Kousaku Matsubara

Pyrrolidine dithiocarbamate, a potent inhibitor of nuclear factor κB (NF-κB) activation, prevents apoptosis in human promyelocytic leukemia HL-60 cells and thymocytes

We examined the effect of pyrrolidine dithiocarbamate (PDTC), which potently blocks the activation of nuclear factor kappa B (NF-kappa B), on the induction of apoptosis by a variety of agents. Treatment of a human promyelocytic leukemia cell line, HL-60, with 10 micrograms/mL etoposide or 2 microM 1-beta-D-arabinofuranosylcytosine induced NF-kappa B activation within 1 hr and subsequently caused apoptosis within 3-4 hr. The simultaneous addition of 50-500 microM PDTC with these agents blocked NF-kappa B activation and completely abrogated both morphologically apoptotic changes and internucleosomal DNA fragmentation for up to 6 hr. However, PDTC failed to inhibit the endonuclease activity contained in the whole cell lysates. The inhibitory effect of PDTC was also observed in etoposide- and dexamethasone-induced apoptosis in human thymocytes at a concentration of 1-10 microM. Since PDTC has both antioxidant and metal-ion chelating activities, we tested the effects of N-acetyl-L-cysteine (NAC) (antioxidant) or o-phenanthroline (OP) (metal-ion chelator) on the induction of apoptosis. Pretreatment of HL-60 cells or thymocytes with 100-500 microM OP for 2 hr, but not 10-60 mM NAC, suppressed subsequent occurrence of apoptosis induced by etoposide. These results suggest that the activation of NF-kappa B plays an important role in the apoptotic process of human hematopoietic cells.

The role of superantigens of group A Streptococcus and Staphylococcus aureus in Kawasaki disease.

Purpose of review Since the first suggestion of a superantigen hypothesis for Kawasaki disease over a decade ago, debate on the aetiology remains inconclusive. This article reviews recent publications that address the role of superantigens of group A Streptococcus and Staphylococcus aureus in the pathogenesis of Kawasaki disease. Recent findings Over the past few years, new superantigens produced by group A Streptococcus and S. aureus have been increasingly identified, bringing the total known number to more than 30. Several studies on T-cell Vβ repertoires and seroloepidemiology have demonstrated evidence for the involvement of single or multiple superantigens produced by the two pathogens. The associated superantigens differed in those studies, including streptococcal pyrogenic toxins A and C, staphylococcal enterotoxins A–C, and toxic shock syndrome toxin 1. These disparate findings suggest that the inflammation of Kawasaki disease does not result from a single agent but rather a final common inflammatory pathway in genetically susceptible individuals after numerous infectious agents. Summary Certain staphylococcal and streptococcal superantigens are suggested to be responsible for the development of Kawasaki disease. A better understanding of the precise role of the causative agents will lead to accurate diagnosis, more targeted therapy and an improvement of coronary outcomes.

Elevation of serum thrombopoietin precedes thrombocytosis in acute infections

Summary.  To clarify the mechanisms underlying thrombocytosis secondary to infections, we longitudinally studied serum levels of thrombopoietin (TPO) and interleukin (IL)‐6 in 15 infants and young children with prominent thrombocytosis (platelets > 700 × 109/l) following acute infections and 116 age‐matched controls using an enzyme‐linked immunosorbent assay. The subjects included nine patients with bacterial infections, three with viral infections and three with non‐determined pathogens. TPO values in the controls were 2·24 ± 0·87 fmol/ml (mean ± SD) with a 95% reference interval of 0·85–4·47 fmol/ml. In the first week of infection, platelet counts were normal, but TPO values increased (∼10·73 fmol/ml). TPO levels peaked on day 4 ± 2 at 6·44 ± 2·37 fmol/ml and then fell gradually. When platelet counts peaked in the second and third weeks, TPO levels were similar to the controls. IL‐6 levels in the first week rose and dropped more rapidly than TPO. Serum TPO values were significantly correlated with C‐reactive protein levels (r = 0·688, P < 0·001) and IL‐6 levels (r = 0·481, P = 0·027). These results suggest that TPO contributes to thrombocytosis following infections in conjunction with IL‐6, arguing for additional regulatory mechanisms of blood TPO levels.

Neurodevelopmental abnormalities associated with severe congenital neutropenia due to the R86X mutation in the HAX1 gene

Objective: Severe congenital neutropenia (SCN), also known as Kostmann syndrome (SCN3, OMIM 610738), includes a variety of haematological disorders caused by different genetic abnormalities. Mutations in ELA2 are most often the cause in autosomal dominant or sporadic forms. Recently, mutations in HAX1 have been identified as the cause of some autosomal recessive forms of SCN, including those present in the original pedigree first reported by Kostmann. We sought to determine the relationship between HAX1 gene mutations and the clinical characteristics of Japanese cases of SCN. Methods: The genes implicated in SCN (ELA2, HAX1, Gfi-1, WAS, and P14) were analysed in 18 Japanese patients with SCN. The clinical features of these patients were obtained from medical records. Immunoblotting of HAX1 was performed on cell extracts from peripheral blood leucocytes from patients and/or their parents. Results: We found five patients with HAX1 deficiency and 11 patients with mutations in the ELA2 gene. In HAX1 deficiency, a homozygous single base pair substitution (256C>T), which causes the nonsense change R86X, was identified in three affected individuals. Two sibling patients showed a compound heterozygous mutation consisting of a single base pair substitution (256C>T) and a 59 bp deletion at nucleotides 376–434. There was no detectable phenotype in any heterozygous carrier. All patients with HAX1 deficiency had experienced developmental delay. Three patients carrying R86X also suffered from epileptic seizures. In contrast, no SCN patient with heterozygous mutations in the ELA2 gene suffered from any neurodevelopmental abnormality. Conclusions: These findings suggest that the R86X mutation in the HAX1 gene is an abnormality in Japanese SCN patients with HAX1 deficiency and may lead to neurodevelopmental abnormalities and severe myelopoietic defects.

Age-related changes in thrombopoietin in children: reference interval for serum thrombopoietin levels

We studied thrombopoietin (TPO, Mpl ligand) values using a sensitive ELISA in 254 serum samples obtained from disease‐free children and adult volunteers. TPO was detected in all samples, and its values ranged widely from 0.25 to 9.18 fmol/ml. When analysed by dividing the subjects into 11 age groups, the mean TPO levels from birth to 1 month of age were increased (3.73–5.92 fmol/ml). The highest values were found 2 d after birth; TPO levels then gradually decreased to adult levels (0.83 fmol/ml). The relationship between TPO values and platelet counts was not significant in all subjects (r = 0.27) or in children alone (r = 0.12). In children > 1 month of age a 95% reference interval for serum TPO values was determined from 0.58 to 3.27 fmol/ml. A significant correlation was found between TPO values in serum and plasma; serum TPO values = −0.257 + 4.039 × plasma TPO values (r = 0.951, P < 0.001, n = 22). This study is the first to report age‐dependent changes in blood TPO levels throughout child development. Serum TPO values were significantly high up to 1 month of age and were correlated with plasma TPO levels.

In vivo administration of granulocyte colony-stimulating factor promotes neutrophil survival in vitro

Abstract: We recently showed that recombinant human granulocyte‐colony stimulating factor (rhG‐CSF) maintained the viability of human neutrophils in incubation for up to 72 hours. However, it is not known whether rhG‐CSF can enhance neutrophil survival in in vivo situations. To clarify this issue, we investigated neutrophil survival in vitro following in vivo injection of rhG‐CSF. Neutrophils were obtained from 4 pediatric patients with malignancies and healthy adult volunteers before and after rhG‐CSF administration. Neutrophils obtained before rhG‐CSF treatment started to undergo apoptosis after 24 h of incubation. In contrast, the survival of neutrophils drawn after rhG‐CSF administration increased by approximately 24 h. Concomitantly, the appearance of typical ladder‐like DNA fragmentation was delayed. Such an increase in neutrophil survival was inhibited by coincubation with either H 7 (10 μmol/1) or H 8 (20 μmol/1), which worked as protein kinase C inhibitors. Although our study did not measure neutrophil survival in vivo directly, it provides us with further evidence that rhG‐CSF may function to prolong neutrophil life expectancy in vivo.

Induction of apoptosis in childhood acute leukemia by chemotherapeutic agents: Failure to detect evidence of apoptosis in vivo

Abstract:  This study is designed to investigate whether apoptosis occurs in vivo in pediatric patients with acute leukemia during induction therapy. When patients with common acute lymphoblastic leukemia (cALL) and acute myeloblastic leukemia (AML) were treated with prednisolone (60 mg/m2/day, p.o. or i.v.) and etoposide (150 mg/m2/day, i.v.), respectively, the blast cell counts fell to below 30% and 5%, respectively, in 1 week. However, during this cytoreduction phase, neither morphologically apoptotic cells nor fragmentation of DNA derived from peripheral blast cells were detected at any preparations. On the other hand, cALL but not AML cells spontaneously undergo apoptosis following their culture in vitro. The addition of autologous serum instead of fetal calf serum substantially prevented apoptosis from occurring spontaneously in cALL cells. When cALL and AML cells freshly obtained from patients before therapy were treated in vitro with 10 μmol/1 prednisolone and 20 μg/ml etoposide, respectively, these cells underwent apoptosis within 6 hours, as determined by a morphological and DNA fragmentation assay. These in vivo and in vitro findings suggest that, although anticancer drugs may induce apoptosis in vivo, these apoptotic cells cannot be detected due to their rapid removal from the circulation.

Age-Dependent Changes in the Incidence and Etiology of Childhood Thrombocytosis

To determine the incidence and etiology of childhood thrombocytosis, over 15,000 platelet counts in 7,539 patients performed at a single regional hospital were reviewed. When thrombocytosis was defined as ≧500 × 109/l of platelet counts, the condition could be diagnosed in 6.0% (456 cases) of the patients. All patients were classified as having secondary thrombocytosis. The incidence of thrombocytosis dramatically changed throughout child development; it was 12.5% in neonates, peaked to 35.8% in 1-month-old infants and then returned to 12.9% in 6- to 11-month-old infants. Thereafter, it gradually decreased with age to only 0.6% in 11- to 15-year-old children. Frequent causes of thrombocytosis included infection (67.5%), Kawasaki disease (9.4%), prematurity (7.7%) and iron deficiency anemia (6.4%). Thrombocytosis was an incidental finding in a substantial population of early infants. Thrombocytosis as a reaction to several types of infection and Kawasaki disease was more common in children under 7 years old, while autoimmune disease and tissue damage were major causes in children aged 11–15 years. No child had thromboembolic complications. These findings indicate that childhood thrombocytosis is a benign condition and its incidence and etiology seem to depend on age.

Loss of function mutations in RPL27 and RPS27 identified by whole-exome sequencing in Diamond-Blackfan anaemia

Diamond‐Blackfan anaemia is a congenital bone marrow failure syndrome that is characterized by red blood cell aplasia. The disease has been associated with mutations or large deletions in 11 ribosomal protein genes including RPS7, RPS10, RPS17, RPS19, RPS24, RPS26, RPS29, RPL5, RPL11, RPL26 and RPL35A as well as GATA1 in more than 50% of patients. However, the molecular aetiology of many Diamond‐Blackfan anaemia cases remains to be uncovered. To identify new mutations responsible for Diamond‐Blackfan anaemia, we performed whole‐exome sequencing analysis of 48 patients with no documented mutations/deletions involving known Diamond‐Blackfan anaemia genes except for RPS7, RPL26, RPS29 and GATA1. Here, we identified a de novo splicing error mutation in RPL27 and frameshift deletion in RPS27 in sporadic patients with Diamond‐Blackfan anaemia. In vitro knockdown of gene expression disturbed pre‐ribosomal RNA processing. Zebrafish models of rpl27 and rps27 mutations showed impairments of erythrocyte production and tail and/or brain development. Additional novel mutations were found in eight patients, including RPL3L, RPL6, RPL7L1T, RPL8, RPL13, RPL14, RPL18A and RPL31. In conclusion, we identified novel germline mutations of two ribosomal protein genes responsible for Diamond‐Blackfan anaemia, further confirming the concept that mutations in ribosomal protein genes lead to Diamond‐Blackfan anaemia.

Invasive group B streptococcal infections in a tertiary care hospital between 1998 and 2007 in Japan

OBJECTIVE To clarify clinical and microbiological features of invasive group B Streptococcus (GBS) disease in Japan. METHODS This was a retrospective review for the period 1998-2007 of patients across all age groups in Nishi-Kobe Medical Center. Invasive GBS disease was defined as GBS isolation from a normally sterile site or skin/soft tissues. RESULTS Six infant and 52 adult cases of invasive infection were identified. Diagnosis was limited to bacteremia and meningitis in infants, but varied widely in adults with skin/soft tissue infections and bacteremia being common. The overall fatality rate was 16%. An approximately 2.8-fold increase was found in the incidence among adult patients from the first to the second 5-year period. The most frequent underlying condition was diabetes, with the majority (18/23) of such patients showing poor control (HbA1c >8.0%). Amputation at the knee, ankle, or toes was performed in six diabetic adults with skin/soft tissue infections. Of the strains serotyped, types Ib and III predominated. All 58 strains were susceptible to penicillin; 2% were resistant to erythromycin and 3% were resistant to clindamycin. CONCLUSION This is the first epidemiological report describing invasive GBS disease in Japan. A significant increase in adult patients was noted, and mortality and morbidity remain substantial.