Methylation of PLK-1 Potentially Drives Bendamustine Resistance in Leukemia Cells.

BACKGROUND: Drug resistance remains a significant impediment in leukemia treatment. While Bendamustine hydrochloride (BH) stands out as a promising therapeutic agent for non-Hodgkin' s lymphoma and mantle cell lymphoma, the mechanisms of resistance to BH are not yet fully understood. Our study focuses on elucidating the mechanisms behind bendamustine resistance in leukemia cells, with a specific emphasis on epigenetics. METHODS: Bendamustine-resistant cells were cultivated from human B cell lymphoblastic leukemia cell lines through systematic and sustained exposure to bendamustine, using the limiting dilution method. Gene expression was assessed via real-time polymerase chain reaction, while the expression of the multidrug resistance protein 1 (MDR1) was evaluated using flow cytometry. RESULTS: Bendamustine-resistant leukemia cells exhibited a decreased RNA expression level for Polo-like kinase-1 (PLK-1). Notably, after treatment with the demethylating agent 5-aza-2'-deoxycytidine, PLK-1 gene expression surged significantly, enhancing bendamustine's cytotoxicity in the resistant leukemia cells. However, MDR1 expression, as determined by flow cytometry, remained consistent between parental and bendamustine-resistant leukemia cells. CONCLUSIONS: Our findings indicate that the methylation of the PLK-1 gene plays a pivotal role in modulating PLK-1 expression and is central to the development of bendamustine resistance in leukemia cells.

A landscape of complex tandem repeats within individual human genomes.

Markedly expanded tandem repeats (TRs) have been correlated with ~60 diseases. TR diversity has been considered a clue toward understanding missing heritability. However, haplotype-resolved long TRs remain mostly hidden or blacked out because their complex structures (TRs composed of various units and minisatellites containing >10-bp units) make them difficult to determine accurately with existing methods. Here, using a high-precision algorithm to determine complex TR structures from long, accurate reads of PacBio HiFi, an investigation of 270 Japanese control samples yields several genome-wide findings. Approximately 322,000 TRs are difficult to impute from the surrounding single-nucleotide variants. Greater genetic divergence of TR loci is significantly correlated with more events of younger replication slippage. Complex TRs are more abundant than single-unit TRs, and a tendency for complex TRs to consist of <10-bp units and single-unit TRs to be minisatellites is statistically significant at loci with ≥500-bp TRs. Of note, 8909 loci with extended TRs (>100b longer than the mode) contain several known disease-associated TRs and are considered candidates for association with disorders. Overall, complex TRs and minisatellites are found to be abundant and diverse, even in genetically small Japanese populations, yielding insights into the landscape of long TRs.

A case of bacteremia and meningitis in a neonate infected with Group B Streptococcus via breastfeeding who survived without neurological sequelae: A case report.

Invasive neonatal infection with Group B Streptococcus (GBS) is a disease of concern that can lead to neurological sequelae. Guidelines for preventing mother-to-child transmission have been introduced to reduce the incidence of early-onset infection, but guidelines for controlling the late-onset form are lacking. Recently, the trans-breastfeeding route of transmission has been highlighted as an example of late-onset infection, but no consensus on how to manage such infections has been reached. In this report, we describe a case of late-onset bacteremia/meningitis in a neonate suspected to have been infected with GBS via breastfeeding. A vaginal culture test of the mother at 35 weeks' gestation was negative for GBS. Since she had symptoms of mastitis, breast milk and nipple cultures were also tested and found to be positive for the strain of GBS identified in the neonate on genetic analysis. Diagnosis of trans-mammary GBS infection is challenging because breastfeeding-related events are difficult to identify. In our case, the diagnosis was based on the mother's history of mastitis, and the patient was treated without escalation to sequelae. When a neonate develops a fever, physicians should consider GBS infection and examine the mother's medical history to facilitate accurate diagnosis, especially if the history includes mastitis. A breast milk culture should be performed if the mother has mastitis, especially in cases of infection in preterm infants and in recurrent cases.

Post-Traumatic Stress Disorder among Children Involved in Traffic Accidents and Their Parents in Japan.

BACKGROUND: Children who survive traffic accidents, and their parents, may develop post-traumatic stress disorder (PTSD) or related symptoms (depression or anxiety), which can hinder the children's development and the parents' ability to provide effective care. In Japan, the PTSD incidence rate after traffic accidents and its related factors remain unclarified. METHODS: The participants were 79 children and 104 parents. The children were aged 3-18 years when injured. From August through December 2015, participants completed a self-reported questionnaire survey that comprised the 15-item Post-traumatic Stress Symptoms for Children and the Japanese version of the Impact of Event Scale-Revised. The children's Injury Severity Score (ISS) was also obtained from their medical records. Correlation analysis, analysis of variance, and multiple regression analysis were conducted. RESULTS: Among the children and parents, 10.1% and 22.1%, respectively, were deemed to be at high risk of PTSD. Their stress scores were significantly positively correlated with each other and negatively correlated with the children's age at the time of the accident. Parents who witnessed their children's accidents and those whose children were hospitalized were more stressed. Neither the children's nor the parents' risk for PTSD was associated with ISS or the amount of time since the accident. CONCLUSIONS: A system that simultaneously works with children and parents to support both parties' psychological recovery is required. To ensure psychological care post-injury, it is necessary to evaluate PTSD risk, regardless of injury severity. Implementing preventive and early interventions can prove more valuable than awaiting natural recovery.

Drug resistance to nelarabine in leukemia cell lines might be caused by reduced expression of deoxycytidine kinase through epigenetic mechanisms.

PURPOSE: Drug resistance is a serious problem in leukemia therapy. A novel purine nucleoside analogue, nelarabine, is available for the treatment of children with T cell acute lymphoblastic leukemia. We investigated the mechanisms of drug resistance to nelarabine. METHODS: Nelarabine-resistant cells were selected by stepwise and continuous exposure to nelarabine using the limiting dilution method in human B and T cell lymphoblastic leukemia cell lines. Expression analysis was performed using real-time polymerase chain reaction, and epigenetic analysis was performed using methylation-specific polymerase chain reaction and chromatin immunoprecipitation. RESULTS: The RNA expression level for deoxycytidine kinase (dCK) was decreased in nelarabine-resistant leukemia cells. There were no differences between the parental and nelarabine-resistant leukemia cells in the methylation status of the promoter region of the dCK gene. In the chromatin immune precipitation assay, decreased acetylation of histones H3 and H4 bound to the dCK promoter was seen in the nelarabine-resistant cells when compared to the parental cells. Furthermore, treatment with a novel histone deacetylase inhibitor, vorinostat, promoted the cytotoxic effect of nelarabine along with increased expression of the dCK gene, and it increased acetylation of both histones H3 and H4 bound to the dCK promoter in nelarabine-resistant leukemia cells. The combination index showed that the effect of nelarabine and vorinostat was synergistic. CONCLUSION: This study reports that nelarabine with vorinostat can promote cytotoxicity in nelarabine-resistant leukemia cells through epigenetic mechanisms.

A Case of Kawasaki Disease with Intussusception.

Kawasaki disease (KD) is a systemic vasculitis of unknown cause and is associated with various digestive disorders, although only a few cases of intussusception associated with KD have been reported. We describe a case of intussusception followed by KD in a 3-year-old boy. The patient was admitted to our hospital for evaluation of severe abdominal pain. Because the target sign was seen on ultrasonography, intussusception was diagnosed and hydrostatic reduction was performed. On the second day after admission, he developed a high fever (38°C) and an irregular rash over his whole body. On the fourth day after admission, the high fever continued, and bilateral nonexudative conjunctivitis, erythema of the lips and oral mucosa, strawberry tongue, indurated edema of the dorsa of the hands and feet, and diffuse erythema of the palms and soles appeared, and KD was ultimately diagnosed. He was treated with intravenous immunoglobulin 2 g/kg, aspirin 30 mg/kg/day, and prednisolone 2 mg/kg/day. The high fever and other clinical symptoms resolved immediately after the start of treatment. There was no relapse of KD symptoms after initial treatment, and periungual desquamation was observed on the 10th day after admission. He was discharged on the 15th day, without abnormalities such as coronary dilatation, 3 months after the onset of KD symptoms. Patients with intussusception and KD were older (≥3 years vs <3 years) than those with intussusception alone. In addition, the site of intussusception in KD was mainly colonic rather than ileocolic. If intussusception precedes development of the characteristic clinical symptoms of KD, diagnosis of KD may be delayed. KD should be considered in children older than 3 years with intussusception at a colonic site.

Growth Hormone Treatment at Nippon Medical School Chiba Hokusoh Hospital.

BACKGROUND: Since 2002, the Department of Pediatrics of Nippon Medical School Chiba Hokusoh Hospital has offered educational activities for children with short stature. We analyzed outcomes of growth hormone (GH) treatment for children with short stature treated at our hospital, particularly outcomes after the growth spurt. METHODS: We analyzed data from children aged 0 to 17 years who were treated with recombinant GH during the period from 2000 through 2016 and were followed for at least 2 years after the start of treatment. RESULTS: Among children with short stature, 85 had GH deficiency, 5 had Turner syndrome, 9 were small for gestational age, and 1 had Noonan syndrome. The outcomes of GH treatment was similar to those previously reported in Japan. Children with GH deficiency who started GH treatment before the growth spurt exhibited marked height catch-up until the second year, but the effect decreased after 3 years. The effect of treatment for GH deficiency that was started after the growth spurt continued for 4 to 5 years after the start of treatment. CONCLUSIONS: Improvement in height standard deviation score was similar when treatment was started before and after the growth spurt.

Interleukin-6-Mediated Inflammation May Cause Methotrexate-Induced Leukoencephalopathy.

Children with leukemia treated with methotrexate (MTX) may develop MTX-induced leukoencephalopathy, which can present as seizures or focal neurological deficits. However, the precise pathophysiology has not been fully elucidated. Differences in cytokine/chemokine profiles in cerebrospinal fluid (CSF) between children with MTX-induced leukoencephalopathy and those with posterior reversible encephalopathy syndrome (PRES), an acute neurological condition associated with hypertension, were investigated. Interleukin (IL)-1ß, 2, 4, 5, 6, 7, 8, 10, 12, 13, and 17, tumor necrosis factor-alpha, interferon-gamma, granulocyte monocyte colony-stimulating factor, granulocyte colony-stimulating factor, macrophage inflammatory protein-1ß, and monocyte chemoattractant protein-1 concentrations were measured in CSF supernatants from 3 children with acute leukemia with MTX-induced leukoencephalopathy, 3 children with acute leukemia with PRES, 6 children with acute leukemia without neurological complications, and 8 children with acute encephalopathy. CSF IL-6 concentrations were higher in children with MTX-induced leukoencephalopathy than in children with acute leukemia with PRES, with acute leukemia without neurological complications, and with acute encephalopathy. We concluded that IL-6 may be involved in the pathogenesis of MTX-induced leukoencephalopathy.

Drug Resistance in Cancer Therapy and the Role of Epigenetics.

Effective leukemia treatment is seriously hampered by drug resistance, and the potential role of epigenetic mechanisms in cancer drug resistance has recently been investigated. With conventional anticancer drugs, including alkylating drugs, anti-metabolite drugs, topoisomerase inhibitors, and microtubule inhibitors-which have been available for half a century-drug resistance often develops because of decreased expression of target enzymes, in conjunction with increased expression of drug export pumps. Alterations of target gene expression and increased export pump function might be caused by epigenetic changes, such as alterations in methylation status, as well as by changes in histone acetylation status. In addition, newly developed anticancer drugs, including small-molecule drugs, such as kinase inhibitors, antibody drugs, and immune modulatory drugs, also resulted in development of drug resistance within 1 year, although these drugs showed significant effectiveness for patients resistant to conventional anticancer drugs. The resistant cells exhibited increased expression of bypass pathways, activation of downstream cascades, decreased expression of antigens of tumor cells, increased DNA repair activity, and increased expression of drug export pumps, which also suggests the presence of epigenetic changes. This article reviews drug resistance in cancer therapy and the possible roles of epigenetic mechanisms.

Long-term outcomes of children with extracutaneous juvenile xanthogranulomas in Japan.

BACKGROUND: Juvenile xanthogranuloma (JXG) is the most common non-Langerhans cell histiocytosis in children. The mortality and morbidity of JXG with extracutaneous lesions remain unclear. METHODS: Data of patients aged < 18 years who were diagnosed with JXG between 2001 and 2010 were retrospectively collected through a nationwide survey. RESULTS: Twenty patients (11 male and nine female) had extracutaneous lesions. The median observation time was 10 years (range, 0-17). Six patients presented with symptoms at birth. The median age at diagnosis was 8.5 months (range, 0 month-13 years). Fifteen patients underwent treatment for JXG, including chemotherapy (n = 11), and five did not receive treatment. All patients except one survived; 17 were disease-free and two survived with disease. One newborn-onset patient with liver, spleen, and bone marrow involvement died of the disease. Permanent sequelae included central diabetes insipidus, growth hormone deficiency, and panhypopituitarism detected at diagnosis in three, one, and two patients, respectively. Four patients had visual impairment (optic nerve compression and intraocular invasion in two each), three had epilepsy, one had mental retardation, and one had a skin scar. Eight patients who had intracranial lesions were older at diagnosis, and had a higher frequency of disease-related comorbidities and permanent sequelae than those without intracranial involvement. CONCLUSIONS: Patients with extracutaneous JXG had good outcomes, although those with intracranial lesions had serious permanent sequelae. Effective and safe treatment regimens for patients with intracranial JXG need to be developed.

Therapy-related Secondary Malignancy After Treatment of Childhood Malignancy: Cases from a Single Center.

BACKGROUND: Therapeutic outcomes for childhood malignancy have dramatically improved. However, secondary malignancies are a major concern, as they greatly affect the quality of life of survivors. This retrospective study evaluated the cumulative incidence, clinical features, and outcomes of secondary malignancies at Nippon Medical School Hospital. METHODS: We examined data from 275 cases of primary childhood malignancy diagnosed between 1980 and 2014. Information regarding treatment of the primary malignancy, including irradiation dose, site, and cumulative dose of anticancer drugs, was assessed. We also collected data on secondary malignancy, including patient sex, age at diagnosis, malignancy site, time from primary to secondary malignancy, and outcomes. RESULTS: Secondary malignancies developed in 11 patients and included acute myeloid leukemia (AML) (4), meningioma (4), Ewing sarcoma (1), germ cell tumor (1), and malignant parotid gland tumor (1). The primary malignancies included acute lymphoblastic leukemia (ALL) (9), non-Hodgkin lymphoma (1) and brain tumor (1). In 7 of the 9 ALL patients, chemoradiotherapy was the primary treatment. The meningiomas and 1 solid tumor developed within the radiation field. All AMLs and meningiomas developed within 5 years and after 20 years, respectively, of the primary diagnosis. The 10- and 20-year cumulative incidence rates for secondary malignancy in our hospital were 1.9% and 5.8%, respectively. CONCLUSIONS: Our results revealed that the type of secondary malignancy depends on the interval after the end of treatment for primary malignancy. Meningioma, notably, develops many years after completion of primary malignancy treatment. Early detection during long-term follow-up is therefore essential.

Estimation of the number of feeding arteries of spinal arteriovenous malformations by using three-dimensional digital subtraction angiography.

PURPOSE: Spinal angiography is the gold standard for evaluation or diagnosis of spinal arteriovenous malformations (AVMs). However, some feeding arteries might be overlooked when multiple feeders exist. This study aimed to retrospectively review cases of spinal intra-dural AVMs, which were identified by three-dimensional digital subtraction angiography (3D-DSA), and attempted to estimate the number of feeding arteries. METHODS: We retrospectively reviewed patients with spinal intra-dural AVMs who underwent 3D-DSA at Hokkaido University Hospital from January 2005 to December 2016. We selected 9 patients in whom we could obtain data of multi-planar reconstruction of 3D-DSA. We measured the computed tomography (CT) values of feeding arteries and draining veins. The CT values represented the averages of maximum CT values of 5 continuous axial slices. The ratio of the CT value of feeders to that of drainers (F/D ratio) was calculated. The correlation between the F/D ratio and the number of feeders was examined with Pearson's correlation coefficient. RESULTS: The average number of feeders was 2.3 (1-4), and the number of feeders was significantly positively correlated with the F/D ratio (r = 0.855, P = .003). CONCLUSIONS: We conclude that the number of feeding arteries of spinal intra-dural AVMs can be estimated by using the F/D ratio obtained from 3D-DSA. These slides can be retrieved under Electronic Supplementary Material.

An 8-Year-Old Girl with Autoimmune Hepatitis Following Aplastic Anemia.

We herein report a case of severe aplastic anemia diagnosed in an 8-year-old girl with a previous diagnosis of autoimmune hepatitis. We found significantly increased CD8+ and CD68+ cell numbers in her bone marrow, which can induce severe organ damage, refractory to immunosuppressive therapy.

Coxsackievirus B5 Aseptic Meningitis in Infants in Chiba Prefecture, Japan, in 2016.

BACKGROUND: Infantile aseptic meningitis is a rare infection of the central nervous system. Coxsackievirus B5 (CVB5) is an enterovirus that is sometimes associated with aseptic meningitis and encephalitis. CASE PRESENTATION: We report on three isolated infants with aseptic meningitis caused by CVB5 in the spring and summer of 2016 with nearly identical 404-bp CVB5 Viral Capsid Protein 1 (VP1) sequences. In addition, viral analysis of sewage samples from Chiba Prefecture in 2016 showed similar 404-bp CVB5 VP1 sequences from May to September 2016. CONCLUSION: These results indicate that viral screening of sewage water may help detect occult outbreaks of viral infection, particularly for enterovirus strains.

Lysosomal Acid Lipase Deficiency in Japan: A Case Report of Siblings and a Literature Review of Cases in Japan.

We report on two siblings with early onset lysosomal acid lipase deficiency or Wolman disease. Their parents had a consanguineous marriage. The children showed evidence of abdominal distension and failed to thrive, despite having regular nutrition. At 3-4 months of age, their abdominal distension and jaundice progressed rapidly and they died of liver failure. Sebelipase alfa, a recombinant form of human lysosomal acid lipase has recently been used as an enzyme replacement therapy in patients with later-onset cholesteryl ester storage disease. Therefore, we investigated cases of lysosomal acid lipase deficiency in Japan and found that the number of cases was extremely low. Only 14 cases of Wolman disease and seven cases of cholesteryl ester storage disease were reported. As it is now possible to treat lysosomal acid lipase deficiency, it is important to increase awareness of this disease among pediatricians and doctors working in internal medicine.

The Incidence of Pediatric Invasive Bacterial Diseases in Nippon Medical School Chiba Hokusoh Hospital before and after the Introduction of Conjugate Vaccines.

The introduction of the Haemophilus influenzae type b (Hib) vaccine and the 7-valent pneumococcal conjugate vaccine (PCV7) has led to dramatic reductions in cases of invasive H. influenzae disease and invasive pneumococcal disease (IPD). After the introduction of the PCV7 and the 13-valent pneumococcal conjugate vaccine (PCV13), the number of children with IPD markedly decreased in our hospital. However, since 2015, three children with IPD have been admitted to our hospital. We analyzed the serotype, multilocus sequence type, and antimicrobial susceptibility of Streptococcus pneumoniae strains isolated in these newly diagnosed cases. The strains were serotypes 7F and 12F. In addition, we analyzed the incidence of invasive bacterial disease before and after the introduction of conjugate vaccines and found no change in the incidences. We found that cases of IPD and invasive H. influenzae disease clearly decreased following the introduction of the PCV7, the PCV13, and the Hib vaccine, as well as disease caused by antibiotic-resistant strains.

Therapeutic Drug Monitoring Simulator for Antibiotic Dosage for Methicillin-Resistant Staphylococcus aureus Sepsis in a Patient with Primary Immunodeficiency on Peritoneal Dialysis.

Bacterial infections often cause fatal systemic infections in patients with primary immunodeficiency. To prevent unfortunate results, the selection, dose, and dosage of antibiotics are extremely important. Here, we report a case of Wiskott-Aldrich syndrome in a patient undergoing peritoneal dialysis because of chronic renal failure in whom methicillin-resistant Staphylococcus aureus sepsis developed. Because of the primary disease and complications, teicoplanin was the only chosen anti-S. aureus drug to prevent side effects. We used parameter estimation and dosage adjustment from a therapeutic drug monitoring simulation software program to overcome the challenges with teicoplanin treatment.

Zonisamide inhibits monoamine oxidase and enhances motor performance and social activity.

Zonisamide (ZNS) is an effective drug for not only motor symptoms but also non-motor symptoms in Parkinson's disease. However, the actions of ZNS as an anti-Parkinsonian drug are not well understood. To clarify the actions of ZNS in vivo, we administered ZNS to mice and examined the effects on neurotransmitter metabolism and behaviors, focusing on motor and non-motor symptoms. Administration of ZNS decreased dopamine (DA) turnover in various brain regions, including the striatum. In behavioral tests, ZNS enhanced locomotor activity and novelty seeking in the open field test, light-dark transition test, and the social interaction test. Consistent with these results of DA metabolism in ZNS-treated mice, monoamine oxidase activity was significantly inhibited by ZNS in primary neurons and astrocytes. Collectively, these data suggest that ZNS inhibits monoamine oxidase activity and decreases DA turnover, which increases locomotor activity and novelty seeking in mice. ZNS is potentially useful to improve not only motor symptoms but also neuropsychiatric non-motor symptoms such as apathy in PD.

Genome-wide DNA methylation profiling of CpG islands in a morpholino anthracycline derivative-resistant leukemia cell line: p38α as a novel candidate for resistance.

Effective leukemia treatment is seriously hampered by drug resistance. We previously showed that aberrant methylation of the topoisomerase II α gene causes altered gene expression and acquired drug resistance in etoposide-resistant leukemia cells. In this study, we analyzed the genome-wide methylation status in resistant leukemia cells. We used MX2, which is a morpholino anthracycline derivative that functions as a topoisomerase II α inhibitor. We established a human myelogenous leukemia cell line (K562/P) and a related cell line with resistance to MX2 (K562/MX2). Using these cell lines, we investigated the genome-wide methylation status, compared expression profiles with a microarray, and analyzed the data using Gene Ontology and key node analysis. We demonstrate that the MX2-resistant cell line was globally hypermethylated. Gene Ontology analysis identified genes involved in the immunological response and gene silencing that were responsible for methylation-related altered gene expression in drug-resistant cells. Key node analysis showed that p38α mitogen-activated protein kinase was a novel enzyme involved in MX2-related resistance. p38 kinase activity in resistant cells was increased compared to MX2-sensitive parent cells. Blocking p38α activity using inhibitors and p38α knock down with small interfering RNA restored the sensitivity to MX2 in resistant cells with a decrease in p38 kinase activity as well as decreased expression of p38α mRNA and phosphorylated p38α protein. These findings may lead to a new strategy for treatment of drug-resistant leukemia cells.

Ten-Year Retrospective Study on the Management of Spinal Arteriovenous Lesions: Efficacy of a Combination of Intraoperative Digital Subtraction Angiography and Intraarterial Dye Injection.

BACKGROUND: The goal of treatment for spinal arteriovenous lesions is to completely obliterate the shunt. In our institution, intraoperative digital subtraction angiography and intraarterial injection of contrast agent have been used to accurately identify the site of arteriovenous shunts. We describe the intraoperative digital subtraction angiography and intraarterial dye injection procedures and how they may improve surgical outcomes. METHODS: We retrospectively investigated 22 patients with intradural arteriovenous lesions (n = 19) or spinal dural arteriovenous fistulas (n = 3). A microcatheter was used during the procedures to avoid catheter migration. RESULTS: There were 29 procedures performed. To support the surgical procedures, indigo carmine was used 17 times and indocyanine green was used 12 times. There were no complications associated with these procedures. The indocyanine green procedure required a lower concentration of dye in the artery than in the vein to clarify the shunt point and visualized complex lesions more clearly. These methods allowed surgeons to orientate the complex vessel structure. CONCLUSIONS: Intraoperative digital subtraction angiography and intraarterial dye injection are useful tools for management of spinal arteriovenous lesions.