A higher CD34 + cell dose correlates with better event-free survival after KIR-ligand mismatched cord blood transplantation for childhood acute myeloid leukemia.

Although killer Ig-like receptor ligands (KIR-L) mismatch has been associated with alloreactive natural killer cell activity and potent graft-versus-leukemia (GVL) effect among adults with acute myeloid leukemia (AML), its role among children with AML receiving cord blood transplantation (CBT) has not been determined. We conducted a retrospective study using a nationwide registry of the Japanese Society for Transplantation and Cellular Therapy. Patients who were diagnosed with de novo non-M3 AML and who underwent their first CBT in remission between 2000 and 2021 at under 16 years old were included. A total of 299 patients were included; 238 patients were in the KIR-L match group, and 61 patients were in the KIR-L mismatch group. The cumulative incidence rates of neutrophil recovery, platelet engraftment, and acute/chronic graft-versus-host disease did not differ significantly between the groups. The 5-year event-free survival (EFS) rate was 69.8% in the KIR-L match group and 74.0% in the KIR-L mismatch group (p = 0.490). Stratification by CD34 + cell dose into four groups revealed a significant correlation between CD34 + cell dose and EFS in the KIR-L mismatch group (p = 0.006) but not in the KIR-L match group (p = 0.325). According to our multivariate analysis, KIR-L mismatch with a high CD34 + cell dose (≥ median dose) was identified as an independent favorable prognostic factor for EFS (hazard ratio = 0.19, p = 0.029) and for the cumulative incidence of relapse (hazard ratio = 0.09, p = 0.021). Our results suggested that higher CD34 + cell doses are crucial for achieving a potent GVL effect in the context of KIR-L-mismatched CBT.

Retinoid Therapy for Neuroblastoma: Historical Overview, Regulatory Challenges, and Prospects.

Retinoids are vitamin A derivatives and include trans-retinoic acid, isotretinoin, tamibarotene, and bexarotene, all of which are currently available for clinical use. The clinical development of retinoid therapy for neuroblastoma has a history spanning more than four decades. The most promising agent is isotretinoin, which can contribute to improving event-free survival in patients with high-risk neuroblastoma by approximately 10% when administered over six months as maintenance therapy. Although isotretinoin is regarded as an essential component in the standard clinical management of high-risk neuroblastoma, its use for this purpose in the US and EU is off-label. To promote isotretinoin use in Japan as a treatment for neuroblastoma, our clinical research team is planning to launch an investigator-initiated, registration-directed clinical trial. The present review article discusses the basic science behind retinoid therapy, pre-clinical/clinical evidence on neuroblastoma, the concept of the proposed clinical trial, and prospects for this therapy.

Synthesis and Identification of decarbamoyloxySaxitoxins in Toxic Microalgae and their Reactions with the Oxygenase, SxtT, Reveal Saxitoxin Biosynthesis.

Saxitoxin (STX, 1) is a representative compound of paralytic shellfish toxins (PSTs) that are produced by marine dinoflagellates and freshwater cyanobacteria. Although several pathways have been proposed for the biosynthesis of STX, the order of ring and side chain hydroxylation, and formation of the tricyclic skeleton have not been well established. In this study, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX, 2), the most reduced STX analogue having the tricyclic skeleton, and its analogues, 12ß-deoxy-doSTX (12ß-d-doSTX, 3), 12α-deoxy-doSTX (12α-d-doSTX, 4), and doSTX (5), were synthesized, and these compounds were screened in the toxic microalgae using high-resolution LCMSMS. dd-doSTX (2) and 12ß-d-doSTX (3) were identified in the PSTs-producing dinoflagellates (Alexandrium catenella, A. pacificum, and/or Gymnodinium catenatum) and in the cyanobacterium Dolichospermum circinale (TA04). doSTX (5), previously isolated from the dinoflagellate G. catenatum, was also identified in D. circinale (TA04). Furthermore, the conversion of 2 to 3, and 4 to 5, by SxtT with VanB, a reported Rieske oxygenase and its redox partner in STX biosynthesis, was confirmed. These results support that 2 is a possible biosynthetic precursor of STX, and that ring and side-chain hydroxylations proceed after cyclization.

Prophylactic bilateral nephrectomy and preemptive kidney transplantation for Denys-Drash syndrome prior to development of kidney failure.

BACKGROUND  : Nephropathy in Denys-Drash syndrome (DDS) develops within a few months of birth, often progressing to kidney failure. Wilms tumors also develop at an early age with a high rate of incidence. When a patient does not have Wilms tumor but develops kidney failure, prophylactic bilateral nephrectomy, and kidney transplantation (KTX) is an optimal approach owing to the high risk of Wilms tumor development. In the case presented here, prophylactic bilateral nephrectomy and KTX were performed in a patient who had not developed Wilms tumor or kidney failure. However, the treatment option is controversial as it involves the removal of a tumor-free kidney and performing KTX in the absence of kidney failure. CASE DIAGNOSIS/TREATMENT: We present the case of a 7-year-old boy, born at 38 weeks gestation. Examinations at the age of 1 year revealed severe proteinuria and abnormal internal and external genitalia. Genetic testing identified a missense mutation in exon 9 of the WT1 gene, leading to the diagnosis of DDS. At the age of 6 years, he had not yet developed Wilms tumor and had grown to a size that allowed him to safely undergo a KTX. His kidney function was slowly deteriorating (chronic kidney disease (CKD) stage 3), but he had not yet developed kidney failure. Two treatment options were considered for this patient: observation until the development of kidney failure or prophylactic bilateral nephrectomy with KTX to avoid Wilms tumor development. After a detailed explanation of options to the patient and family, they decided to proceed with prophylactic bilateral nephrectomy and KTX. At the latest follow-up 4 months after KTX, the patient's kidney functioned well without proteinuria. CONCLUSION: We performed prophylactic bilateral nephrectomy with KTX on a DDS patient who had not developed kidney failure or Wilms tumor by the age of 7 years. Although the risk of development of Wilms tumor in such a patient is unclear, this treatment may be an optimal approach for patients who are physically able to undergo KTX, considering the potentially lethal nature of Wilms tumor in CKD patients.

A Japanese retrospective study of non-tuberculous mycobacterial infection in children, adolescents, and young adult patients with hematologic-oncologic diseases.

Non-tuberculous mycobacterial infection (NTM) is rare in healthy children, with lymphadenitis being the most common presentation. Immunocompromised populations are known to be at high risk, but the clinical picture of NTM infection in pediatric hematology/oncology patients is unclear. In this nationwide retrospective analysis of patients under the age of 40 treated in Japanese pediatric hematology/oncology departments who developed NTM infection between January 2010 and December 2020, 36 patients (21 patients with hematopoietic stem cell transplantation (HSCT) and 15 nontransplant patients) were identified. Post-transplant patients were infected with NTM at 24 sites, including the lungs (n = 12), skin and soft tissues (n = 6), bloodstream (n = 4), and others (n = 2). Nine of twelve patients with pulmonary NTM infection had a history of pulmonary graft-versus-host disease (GVHD), and rapid-growing mycobacteria (RGM) were isolated from five of them. In nontransplant patients, the primary diseases were acute lymphoblastic leukemia (ALL; n = 5), inborn errors of immunity (IEI; n = 6), and others (n = 4). All cases of ALL had bloodstream infections with RGM, whereas all cases of IEI were infected with slow-growing mycobacteria (SGM). In summary, three typical clinical scenarios for pediatric hematology/oncology patients have been established: RGM-induced pulmonary disease in patients with pulmonary GVHD, RGM bloodstream infection in patients with ALL, and SGM infection in patients with IEI. Our findings suggest that NTM must be regarded as a pathogen for infections in these high-risk patients, especially those with pulmonary GVHD, who may require active screening for NTM.

Investigation of Fertility Preservation Education Videos for Pediatric Patients Based on International and Historical Survey.

Purpose: Recently, direct communication with children about cancer seems to have shifted, but little is known about communication regarding discussions of future infertility risk due to cancer therapy. This study conducted cross-cultural comparisons between Japan and the United States to clarify communication patterns about cancer notification and develop appropriate information about fertility issues. Methods: An online survey was distributed to members of the Japanese Society of Pediatric Hematology/Oncology in July 2019 and the American Society of Pediatric Hematology/Oncology in July 2020. Based on the results from the survey, we developed three types of educational videos: a prepubertal version A, B, and a pubertal version. Next, we conducted a survey to assess whether these were appropriate for clinical practice. Results: We analyzed 325 physicians in Japan and 46 in the United States. In Japan, 80.5%, 91.7%, and 92.1% of the physicians notified patients aged 7-9, 10-14, and 15-17 years of their cancer diagnosis directly, respectively, compared within the United States, where the rate was 100%, regardless of age. Further, 9% and 45% of physicians in Japan and the United States, respectively, discuss fertility issues directly with patients aged 7-9 years. In the survey to assess the educational videos, 85% of the physicians preferred to use the educational videos in clinical practice. Conclusion: This is the first step in bringing concordance to communication patters for emerging cancer care around the globe and that this study and its intervention arm provide guidance in ways that ensure global equity in care.

The quite low cross-reactivity of Kawatsu's anti-tetrodotoxin monoclonal antibody to 5,6,11-trideoxytetrodotoxin, 11-nortetrodotoxin-6(S)-ol, and 11-oxotetrodotoxin, the major tetrodotoxin analogues in pufferfish.

The monoclonal antibody against tetrodotoxin (TTX), prepared by Kawatsu et al. (1997), has been used in several TTX-related studies. Herein, we confirmed the quite low cross-reactivity of this antibody to three major TTX analogues in pufferfish using competitive ELISA: 5,6,11-trideoxyTTX (<2.2%), 11-norTTX-6(S)-ol (<0.3%), and 11-oxoTTX (<1.5%), with reactivity against TTX being 100%. We further confirmed that the presence of these analogues did not cause a marked overestimation of TTX in pufferfish extracts using competitive ELISA.

Metabolic inhibitor induces dynamic changes in saxitoxin biosynthesis and metabolism in the dinoflagellate Alexandrium pacificum (Group IV) under in vivo labeling condition.

In paralytic shellfish toxin-producing dinoflagellates, intracellular levels of saxitoxin and its analogues (STXs) are controlled by a balance between degradation and biosynthesis in response to marine environmental fluctuations and stresses. The purpose of this study was to demonstrate the utility of statistical analysis of in vivo labeling data for the dynamic analysis of variations in toxin production under stress. A toxic strain of the dinoflagellate Alexandrium pacificum (Group IV) was cultured in colchicine-containing 15N-labeled sodium nitrate-medium and metabolite levels were analyzed over time by liquid chromatography-mass spectrometry. Quantitative values of all isotopomers of precursor amino acids, biosynthetic intermediates, and major STXs were subjected to statistical analysis. The decrease of the nitrogen incorporation rates for all compounds suggested that colchicine decreased nitrate assimilation upstream of glutamate biosynthesis. In colchicine-treated cultures, the per-cell content of total STX analogues did not change significantly over time; however, the production rate of each pathway varied greatly. De novo STX biosynthesis was decreased by colchicine until Day 3, while the salvage pathway was not. Subsequently, biosynthesis by both pathways was enhanced. This analysis of dynamic metabolism provides new insights into the complex mechanisms regulating STX metabolism in dinoflagellates.

State-Dependent Inhibition of Voltage-Gated Sodium Channels in Neuroblastoma Neuro-2A Cells by Arachidonic Acid from Halichondria okadai.

Voltage-gated sodium channels (Nav) are closely associated with epilepsy, cardiac and skeletal muscle diseases, and neuropathic pain. Several toxic compounds have been isolated from the marine sponge Halichondria okadai; however, toxic substances that modulate Nav are yet to be identified. This study aimed to identify Nav inhibitors from two snake venoms and H. okadai using mouse neuroblastoma Neuro-2A cells (N2A), which primarily express the specific Nav subtype Nav1.7, using whole-cell patch-clamp recordings. We successfully isolated arachidonic acid (AA, 1) from the hexane extract of H. okadai, and then the fatty acid-mediated modulation of Nav in N2A was investigated in detail for the first time. Octanoic acid (2), palmitic acid (3), and oleic acid (4) showed no inhibitory activity at 100 µM, whereas AA (1), dihomo-γ-linolenic acid (DGLA, 5), and eicosapentaenoic acid (EPA, 6) showed IC50 values of 6.1 ± 2.0, 58 ± 19, and 25 ± 4.0 µM, respectively (N = 4, mean ± SEM). Structure and activity relationships were investigated for the first time using two ω-3 polyunsaturated fatty acids (PUFAs), EPA (6) and eicosatetraenoic acid (ETA, 7), and two ω-6 PUFAs, AA (1) and DGLA (5), to determine their effects on a resting state, activated state, and inactivated state. Steady-state analysis showed that the half inactivation potential was largely hyperpolarized by 10 µM AA (1), while 50 µM DGLA (5), 50 µM EPA (6), and 10 µM ETA (7) led to a slight change. The percentages of the resting state block were 24 ± 1, 22 ± 1, 34 ± 4, and 38 ± 9% in the presence of AA (1), DGLA (5), EPA (6), and ETA (7), respectively, with EPA (6) and ETA (7) exhibiting a greater inhibition than both AA (1) and DGLA (5), and their inhibitions did not increase in the following depolarization pulses. None of the compounds exhibited the use-dependent block. The half recovery times from the inactivated state for the control, AA (1), DGLA (5), EPA (6), and ETA (7) were 7.67 ± 0.33, 34.3 ± 1.10, 15.5 ± 1.10, 10.7 ± 0.31, and 3.59 ± 0.18 ms, respectively, with AA (1) exhibiting a distinctively large effect. Overall, distributed binding to the resting and the inactivated states of Nav would be significant for the inhibition of Nav, which presumably depends on the active structure of each PUFA.

Isolation and Biological Activity of 9-epiTetrodotoxin and Isolation of Tb-242B, Possible Biosynthetic Shunt Products of Tetrodotoxin from Pufferfish.

Tetrodotoxin (TTX, 1) is a potent voltage-gated sodium channel blocker detected in certain marine and terrestrial organisms. We report here a new TTX analogue, 9-epiTTX (2), and a TTX-related compound, Tb-242B (4), isolated from the pufferfish Takifugu flavipterus and Dichotomyctere ocellatus, respectively. NMR analysis suggested that 2 exists as a mixture of hemilactal and 10,8-lactone forms, whereas other reported TTX analogues are commonly present as an equilibrium mixture of hemilactal and 10,7-lactone forms. Compound 2 and TTX were confirmed not to convert to each other by incubation under neutral and acidic conditions at 37 °C for 24 h. Compound 4 was identified as the 9-epimer of Tb-242A (3), previously reported as a possible biosynthetic precursor of TTX. Compound 4 was partially converted to 3 by incubation in a neutral buffer at 37 °C for 7 days, whereas 3 was not converted to 4 under this condition. Compound 2 was detected in several TTX-containing marine animals and a newt. Mice injected with 600 ng of 2 by intraperitoneal injection did not show any adverse symptoms, suggesting that the C-9 configuration in TTX is critical for its biological activity. Based on the structures, 2 and 4 were predicted to be shunt products for TTX biosynthesis.

Alterations of choroidal circulation and vascular morphology in a patient with chronic myeloid leukemia before and after chemotherapy.

BACKGROUND: Chronic myeloid leukemia (CML) is known to cause leukemic retinopathy due to leukemia cell invasion into the choroid; however, details of the circulatory dynamics and morphological changes in the choroid are unknown. The aim of this study was to present a case of leukemic retinopathy and examine choroidal circulatory and structural analyses using laser speckle flowgraphy (LSFG) and optical coherence tomography with a binarization method, respectively. CASE PRESENTATION: A 15-year-old male diagnosed with CML complained of blurred vision in his right eye. He was ophthalmologically diagnosed with leukemic retinopathy due to retinal hemorrhage in both eyes. Tyrosine kinase inhibitors achieved complete cytogenetic remission and resolution of retinal hemorrhages at 6 months after treatment. After the treatment, the best-corrected visual acuity had recovered from 0.1 to 1.2 oculus dexter (OD) and remained at 1.5 oculus sinister (OS). The rate of change in macular blood flow assessed by the mean blur rate on LSFG was 18.3% increase OD and 25.2% decrease OS 19 months after treatment. The central choroidal thickness showed 0.4 and 3.1% reductions OD and OS, respectively. The binarization technique demonstrated that the rate of luminal areas in choroidal areas exhibited 3.2% increase OD but 4.8% decrease OS. CONCLUSION: Choroidal blood flow improved OD after treatment for CML, while it deteriorated OS, together with choroidal thinning due to reduction of luminal areas. The degrees of leukemia cell invasion into the choroidal tissue and tissue destruction might be different between the eyes in this case.

First Identification of 12ß-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12ß-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC).

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12ß-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12ß-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12ß-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.

Alteration in meibum lipid composition and subjective symptoms due to aging and meibomian gland dysfunction.

PURPOSE: To investigate the alteration in lipid composition of meibum, objective clinical signs, and subjective symptoms associated with aging and meibomian gland (MG) dysfunction (MGD). METHODS: In 10 MGD patients [4 males/6 females, mean age: 65.6 ± 7.9 years (range: 50-79 years)] and 24 healthy volunteer subjects [young subjects: 6 males/6 females, mean age: 25.7 ± 3.8 years (range: 20-35 years), elderly subjects: 6 males/6 females, mean age: 58.4 ± 7.5 years (range: 50-79 years)], three objective clinical signs were evaluated: MG orifice obstruction, meibum score, and tear film lipid layer interference pattern. Subjective symptoms were analyzed via a 15-item questionnaire. After careful collection of meibum samples, comprehensive lipid analysis was performed via liquid chromatography-mass spectrometry. Data was analyzed via JMP® ver. 13 (SAS Institute, Inc., Cary, NC) statistical analysis software. RESULTS: In the MGD patients and elderly subjects, there was a significant decrease in non-polar lipids such as cholesterol esters (ChEs), while a significant increase in polar lipids [cholesterol (Ch), (O-acyl)-ω-hydroxy fatty acid (OAHFA), and free fatty acid (FA)] in total lipids (Tukey-Kramer test: p < 0.05). Triglyceride was significantly increased only in MGD patients (p < 0.05). Symptom scores representative of vision quality (i.e., blurred vision/haziness) were significantly negatively-correlated with the ratio of the non-polar lipid ChE, while significantly positively correlated with the polar lipids Ch, OAHFA, and FA (Spearman's rank correlation coefficient: p < 0.05). CONCLUSIONS: Our findings revealed that both MGD and aging affect the composition ratio of major meibum lipids, resulting in the appearance of subjective symptoms.

Preparation of domoic acid analogues using a bioconversion system, and their toxicity in mice.

Domoic acid (1, DA), a member of the natural kainoid family, is a potent agonist of ionotropic glutamate receptors in the central nervous system. The chemical synthesis of DA and its derivatives requires considerable effort to establish a pyrrolidine ring containing three contiguous stereocenters. Recently, a biosynthetic cyclase for DA, DabC, was identified. This enzyme cyclizes the linear precursor of isodomoic acid A (IA) to IA, a bioactive DA analogue. In this study, we developed a bioconversion system to obtain DA analogues from linear substrates prepared by simple chemical synthesis using DabC expressed in Escherichia coli, in vivo. Three IA analogues with various substitutions at the C7'-geranyl terminus were prepared using this system: two minor natural analogues, 7'-methyl-IA (5) and 7'-hydroxy-IA (6), and one new unnatural analogue, 7'-amide-IA (7). In addition, the toxicity of these DA analogues in mice was examined by intracerebroventricular injection. Most of the mice injected with 5 (3 nmol) and 6 (3 nmol) did not show any adverse symptoms, whereas the mice injected with 7 (3 nmol) showed typical symptoms induced by DA (1, 0.7 nmol) and IA (2, 3 nmol). These results suggest that the 7'-carbonyl group in the side chain of IA (2) is crucial for its toxicity. The docking studies of DA, IA (2), 5, 6, and 7 to GluK1 supported these results.

Two new skeletal analogues of saxitoxin found in the scallop, Patinopecten yessoensis, as possible metabolites of paralytic shellfish toxins.

The scallop, Patinopecten yessoensis, was screened for new saxitoxin analogues to study the metabolism of paralytic shellfish toxins (PSTs), and this resulted in the discovery of two new analogues: M5-hemiaminal (HA) and M6-HA. M5-HA was isolated and its structure was determined by using NMR spectroscopy. It contains hydrogen at C-4 with opposite stereochemistry to that in saxitoxin, and a hemiaminal was formed between 9-NH2 and the hydrated ketone at C-12 in α-orientation. This is the first reported structural feature in a natural saxitoxin analogue, whereas the same ring system has previously been reported in a synthetic saxitoxin analogue, FD-saxitoxin. Acid hydrolysis of the carbamoyl N-sulfate in M5-HA produced M6-HA which was also identified in P. yessoensis by using LC-MSMS. M5-HA was not synthetically produced from M1 (11-hydroxy gonyautoxin-5) and M3 (11,11-dihydroxy gonyautoxin-5) through incubation in aqueous buffers. Furthermore, PSTs in the hepatopancreas of P. yessoensis, cultured in a bay located in northeastern Japan, were chronologically analyzed in 2018. The highest concentrations of M1/M3/M5-HA were observed two weeks after C-toxins had reached their highest concentrations, which provides evidence that M1/M3/M5-HA are metabolites of C-toxins. The voltage-gated sodium channel blockage activity of M6-HA was not detected at the concentration of 140 nM by using the Neuro-2A veratridine/ouabain assay.