level (mg/dL) / Plt level (nL). EASIX-pre was evaluated 7-10 days before conditioning. EASIX-1year was evaluated within 1 month of 1 year after allo-HCT. Landmark analysis was used to perform statistical analysis of late NRM and OS starting 1 year after allo-HCT. Late NRM and OS assessments using EASIX-pre and EASIX-1year were performed with two risk groups based on the cutoff values from the receiver operating characteristic curve. Forty-four patients had acute myeloid leukemia, 24 had acute lymphoblastic leukemia, 14 had myelodysplastic syndrome, 8 had malignant lymphoma, and 12 had other diseases. The median age of the patients was 40 years (range: 16-66 years). Fifty-seven patients received myeloablative conditioning and the others received reduced intensity conditioning regimens. The number of patients in each HCT-comorbidity index (HCT-CI) risk group was as follows: low risk: 51, intermediate risk: 28, and high risk: 23. This study was performed in accordance with the Japanese Ethical Guidelines for Medical and Health Research Involving Humans and approved by the Ethical Committee of our facility. Results Median EASIX-pre was0.98 (0.12-24.1). The C-statistic of EASIX-pre for late NRM and OS were 0.561 (cutoff value: 0.595) and 0.591 (cutoff value: 0.766), respectively. Univariate analysis revealed that a high EASIX-pre value was not significantly associated with late NRM (5-year NRM 3.4% vs. 0%, p=0.24) and OS (5-year OS 91.3% vs. 93.5%, p=0.22). Moreover, the HCT-CI at pre-transplantation was not an indicator of late NRM (5-year NRM 10.2 % vs. 0%, p=0.29, 5-year OS 79.8% vs. 96.2%, P=0.19). Median EASIX-1year was 0.98 (0.15-21.8). The C-statistic of EASIX-1year for late NRM and OS were 0.63 (cutoff value 2.396) and 0.663 (cutoff value 1.159), respectively. Univariate analysis revealed that a high EASIX-1year value was significantly associated with late NRM (5-year NRM 10.1% vs. 1.3%, p By adjusting age, donor source, HCT-CI, chronic graft versus host disease, and conditioning in multivariate analysis, a high of EASIX-1year was extracted as the risk factor for late NRM (Hazard ratio 4.26, p Conclusion The present study indicated that EASIX-1year may be useful as a prognostic index for late NRM. Otherwise, pre-transplant conditions, such as EASIX-pre and pre-transplant HCT-CI, may have limited effects on late NRM. Disclosures No relevant conflicts of interest to declare.

Introduction Despite advances in the genetic analysis of acute myeloid leukemia (AML), Wilms' tumor oncogene 1 (WT1) mRNA remains an important pan-marker of AML. A log reduction in WT1 mRNA expression after chemotherapy is a predictor of prognosis, and WT1 mRNA can be used as a marker of minimal residual disease or relapse. In the 1990s, a high expression level of WT1 mRNA at diagnosis was considered a poor prognostic factor. However, recent analyses have found that WT1 mRNA expression varies with AML type. Since the prognosis is affected by cytogenetic abnormalities and therapeutic intensity, we re-evaluated the clinical significance of WT1 mRNA expression at diagnosis in the context of the European Leukemia Net (ELN) risk classification and treatment. Method We retrospectively analyzed 216 patients at five institutions between 2011 and 2020. The expression level of WT1 mRNA was measured for all patients at diagnosis using bone marrow (BM) samples from 191 patients and peripheral blood (PB) samples from 25. WT-1 mRNA expression was measured using real-time quantitative polymerase chain reaction and the measured values were converted in normal logarithm. The median age at diagnosis was 62 (range: 23-93) years. The cytogenetic risk of ELN was classified as favorable (n = 41), intermediate (n = 123), and adverse risk (n = 41). Selected therapeutics were standard chemotherapy (n = 182, 84.3%, including CAG regimen; cytarabine, aclarubicin and G-CSF), hypomethylating agents or low-dose cytarabine (n = 19, 8.8%), and best supportive care (n = 15, 6.9%). Also, 143 patients (66.2%) received one or more courses of standard consolidation chemotherapy and 61 (28.3%) underwent allogeneic hematopoietic stem cell transplantation (allo-HCT). The median observation period was 518 [1-3418] days (Table 1). The overall survival (OS) and event-free survival (EFS) rates were assessed. Relapse or death was defined as an event, and OS was evaluated on the date of death. Result The median expression level of WT1 mRNA was 4.68 (range: 1.0-5.72) [log copies/µg RNA, units are omitted thereafter] in BM and 3.66 (range: 1.34-5.20) in PB (Table 1). Favorable-risk AML had the highest WT1 mRNA expression level in BM (4.95, p = 0.0054), whereas there was no difference between the expression levels in intermediate- and adverse-risk AML in BM (4.63 and 4.47, p = 0.711, Fig. 1A). WT1 mRNA expression in PB were 4.04, 3.84, and 3.05 for favorable-, intermediate-, and adverse-risk AML, respectively, and were higher for those with a favorable-risk AML (vs. adverse risk, p = 0.048, Fig. 1B). When WT1 mRNA expression level in BM was compared between the two groups, i.e., Since AML prognosis is affected by ELN risk and selected therapeutics, we evaluated 109 intermediate-risk patients who had received at least one course of standard chemotherapy. Fifty of them (45.9%) underwent subsequent allo-HCT. The 2-year EFS rates of PB or BM-WT1 low (n = 25, PB = 2 and BM = 23) vs. PB or BM-WT1 high (n = 84, PB = 23 and BM = 61) were 23.9% and 50.4%, respectively (p = 0.023, Fig. 2A) and the 2-year OS rates were 51.2% and 64.6%, respectively (p = 0.03, Fig. 2B). PB or BM-WT1 low of intermediate-risk AML had a poor prognosis even with standard chemotherapy. Multivariate analysis adjusted for ages, the reduction rate of WT1 mRNA after induction chemotherapy, duration of receiving consolidation chemotherapy, allo-HCT, and PB or BM-WT1 low at diagnosis were independent poor prognosis factors for EFS in intermediate-risk AML (HR: 3.32, 95%CI: 1.29-8.53, p = 0.013). The OS rate of PB or BM-WT1 low also worsened (HR: 2.33, 95%CI: 0.88-6.18, p = 0.089) (Table 2). Whereas, the outcome of adverse-risk AML was not affected by PB or BM-WT1 low/high. Conclusion The expression level of WT1 mRNA at diagnosis was negatively correlated with prognosis. Favorable-risk AML had higher expressions of WT1 mRNA. PB or BM-WT1 low in intermediate-risk AML was associated with a poor prognosis. In standard-risk AML, WT1 mRNA may be a useful pan-marker to predict prognosis at diagnosis. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

[Introduction] Remarkable advances have been made in cancer immunotherapy, including the development of peptide-based cancer vaccines. Wilms' tumor 1 (WT1) is one of the cancer-testis antigens, and the WT1 gene is overexpressed in hematologic malignancies. Several clinical trials of WT1 peptide vaccines showed promising efficacy and high safety of the vaccines for hematologic malignancies. In these trials, immunological responses were assessed within 2 years after vaccination, and the transient WT1-specific immune response observed in many cases early after vaccination was confirmed. However, the long-term durability of the response of WT1-specific CD8+ cytotoxic T lymphocytes (CTLs) after peptide vaccine therapy and the T-cell receptor (TCR) diversity in those CTLs has not been clarified. More than 10 years ago, a patient with chronic myeloid leukemia (CML) received WT1 peptide vaccination after the failure of tyrosine kinase inhibitor therapy. After vaccination, WT1-specific CD8+ CTLs were observed. We continued the immunological assessment of the patient for more than 10 years after the WT1 peptide vaccination. Herein, we report our findings from the long-term monitoring of WT1-specific CTLs in the patient with CML and describe the results of our detailed analysis, including the functionality and clonality of the CTLs. [Methods] After obtaining written consent from a patient whose CML was difficult to control by imatinib, HLA-A*24:02-restricted modified-type WT1 peptide (WT1 peptide; 9 mer peptide of CYTWNGMNL) was administered to the patient. Post-vaccination, we followed up with the patient. Immune monitoring was performed using a WT1 tetramer assay after mixed lymphocyte peptide culture (MLPC assay). The limiting-dilution (mononuclear cells divided into 20 wells or more, equally containing 3 × 10 5 cells), 2-week cultures with WT1 peptide stimulation and counting of "positive wells" containing expanded WT1 tetramer+ CD8+ T cells were performed for the MLPC assay. The MLPC assay was used to detect functional WT1-specific CD8+ T cells that can expand in response to the WT1 peptide and estimate the frequency of these WT1-specific CD8+ T cells among all CD8+ T cells. For the functionality of WT1-specific CD8+ T cells, we evaluated WT1-specific cytotoxicity and cytokine production in the presence and absence of WT1 peptide pulse to T2A24 cells transfected with the GFP gene (T2A24-GFP). The phenotype and TCR of the WT1-specific CD8+ T cells expanded by MLPC were analyzed using flow cytometry and next-generation sequencing, respectively. [Results] After the WT1 peptide vaccination, the copy numbers of major bcr-abl transcripts gradually decreased, and a therapy free remission was achieved in the patient. No severe adverse effects were observed. The estimated frequency of WT1-specific CD8+ T cells peaked in the third year after vaccination (27 cells per 10 6 CD8+ T cells, 0.00027%) and then declined to 1 - 5 per 10 6 CD8+ T cells at 13 years after vaccination. The WT1-specific CD8+ T cells showed that WT1 peptide-specific cytotoxicity and WT1 peptide-specific IFN-γ release in vitro. These WT1-specific CTLs had different TCRs in each MLPC well. This result was confirmed by three independent analyses, and no common TCRs were detected. Twelve different TCRs were detected in the three analyses. [Conclusion] The WT1 peptide vaccine successfully generated long-lasting and diverse WT1-specific immune responses in a patient with CML. The WT1 peptide vaccine may be a efficient immune therapy for CML patients. Disclosures No relevant conflicts of interest to declare.

Introduction: Severe acute graft versus host disease (GVHD) represents a major risk associated with allogeneic hematopoietic cell transplantation (HSCT). Both acute and progressive GVHD following HSCT are in large part attributable to responses of donor T cells to host allo-antigens. Alloreactive T cell activation can be modulated by signals from the tissue microenvironment (TME). Glycosaminoglycans (GAGs) in the TME or modifying T cell surface proteoglycans are known to regulate T cell function. However, the roles of GAGs in acute GVHD following allogeneic HSCT remain to be elucidated, since a suitable murine model has not been available. We previously established a unique mouse model (T1KO), in which knockout of the chondroitin sulfate (CS) N-acetylgalactosaminyltransferase-1 (T1) gene - encoding the rate-limiting CS-synthesizing enzyme - decreases CS production. We also reported a role for CS in murine hematopoietic stem cells. In the present study, we focus on donor T cell CS levels to assess the role of T cell CS in acute GVHD following allogeneic HSCT. We were able to mitigate the clinical features of GVHD in a murine model using T1KO donors. Methods: Eight- to 12-week-old T1KO mice were generated from a C57BL/6N (WT) strain as donors (H-2b) for allogeneic bone marrow transplantation (BMT). Donor BM cells were then transplanted into eight- to 12-week-old BALB/c recipients (H-2d). Prior to transplantation, recipients were irradiated at a dose of 7 Gy. They then received 5 x 106 BM cells and splenocytes of identical genotype, as well as low (5 x 105), intermediate (1 x 106), or high (4 x 106) doses of CD90.2+ cells from either WT or T1KO donors. For five weeks following BMT, survival was monitored daily and clinical GVHD scores (incorporating body weight, activity, fur condition, alopecia, and tortoiseshell-like mucosae) were calculated three times per week. Results: The peripheral blood CD4+/CD8+ T cell ratio of T1KO mice was equivalent to that of WT mice. Following allogeneic BMT, histopathologic analysis confirmed onset of acute recipient GVHD on day 49 when donors were WT mice. As a result, significant lymphocyte infiltration was observed in target organs (liver, colon, and skin). High-dose transplantation of splenocytes from T1KO rather than WT mice significantly prolonged recipient median survival (27.0 versus 20.5 days, p = 0.02). A similar trend was observed for low- and intermediate-dose transplantation (low: 53.0 versus 29.0 days, p = 0.09; intermediate: 50.5 versus 30.5 days, p = 0.13. Similarly, a significant improvement in day 11 GVHD score was observed following high-dose splenocyte transplantation when donors were T1KO mice (mean scores: 1.00 versus 2.75, p < 0.01). However, no significant differences in GVHD score were observed following low- and intermediate-dose transplantation. Taken together, our results suggest that donor T cells producing lower CS levels alleviate acute GVHD following allogeneic HSCT in a murine model. Conclusion: Alloreactive T cell activation following allogeneic HSCT may be down-regulated by lower CS levels, thereby mitigating GVHD severity. Figure 1 Disclosures No relevant conflicts of interest to declare.

Introduction: Glycosaminoglycans (GAGs), such as heparan sulfate and hyaluronic acid, have been implicated in several hematopoietic processes. GAGs are abundant in the extracellular matrix (ECM) and interact with several cell surface proteins and chemokines. However, the effects of chondroitin sulfate (CS), another species of GAG, in hematopoiesis remain unclear. We examined CS in hematopoiesis by genetically reducing CS in mice by disruption of a gene encoding the rate-limiting CS-synthesizing enzyme N-acetylgalactosaminyltransferase-1 (T1). Methods: T1 knockout (T1KO) mice were generated from the C57BL/6N strain (WT). We evaluated hematopoietic recovery after sublethal irradiation (a 5 Gy dose) to understand the role of CS in hematopoiesis after radiation stress. In addition, we evaluated the effects of each CS on hematopoietic cells and on the stromal microenvironment by creating conditions of CS deficiency in hematopoietic cells or in the stromal microenvironment using hematopoietic stem cell transplantation. In particular, BM cells from WT or T1KO mice were transplanted into 8-10-week-old recipient WT or T1KO mice irradiated at a dose of 9 Gy, and mice were analyzed 5 weeks after transplantation. Furthermore, we examined the role of CS on long-term reconstructive function using a CRU assay in serial transplantation. BM cells from WT or T1KO (CD45.2) mice were transplanted into recipient mice (CD45.1) irradiated at a dose of 9 Gy with BM competitor cells from CD45.1 mice, and PB and BM cell chimerism were analyzed 6 weeks and 12 weeks after transplantation. For serial transplantation, BM cells were collected from recipient mice 12 weeks after transplantation and were transplanted into CD45.1 mice irradiated at a dose of 9 Gy without competitor cells. For evaluating the effect of CS on the stromal microenvironment, BM cells from WT mice were serially transplanted into WT or T1KO recipient mice irradiated at a dose of 9 Gy 12 weeks after transplantation. Results: The amount of CS in BM of T1KO mice was 50-66% of that in WT mice. At steady state, there were no significant differences in the number of PB cells, such as neutrophils, lymphocytes, RBCs and platelets, and total BM cells in T1KO and WT mice. T1KO mice had a significantly higher number of BM LSK cells compared to that of WT mice (WT: 0.213 ± 0.044%; T1KO: 0.282 ± 0.046%, p < 0.01). The corresponding number of CFU-GM of BM cells was also higher in the T1KO mice group (WT: 29.6 ± 3.60; T1KO: 45.4 ± 2.37, p < 0.01). However, hematopoietic recovery (PB cells, total BM cells, and LSK cells) after sublethal irradiation was significantly delayed in T1KO mice. CS deficiency in hematopoietic cells resulted in a lower number of LSK cells compared to that of WT hematopoietic cells after transplantation (WT: 0.176 ± 0.078%; T1KO: 0.131 ± 0.046% p < 0.05). Conversely, no significant difference was observed in mice with CS-reduced stroma. To reveal the effect of CS in hematopoietic cells on long-term reconstructive function, we evaluated the chimerism of PB Gr1+CD11b+cells, B220+ cells, CD3+ cells, and BM LSK cells by a CRU assay. In the first transplantation, there were no significant differences in short-term reconstitution (after 6 weeks) and long-term reconstitution (after 12 weeks). In the second transplantation, hematopoietic cells derived from T1KO mice had lower chimerism in all PB cell lineages. Next, we evaluated the role of CS on the stromal microenvironment by serial transplantation. In the first transplantation, there were no significant differences between PB and BM cells. In the second transplantation, the proportion of BM LSK cells was higher in T1KO recipient mice (CS deficiency in the stroma). Conclusion: CS may have an important role in hematopoiesis. CS in hematopoietic cells and the stromal microenvironment had different effects on BM hematopoiesis. Disclosures No relevant conflicts of interest to declare.

[Background] Chromosome analysis is indispensable for the diagnosis and risk classification of acute myeloid leukemia (AML). A marker chromosome (MC) is a fragmented chromosome that cannot be identified as originating from an existing autosomal or sex chromosome. Although often observed, the significance of MC in hematological malignancies is unknown. Recently, MC was found to be the result of chromothripsis (CT). CT is a catastrophic phenomenon by which chromosomes are shattered into tens to hundreds of fragments. Half of all CT cases are related to TP53 mutation. In addition to MC, complex karyotype (CK), monosomal karyotype (MK) and existing sub-clone (SC) have also been noted as the result of CT. Previous studies reported that MC was a poor prognostic factor in AML. These studies included AML patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT). However, the influence of MC on AML after allo-HCT was not clarified. [Purpose] In this study, we evaluated the impact of MC on the outcome of AML patients after allo-HCT. [Method] This retrospective analysis included 166 AML patients who received allo-HCT at Niigata University Hospital (n=129) or Nagaoka Red Cross Hospital (n=37) between 1990 and 2017. The median age of patients at allo-HCT was 38 years old (range 14-67 y). The median follow-up period was 2.0 y (range 0.0-22.2 y). According to the revised Medical Research Council (rMRC) criteria for cytogenetic risk categories, 26 (15.7%), 104 (62.7%) and 36 (21.7%) patients were categorized as favorable, intermediate and adverse-risk, respectively. Myeloablative conditioning was used for 128 (77.1%) and reduced-intensity was used for 38 (22.9%) patients. Donors were related for 83 (59.3%) and unrelated for 57 (40.7%) patients. The Kaplan-Meier method (log-rank test) and Gray's test were used to estimate the probabilities of overall survival (OS) and cumulated incidence of relapse (CIR). The impact of several variables was assessed by multivariate analysis using a Cox regression model and Fine-Gray test with backward stepwise selection based on the p-value. For the comparison of clinical phenotypes, category variables were evaluated by Fisher's exact test. [Results] MC was detected in 14 (8.4%) of 166 patients. Eleven (78.6%) were grouped as adverse-risk by rMRC criteria. CK, MK and SC were observed in 26 (16.3%), 20 (12.0%) and 23 (13.9%) patients, respectively. The median age of AML/MC+ (n=14) and AML/MC-(n=152) patients were similar (38.5 y, range 19-64 y vs 38 y, range 14-64 y, P=0.812). Twelve AML/MC+ patients (85.7%) received allo-HCT at the advanced stage (³3 CR or non-remission) and 10 (71.4%) had primary induction failure (PIF). Compared with AML/MC- patients, those with AML/MC+ had a higher incidence of MK (78.6% vs. 5.9%, p [Conclusion] It is very difficult for AML/MC+ patients to achieve complete remission, leading to a markedly poor prognosis after allo-HCT. The outcome for AML/MC+ was inferior to that for AML with CK, MK, SC or the current adverse-risk karyotype by rMRC. This analysis revealed MC as a new independent factor that further indicates a poor prognosis after allo-HCT, especially in high-risk AML patients. Disclosures No relevant conflicts of interest to declare.

[Introduction] Bloodstream infection (BSI) is a serious complication of HCT that may be life-threatening. BSI frequently occurs before neutrophil engraftment (pre-engraftment BSI), but has also been reported after neutrophil recovery (post-engraftment BSI). In contrast to pre-engraftment BSI, the clinical features and risk factors of post-engraftment BSI remain unclear. [Aims] We investigated the clinical characteristics of and risk factors for post-engraftment BSI. [Methods] This retrospective study included 176 adult patients who underwent HCT and achieved neutrophil engraftment between 2006 and 2017 at our institute. Diagnoses consisted of AML (n=86), ALL (n=36), MDS (n=21), MPN (n=4), CML (n=1), ATL (n=6), aplastic anemia (n=6), and malignant lymphoma (n= 16). The median age at HCT was 42 y (range 16-67 y). Graft sources were BM (n=69), PB (n=57), and CB (n=50). Fifty-five patients (31.2%) had a high (≥3) HCT-CI score on HCT. Sixty-four patients (36.4%) received HCT with an advanced disease status. Fluoroquinolone (FQ) as prophylaxis was administered to 89 patients (50.6%). Central venous catheters (CV) were inserted in all patients before HCT. All patients consulted a dentist before HCT and received guidance on appropriate oral self-care to prevent severe oral mucositis; 92 (52.3%) continuously received intensive oral care after HCT (I-care; visit to a dentist at least once a week until neutrophil engraftment for the assessment of oral mucositis and cleaning), whereas 84 (47.7%) only performed oral self-care (S-care; according to guidance by a dentist). In the present study, BSI was defined as an infectious state with fever (≥38°C) and the isolation of a pathogen on at least 1 blood culture or on 2 or more if a common skin contaminant was isolated. Post-engraftment BSI was evaluated until day 180. [Results] Seventy-five events of BSI (in 69 patients) occurred until day 180. The total cumulative incidence of BSI (CIB) was 39.2%. The CIB of pre- and post-engraftment BSI were similar at 21.6% (n=38) and 21.0% (n=37) (p=1.0), respectively. Six patients developed pre- and post-engraftment BSI. CV was inserted in all patients when BSI occurred. Twenty-five pathogens were isolated in the present study. Regarding the type of pathogen, Gram-positive cocci were the most common in pre-/post-engraftment BSI (63.2%/69.0%). Gram-negative (29.0%/14.3%) and -positive rods (15.8%/19.0%) were detected. Staphylococcus epidermidis was the most frequently detected species in pre/post-engraftment BSI (31.6%/57.1%). Similar to CIB, no significant difference was observed in the pathogens identified between pre-/post-engraftment BSI (p=0.34). We performed further analyses to identify risk factors for post-engraftment BSI. In Fisher's exact test as a univariate analysis, HCT-CI≥3 (p=0.045), TBI≥3 Gy (p=0.041), not administered FQ (p=0.042), no I-care (p=0.003), and a graft source of BM (p=0.002) were identified as risk factors for post-engraftment BSI. Age (p=0.25), conditioning (p=0.197), repeated HCT (0.607), disease stage prior to HCT (p=0.701), and a history of pre-engraftment BSI (p=0.501) did not contribute to post-engraftment BSI. A logistic regression test with backward stepwise selection based on p-values as the multivariate analysis revealed that I-care (OR 0.358, 95%CI: 0.16-0.801, p=0.0124) and a graft source of PB (OR 0.322, 95%CI: 0.124-0.837, p=0.02) reduced the risk of post-engraftment BSI. Furthermore, to confirm the impact of the oral care type on post-engraftment BSI, we compared the CIB of the S- and I-care groups. The CIB of pre-engraftment BSI was similar between the I- and S-care groups (21.4% vs 21.7%, p=1), whereas that of post-engraftment BSI was significantly lower in the I-care group (12.0% vs 29.8%, p [Conclusion] CIB and isolated pathogens were similar between pre- and post-engraftment BSI, even if neutrophils had recovered sufficiently. Since BSI may occur at any time during transplantation, careful follow-ups are needed. Intensive oral care by a dental specialist may reduce the risk of post-engraftment BSI. Disclosures No relevant conflicts of interest to declare.

In the course of our search for new melanin synthesis inhibitors from plants, 40 new flavonoids and 11 known flavonoids were isolated from the roots of Lespedeza floribunda Bunge. The structures of the new compounds were determined by MS and NMR analyses, and the absolute configurations by CD spectra. Many of the compounds inhibited melanin synthesis in normal human epidermal melanocytes (NHEM), and compounds 3, 7, 8, 11, 16, 24, 27, 29, 33, 43, 45, and 51 were particularly inhibitory. Their activities were stronger than that of hydroquinone, which is known as a major skin-lightening drug.

[Introduction] The launch of the oral iron chelator "Deferasirox" has improved the outcomes of blood transfusion-dependent patients with iron overload in the last decade. Although serum ferritin (SF) remains the mostly commonly used metric to monitor body iron stores for decisions regarding the indication of iron chelate therapy, it is known to be affected by many factors. The liver iron concentration (LIC) is considered to be an indicator of total body iron stores, and the MR imaging-based R2 technique is the standard non-invasive technique used to evaluate LIC. However, this technique is not used in every institution due to some limitations such as its high cost and the requirement for special software. Although the application of CT, which is easy to use and inexpensive, needs to be considered for the evaluation of LIC, the use of conventional single energy CT (SECT) to measure LIC is also limited by normal variations in CT attenuation, predominantly in patients with mild iron overload. Moreover, SECT fails to detect iron in fatty livers, which has an inverse effect on attenuation by lowering CT numbers. Dual-energy CT (DECT) is a technique that is employed to obtain precise information on tissue composition and may be useful for monitoring LIC. It is based on substances showing different densities with two different energies, with each substance displaying its own energy-dependent change in CT attenuation. The role of DECT in monitoring LIC has not yet been clarified in blood transfusion-dependent patients with iron overload. We herein evaluated iron deposition in the livers of blood transfusion-dependent patients using DECT. [Patients and Methods] Seventeen blood transfusion-dependent patients underwent liver DECT using a dual-source 128-slice CT system, and SF levels were measured at same time. DECT images were acquired using a tube voltage pair of 140 kV and 80 kV or 140 kV and 100 kV, and the three-material decomposition of fat, soft tissue, and iron. [Results] The median age of patients was 52 years (range, 25 to 66), and 8 patients were male. Eight patients with AML, 3 with MDS, and 1 each with ALL, lymphoma, aplastic anemia, Evans syndrome, congenital dyserythropoietic anemia, and chronic renal failure underwent DECT. Nine patients had undergone stem cell transplantation before DECT, and 3 were receiving iron chelate therapy. The total number of units of blood transfused was available in 11 out of 17 patients. The median number of units given was 66 (range, 36 to 150). The median SF level was 2346 ng/ml (range, 569 to 7875). We divided patients into three groups based on SF levels: high >3000 ng/ml, intermediate 1000~3000 ng/ml, low [Conclusion] Discrepancies between SF levels and DECT images indicate that DECT is a useful technique for the accurate evaluation of LIC, and the detection of focal iron deposition in the liver may be useful for optimizing iron chelation therapy. Disclosures No relevant conflicts of interest to declare.

Background: For patients with chemotherapy refractory acute leukemia (refractory AL), allogeneic hematopoietic stem cell transplantation (allo HSCT) is the only treatment that has curative potential. However, the long term overall survival (OS) of patients with refractory AL who received allo HSCT was reported to be less than 25% from CIBMTR. In contrast, the OS of patients who received allo HSCT on complete remission (CR) was reported to be approximately 60%. Although some refractory AL patients can achieve CR and survive for a long time, their characteristics remain to be elucidated. These are required in order for efficient use of transplant resources to find the subpopulation of refractory AL patients who can receive larger benefits from allo HSCT. Objectives: The purpose of this study was to find the predictive factors of favorable prognosis among patients with refractory AL prior to allo HSCT. Patients and Methods: Fifty refractory AL patients (AML n=40, ALL n=10), who underwent allo HSCT between January 2000 and January 2016 at Niigata University Hospital and Nagaoka Red Cross Hospital, were analyzed retrospectively. The median age of the patients at the time of allo HSCT was 38 years (range: 18-64), and the median follow up period was 8.3 months (range: 0.5-144.6). All patients were evaluated with bone marrow (BM) aspiration within one month prior to allo-HSCT. Thirty-two patients received myeloablative conditioning and 18 patients received reduced-intensity conditioning. Donor sources were siblings (n=12), unrelated (n=16), haploidentical (n=12) or cord blood (n=10). According to the NCCN guidelines of cytogenetic risk status, AML patients were classified as low risk (n=2), intermediate risk (n=22) or high risk (n=16). ALL patients were classified as standard risk (n=7), high risk (n=2) or unknown (n=1). Relapse free survival (RFS) and OS were estimated by the Kaplan-Meier method. Multivariate Cox regression was used to identify the independent prognostic factors. Results: The median blast percentage in BM before allo HSCT was 18.0% (range: 0.8-93.6%). Non-relapse mortality was 18.0%. The 1y-RFS and OS were 32.2% and 45.8%. The 5y-RFS and OS were 25.3% and 25.7%, respectively. First, to predict relapse based on the optimal threshold value of blast percentage in BM, we calculated receiver operating characteristics (ROC) analysis and the largest areas under the curve (AUC). ROC analysis for blast percentage in BM and relapse revealed that ≤32% was the optimal threshold value (AUC 0.677) to predict relapse. Univariate analysis revealed that patients with HCT-CI ≦2 (p To investigate the correlation between multiple factors and the outcome, we scored the patients according to these three favorable factors (AML, HCT-CI≦2 and BM blast ≤32%), and stratified them into 3 groups as follows; score=3 (n=23), score=2 (n=20) and score=0-1 (n=7). The patients with score=3 exhibited better 1y-RFS compared with the other groups (p On multivariate analysis adjusted by age, cytogenetic risk, primary refractory or not, donor source and conditioning regimen, score=3 was an independent favorable prognosis factor for RFS (HR 0.17 (p Conclusions: We found three favorable prognostic factors (AML, HCT-CI≦2 and BM blast ≤32%) in this study. Refractory AL patients with all three factors may receive larger benefits from allo-HSCT and the 5y-OS may be comparable to allo HSCT with CR. Disclosures No relevant conflicts of interest to declare.

After allogeneic hematopoietic stem cell transplantation (HSCT), immune recovery is important to protect the patient from relapse and co-morbidities such as graft-versus-host disease (GVHD) and infection. Various numbers of low-frequency immunocompetent cells are known to exist among T cells and each subset shows different immunological action. Among them, γδ T cells were reported to facilitate a graft-versus-leukemia (GVL) effect and regulatory T cells (Tregs) were reported to prevent acute GVHD. In this study, we focused on the clinical relevance of γδ T cells and Tregs in peripheral blood (PB) after allogeneic HSCT in patients with hematological neoplasm to outcome. We retrospectively analyzed 33 adult patients with hematological neoplasms who underwent allogeneic HSCT between July 2011 and February 2015 at Niigata University Medical and Dental Hospital, including 17 with acute myeloid leukemia, 8 with acute lymphoblastic leukemia, 4 with myelodysplastic syndromes, 2 with Epstein-Barr virus-associated lymphoproliferative disorder, 1 with adult T-cell leukemia/lymphoma and 1 with primary myelofibrosis. Circulating γδ T cells and Tregs were analyzed by flow cytometry within 30-100 days after allogeneic HSCT. γδ T cells were identified as CD3+/γδTCR+ cells. Tregs were identified as CD4+/Foxp3+/CD25+ cells. The percentage of γδ T cells was calculated by dividing by CD3+ cells. The percentage of Tregs was calculated by dividing by CD4+ cells. The Kaplan-Meier method was used to estimate the probability of disease-free survival (DFS). The Mann-Whitney U test was used to compare the percentage of γδ T cells in PB or Tregs in PB and any grade of acute GVHD or grade II-IV acute GVHD. Cumulative relapse rate and non-relapse mortality (NRM) were based on Gray's estimates. Fine-Gray proportional hazards models were used for assessment by multivariate analysis of relapse rate. Factor adjustment was performed for age, conditioning regimen, disease status and HLA compatibility. The median percentage of γδ T cells divided by CD3+ cells in PB was 3.3% (0-28.4%). The median percentage of Tregs divided by CD4+ cells in PB was 1.9% (0-17.3%). The percentage of γδ T cells in PB was not associated with the incidence of acute GVHD. In addition, the percentage of Tregs in PB was not associated with the incidence of acute GVHD. Next, we established an immune scoring system according to the percentage of γδ T cells and Tregs in PB. Less than 4% of γδ T cells as a proportion of CD3+ cells in PB was scored as 1 point and more than 4% of Tregs as a proportion of CD4+ cells in PB was scored as 1 point. The patients with 1 point for γδ T cells did not show a significant difference to the patients with 0 points in terms of cumulative relapse rate or NRM. In addition, the patients with 1 point for Tregs did not show a significant difference to the patients with 0 points in terms of cumulative relapse rate or NRM. We classified the patients into score 0-1 and score 2 upon adding the points. Patients with score 2 showed a higher relapse rate in univariate analysis (p=0.002) and multivariate analysis (hazard ratio 3.65, p=0.017) than patients with score 0-1. Moreover, patients with score 2 showed higher DFS in univariate analysis (p=0.001) and multivariate analysis (hazard ratio 3.50, p=0.027) than patients with score 0-1. Our study suggests that the combination of a low rate of γδ T cells and a high rate of Tregs in PB after allogeneic HSCT is a poor prognostic factor for patients with hematological neoplasm. In addition, it suggests that the balance of immunosuppression and immunoactivation may be important for the outcome of patients after allogeneic HSCT. Disclosures No relevant conflicts of interest to declare.

The toxicity of melamine and its analogue in man and animals has been reported widely. The aim of the present study was to examine the pathological effects of feeding melamine and cyanuric acid, separately or in combination, to walking catfish (Clarius batrachus). The catfish developed darkening of the skin as early as 3 days post feeding. Melamine-related crystals were distributed multifocally throughout the liver, kidney, heart, spleen and corpuscle of Stannius of fish fed melamine and cyanuric acid in combination. Oil red O staining and electron microscopy revealed that the melamine-related crystals had structure resembling that of plastic polymer crystals. Elevations in the serum concentrations of alanine transaminase, aspartate transaminase, creatinine and uric acid were related to the crystal-associated granulomatous inflammation in the liver and kidney of affected fish. None of the catfish died during the 2-week experiment. Melamine and cyanuric acid are therefore systemically toxic to fish in addition to causing renal crystal formation and renal damage as seen in man and animals. The finding of extrarenal crystals implies that the metabolism and biotransformation of these toxic compounds should be further investigated in aquatic animals.

In ultraviolet-induced tanning, the protein levels of various gene products critical for pigmentation (including tyrosinase and tyrosinase-related protein-1) are increased in response to ultraviolet B irradiation, but changes in mRNA levels of these factors have not been investigated in vivo . We have established an in situ hybridization technique to investigate mRNA levels of pro-opiomelanocortin, tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, P-protein, Pmel-17/gp100, and microphthalmia-associated transcription factor, and have analyzed the changes in mRNA levels in the ultraviolet B-exposed skin in vivo . The right or left forearm of each volunteer was irradiated with ultraviolet B, and skin biopsies were obtained at 2 and 5 d postirradiation. mRNA level of pro- opiomelanocortin was increased 2 d after ultraviolet B irradiation, and returned to a near-basal level after 5 d, whereas the mRNA levels of tyrosinase, tyrosinase-related protein-1, dopachrome tautomerase, P-protein, and Pmel-17/gp100 showed some or no increase at 2 d, but were significantly increased 5 d after ultraviolet B irradiation. Microphthalmia-associated transcription factor mRNA was slightly increased on days 2 and 5 after ultraviolet B irradiation. Our results suggest that the mechanism of the tanning response of human skin may involve the transcriptional regulation of certain pigmentary genes, and that pro-opiomelanocortin-derived melanocortins such as α-melanocyte-stimulating hormone and adrenocorticotropic hormone may play a part in regulating these genes in vivo .

Initial step of elemental sulfur (S0) oxidation by a purified sulfur:ferric ion oxidoreductase from Thiobacillus ferrooxidans was investigated. When S0 and reduced glutathione (GSH), whichwas absolutely required for S0 oxidation by sulfur:ferric ion oxidoreductase, were incubated in a buffer solution (pH 6.5), hydrogen sulfide (H 2S) and GSSG were chemically produced at the rate of 0.021 and 0.082 μmol/ml per h, respectively. If sulfur:ferric ion oxidoreductase was addedto the incubation mixture, H2S production immediately stopped and sulfite production opened, suggesting that H2S is an actual substrate of sulfu:ferric ion oxidoreductase. Amongthe reduced sulfur compounds tested, S0, H2S and FeS were utilized as an electron donorof sulfur:ferric ion oxidoreductase and a mechanism of initial steps of S0 oxidation was proposed. It was also found that when S0 was oxidized by sulfur:ferric ion oxidoreductase in the presence of GSH, contact of sulfur:ferric ion oxidoreductase with solid element sulfur was unnecessary.

In the presence of phosphate ions, molybdic ions (Mo6+) were reduced enzymatically with elemental sulfur by washed intact cells of Thiobacillus ferrooxidans to give molybdenum blue. The whole-cell activity that reduced Mo6+ was totally due to cellular sulfur:ferric ion oxidoreductase (SFORase) (T. Sugio, W. Mizunashi, K. Inagaki, and T. Tano, J. Bacteriol. 169:4916-4922, 1987). The activity of M06+ reduction with elemental sulfur was competitively inhibited by Fe3+, Cu2+, and Co2+. The Michaelis constant of SFORase for Mo6+ was 7.6 mM, and the inhibition constants for Fe3+, Cu2+, and Co2+ were 0.084, 0.015, and 0.17 mM, respectively, suggesting that SFORase can reduce not only Fe3+ and Mo6+ but also Cu2+ and Co2+ with elemental sulfur.