Nationwide population-based surveillance of invasive pneumococcal disease in Japanese children: Effects of the seven-valent pneumococcal conjugate vaccine.

BACKGROUND: In Japan, the seven-valent pneumococcal conjugate vaccine (PCV7) was introduced in 2010. PCV13 has replaced PCV7 since November 2013. METHODS: The effectiveness of PCV7 in protecting against invasive pneumococcal disease (IPD) in children aged <5 years was evaluated in a nationwide active population-based surveillance of IPD in 2008-2013 in 10 prefectures in Japan. RESULTS: 1181 cases were identified; 711 pneumococcal strains were analyzed for serotyping and antimicrobial resistance. Compared with the baseline IPD incidence (25.0 per 100,000), a 98% decline in IPD caused by PCV7 serotypes was found after the introduction of PCV7. This was partially offset by an increased incidence of IPD caused by PCV13 minus PCV7 and non-PCV13 serotypes, resulting in a 57% decline in overall IPD incidence. Absolute increases in the incidence rates of IPD caused by PCV13 minus PCV7 and non-PCV13 serotypes were 2.1 and 2.8 per 100,000 during the study period, respectively. The proportion of meropenem-nonsusceptible strains, especially with serotypes 19A and 15A, increased significantly after PCV7 introduction. CONCLUSIONS: Our data confirmed a 98% decline in IPD incidence caused by PCV7 serotypes in children aged <5 years and serotype replacement after PCV7 introduction. This shows the importance of continuing surveillance of serotypes responsible for IPD and their antimicrobial resistance in Japan.

PCR array analysis of gene expression profiles in chronic active Epstein-Barr virus infection.

To determine the host cellular gene expression profiles in chronic active Epstein-Barr virus infection (CAEBV), peripheral blood samples were obtained from three patients with CAEBV and investigated using a PCR array analysis that focused on T-cell/B-cell activation. We identified six genes with expression levels that were tenfold higher in CAEBV patients compared with those in healthy controls. These results were verified by quantitative reverse transcription-PCR. We identified four highly upregulated genes, i.e., IL-10, IL-2, IFNGR1, and INHBA. These genes may be involved in inflammatory responses and cell proliferation, and they may contribute to the development and progression of CAEBV.

Current research on chronic active Epstein-Barr virus infection in Japan.

Epstein-Barr virus (EBV) infection is usually asymptomatic and persists lifelong. Although EBV-infected B cells have the potential for unlimited proliferation, they are effectively removed by the virus-specific cytotoxic T cells, and EBV-associated lymphoproliferative disease develops only in immunocompromised hosts. Rarely, however, individuals without apparent immunodeficiency develop chronic EBV infection with persistent infectious mononucleosis-like symptoms. These patients have high EBV-DNA load in the peripheral blood and systemic clonal expansion of EBV-infected T cells or natural killer (NK) cells. Their prognosis is poor with life-threatening complications including hemophagocytic lymphohistiocytosis, organ failure, and malignant lymphomas. The term "chronic active EBV infection" (CAEBV) is now generally used for this disease. The geographical distribution of CAEBV is markedly uneven and most cases have been reported from Japan and other East Asian countries. Here we summarize the current understanding of CAEBV and describe the recent progress of CAEBV research in Japan.

Prognostic factors of Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis in children: report of the Japan Histiocytosis Study Group.

BACKGROUND: Despite several advances in the treatment of Epstein-Barr virus (EBV) in recent years, patients with Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH) do not always show satisfactory outcomes. We here conducted a nationwide survey in Japan to identify prognostic factors of EBV-HLH in children with this disease in an effort to improve the management and the outcomes of these patients. PROCEDURE: Between January 2003 and June 2008, we enrolled 98 children younger than 18 years of age who were diagnosed with EBV-HLH. We then studied the clinical characteristics and laboratory findings at the time of diagnosis with the aim to identify prognostic factors for EBV-HLH. RESULTS: The mean age of onset of EBV-HLH was 3.9 ± 2.8 years. Most of our patients presented with fever, hepatosplenomegaly, lymphadenopathy, and hemophagocytosis of bone marrow. Sixty-two percent of patients showed T cell clonality, and 97% had EBV infection in either T or natural killer cells. Most patients (60%) were treated with a multi-agent chemotherapeutic regimen, including corticosteroid, etoposide, and cyclosporine. After initial treatment, 90.3% of patients were in remission, and 7 patients (8.2%) experienced recurrence of EBV infection. Among several prognostic factors, patients with both hyperbilirubinemia (>1.8 mg/dl) and hyperferritinemia (>20,300 ng/ml) at the time of diagnosis had significantly poorer outcomes than those with low serum bilirubin and ferritin levels. CONCLUSIONS: These findings suggest that the therapeutic strategy for children with EBV-HLH could be tailored according to the laboratory findings at diagnosis.

Molecular diagnosis of mitochondrial respiratory chain disorders in Japan: focusing on mitochondrial DNA depletion syndrome.

BACKGROUND: Although mitochondrial respiratory chain disorders (MRCD) are one of the most common congenital metabolic diseases, there is no cumulative data on enzymatic diagnosis and clinical manifestation for MRCD in Japan and Asia. METHODS: We evaluated 675 Japanese patients having profound lactic acidemia, or patients having symptoms or signs of multiple-organ origin simultaneously without lactic acidemia on respiratory chain enzyme activity assay and blue native polyacrylamide gel electrophoresis. Quantitative polymerase chain reaction was used to diagnose mitochondrial DNA depletion syndrome (MTDPS). Mutation analysis of several genes responsible for MTDPS was also performed. RESULTS: A total of 232 patients were diagnosed with a probable or definite MRCD. MRCD are common, afflicting one in every several thousand people in Japan. More than one in 10 of the patients diagnosed lacked lactic acidemia. A subsequent analysis of the causative genes of MTDPS identified novel mutations in six of the patients. A 335 bp deletion in deoxyguanosine kinase (DGUOK; g.11692_12026del335 (p.A48fsX90)) was noted in two unrelated families, and may therefore be a common mutation in Japanese people. The proportion of all patients with MTDPS, and particularly those with recessive DNA polymerase γ (POLG) mutations, appears to be lower in Japan than in other studies. This is most likely due to the relatively high prevalence of ancient European POLG mutations in Caucasian populations. No other significant differences were identified in a comparison of the enzymatic diagnoses, disease classifications or prognoses in Japanese and Caucasian patients with MRCD. CONCLUSION: MTDPS and other MRCD are common, but serious, diseases that occur across all races.

Clinical characteristics and outcomes of chédiak-Higashi syndrome: a nationwide survey of Japan.

BACKGROUND: Chédiak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by immunodeficiency, neurological dysfunction, and oculocutaneous albinism. Recently, several clinical CHS phenotypes have been reported. Here, we report results of a nationwide survey performed to clarify clinical characteristics and outcomes of CHS patients in Japan. METHODS: Questionnaires were sent to 287 institutions to collect data regarding CHS patients diagnosed between 2000 and 2010, including results of lysosomal trafficking regulator (LYST) gene analysis. Cytotoxicity and degranulation activity of cytotoxic T lymphocytes were analyzed in available patient samples. RESULTS: A total of 15 patients diagnosed with CHS were eligible for enrollment in this study. Of these, 10 (67%) had recurrent bacterial infections, five (33%) developed life-threatening hemophagocytic lymphohistiocytosis (HLH), and one patient had complicated malignant lymphoma. Hematopoietic stem cell transplantation (HSCT) was performed for six patients including three with HLH, and 10 of the enrolled patients have survived at the time of this writing. LYST analysis was performed for 10 patients; seven different mutations were detected in seven patients, whereas no mutation was identified in three patients. Cytotoxicity and degranulation activity were impaired in patients with and without LYST mutation. DISCUSSION: Results of this survey indicate that one or two patients with CHS were newly diagnosed each year in Japan. The incidence of HLH was not as high as expected. Mutations of genes other than LYST were suspected in some cases. We conclude that determining indication for HSCT for CHS patients should be based on genetic and cytotoxic analysis.

Effect of single-dose oral mizoribine pulse therapy twice per week for frequently relapsing steroid-dependent nephrotic syndrome.

AIM: To evaluate the efficacy of single-dose oral mizoribine (MZB) pulse therapy given twice weekly for frequently relapsing steroid-dependent nephrotic syndrome (FR-SDNS). METHODS: The subjects were 8 patients with FR-SDNS with a median age of 6.9 years old (range 3.1 - 18.0 y). The study was performed as a Phase II trial. The MZB dose was adjusted to achieve a peak blood level of 3 - 5 µg/ml (3.9 - 15.9 mg/kg/d, maximum dose: 750 mg) using a single dose given twice weekly before a meal. The therapeutic benefits of MZB pulse therapy were assessed based on a comparison of the incidence of relapse and the required daily dosage of prednisolone (PSL) in the 12 months prior to and following therapy. RESULTS: The incidence of relapse after therapy was significantly lower than that before therapy (2.5 ± 1.4 vs. 4.3 ± 0.5, p < 0.01) and the required daily dosage of prednisolone (PSL) after therapy was lower than that before therapy (0.48 ± 0.23 vs. 0.52 ± 0.32 mg/kg/d, not significant). However, this therapy was not effective for 3 out of 4 patients treated with cyclosporine. During follow-up, discontinuation of PSL was possible in 4 of 5 patients who showed a decreased rate of relapse after therapy. The peak blood concentration of MZB in these patients was significantly higher than that in 3 patients who did not show a decreased rate of relapse (3.95 ± 0.11 vs. 3.05 ± 0.21 µg/ml, p < 0.01). No adverse effects were observed in any patients. CONCLUSION: Our results show that single-dose oral MZB pulse therapy is effective in decreasing the frequency of relapse in some pediatric patients with FR-SDNS. A peak concentration of MZB of ~3.8 - 4.0 µg/ ml may be required for FR-SDNS therapy.

Characterization of Epstein-Barr virus (EBV) BZLF1 gene promoter variants and comparison of cellular gene expression profiles in Japanese patients with infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis.

Epstein-Barr virus (EBV) genotypes can be distinguished based on gene sequence differences in EBV nuclear antigens 2, 3A, 3B, and 3C, and the BZLF1 promoter zone (Zp). EBV subtypes and BZLF1 Zp variants were examined in Japanese patients with infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis. The results of EBV typing showed that samples of infectious mononucleosis, chronic active EBV infection, and EBV-associated hemophagocytic lymphohistiocytosis all belonged to EBV type 1. However, sequencing analysis of BZLF1 Zp found three polymorphic Zp variants in the same samples. The Zp-P prototype and the Zp-V3 variant were both detected in infectious mononucleosis and chronic active EBV infection. Furthermore, a novel variant previously identified in Chinese children with infectious mononucleosis, Zp-V1, was also found in 3 of 18 samples of infectious mononucleosis, where it coexisted with the Zp-P prototype. This is the first evidence that the EBV variant distribution in Japanese patients resembles that found in other Asian patients. The expression levels of 29 chronic active EBV infection-associated cellular genes were also compared in the three EBV-related disorders, using quantitative real-time reverse transcription polymerase chain reaction analysis. Two upregulated genes, RIPK2 and CDH9, were identified as common specific markers for chronic active EBV infection in both in vitro and in vivo studies. RIPK2 activates apoptosis and autophagy, and could be responsible for the pathogenesis of chronic active EBV infection.

Hemophagocytic lymphohistiocytosis after hematopoietic stem cell transplantation in children: a nationwide survey in Japan.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is associated with hypercytokinemia in children. Although HLH can be also observed after hematopoietic stem cell transplantation (HSCT), the incidence and clinical features of HLH after HSCT remain obscure. PROCEDURE: The clinical features of HLH after HSCT (post-HSCT HLH) were investigated in children with malignancies, immune deficiencies, or aplastic anemia. The HLH/Langerhans Cell Histiocytosis (LCH) Committee of the Japanese Society of Pediatric Hematology (JSPH) sent questionnaires to hospitals with JPSH members asking for details of cases in which HLH occurred after HSCT between 1998 and 2008. RESULTS: Among 42 children who were diagnosed with post-HSCT HLH between 1998 and 2008 in Japan, 37 fulfilled our inclusion criteria; of these, 26 were classified as early-onset (onset <30 days after HSCT) and 11 were classified as late-onset (onset >30 days after HSCT). In the early-onset group, the presence of respiratory symptoms, high levels of total bilirubin, and triglycerides at onset and the lack of control of GVHD with tacrolimus were significantly associated with non-resolution of HLH (P < 0.05). The survival rate was significantly higher in patients with resolution of HLH than in those without resolution (59% vs. 14%, P < 0.05). CONCLUSIONS: These findings suggest that early-onset post-HSCT HLH is a specific entity of HLH, and appropriate diagnosis and prompt management need to be established.

A novel anti-peroxiredoxin autoantibody in patients with Kawasaki disease.

Antibodies to the anti-oxidative peroxiredoxin (Prx) enzymes occur in both systemic autoimmune disease and vasculitis in adulthood. Because increased oxidative stress induces vasculitis in Kawasaki disease (KD), autoimmunity to Prxs in patients with KD was investigated. The presence of antibodies to Prx 1, 2 and 4 was analyzed by ELISA and Western blot. Of 30 patients with KD, 13 (43.3%) possessed antibodies to Prx 2, whereas these antibodies were present in only 1 of 10 patients (10.0%) with sepsis (4 with purulent meningitis and 6 with septicemia). In contrast, antibodies to Prx 1 and 4 were not detected in either group. There was no significant correlation among the titers of the three antibodies. Clinical parameters were compared between anti-Prx 2-positive and -negative patients. The presence of anti-Prx 2 antibodies correlated with a longer period of fever and poor response to high-dose γ-globulin therapy in patients with KD. Anti-Prx 2-positive patients had significantly greater excretion of urinary 8-isoprostaglandin than did anti-Prx 2-negative patients. These results provide the first evidence for an antibody to Prx 2 in patients with KD. They also suggest that this antibody might serve as a marker of disease severity and be involved in the pathophysiology of vasculitis in some patients with KD.

Neonatal lupus erythematosus in identical twins, showing transient bullous lesions.

One of identical twin girls was born with ulcers on her leg, and shortly after birth developed a flaccid blister on the leg. Subepidermal blister with vacuolar degeneration of basal cell layer and the heavy infiltration of mononuclear cells in the upper dermis were observed in the blister lesion. She also had generalized livedo. Her identical twin sister did not exhibit ulcers or blisters, but was born with milia on her limbs. Their mother was found to have lupus erythematosus with positive anti-Ro/SSA antibodies and developed Sjögren syndrome. We emphasize neonatal blistering and congenital milia unique manifestations of neonatal lupus erythematosus.

Autoimmune lymphoproliferative syndrome mimicking chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus infection (CAEBV) is defined as a systemic EBV-associated lymphoproliferative disease characterized by fever, lymphadenopathy, and splenomegaly in apparently immunocompetent persons. Recent studies have revealed that EBV infects T or natural killer cells in most patients with CAEBV; the etiology of CAEBV, however, remains unknown. Autoimmune lymphoproliferative disorder (ALPS) is an inherited disorder associated with defects in apoptosis, and clinically characterized by lymphadenopathy, splenomegaly, hypergammaglobulinemia, and autoimmune disease. ALPS is most often associated with mutations in the FAS gene, which is an apoptosis-signaling receptor important for homeostasis of the immune system. Based on the clinical similarity between ALPS and CAEBV with respect to lymphoproliferation, we have examined the possibility of the co-occurrence of ALPS in patients with a diagnosis of CAEBV. In this study, we have identified FAS gene mutations in three Japanese patients with lymphadenopathy, hepatosplenomegaly, and unusual EBV infection, who were diagnosed with CAEBV. These observations, which indicate that the clinical development of ALPS may be associated with EBV infection, alert us to a potential diagnostic pitfall of CAEBV.

Conditional VHL gene deletion activates a local NO-VEGF axis in a balanced manner reinforcing resistance to endothelium-targeted glomerulonephropathy.

BACKGROUND/AIMS: We have reported that tubular epithelial cell injury caused by renal ischemia-reperfusion is attenuated in conditional VHL knockout (VHL-KO) mice and also that induction of hypoxia-inducible factor (HIF) suppresses angiotensin II-accelerated Habu snake venom (HV) glomerulonephropathy in rats. However, it remains unknown whether VHL knockdown protects glomerular endothelial cells from endothelium-targeted glomerulonephritis. METHODS AND RESULTS: VHL-KO mice with HV glomerulonephropathy (HV GN) had fewer injured glomeruli, a lower mesangiolysis score and reduced blood urea nitrogen levels. Immunoreactivity of vascular endothelial growth factor (VEGF) in the glomerular capillaries was enhanced by VHL knockdown and was conserved even in VHL-KO mice with HV GN, despite HV-attenuating endothelial VEGF expression in vitro. VHL-KO mice showed enhanced nitric oxide (NO) production in glomerular endothelial cells and tubular cells, associated with activated VEGF expression in the kidney (i.e. an activated NO-VEGF axis). The levels of NO in glomeruli and tubules were conserved even in mice with HV GN. In contrast, suppressing NO production in glomerular endothelial cells by an NO synthase inhibitor, N(ϖ)-nitro-L-arginase, completely blunted the protection of VHL-KO from HV GN. The activated NO-VEGF axis in the kidney of VHL-KO mice was also associated with an elevation in Flk-1 phosphorylation and increased levels of IL-10 and IP-10. CONCLUSION: Conditional VHL knockdown may enhance the NO-VEGF axis and protect glomerular endothelial cells from HV GN, thereby providing resistance to injury of tubular epithelial cells and glomerular endothelial cells.

Asymptomatic high Epstein-Barr viral load carriage in pediatric renal transplant recipients.

High viral load carriage of EBV is one of the risks for PTLD in transplant recipients. We reviewed retrospectively in pediatric renal transplant recipients with EBV seronegative. EBV loads in peripheral blood and EBV-CTLs were measured every 1-3 months in 13 patients after grafting. Immunosuppressants were reduced when the patients were considered to have persistent high EBV loads (>1000 copies/µgDNA for over six months). All showed primary EBV infection: six with asymptomatic persistent high EBV loads (group A) and seven with neither EBV-associated symptoms nor persistent high EBV loads (group B). No patient developed PTLD in either group. Chronic rejection occurred in one patient in group A after immunosuppressants' reduction. There was no difference in renal dysfunction rates between the two groups. The maximum and increase rates in EBV loads were significantly higher in group A. The CTLs' percentage was significantly lower in group A when EBV loads first rose above 100 copies/µg DNA. This study suggests the possibility that EBV loads and CTLs' monitoring may be useful for avoidance of PTLD, as patients with asymptomatic persistent high EBV loads had higher EBV loads and lower percentages of CTLs.

[A case of persistent cerebellar ataxia complicated by conversion disorder--confirmed by positive cerebrospinal fluid glutamate receptor delta2 and epsilon2 antibodies].

We recently encountered a 13-year-old girl who developed persistent cerebellar symptoms one month after mixed measles/rubella vaccination, making it difficult to distinguish this condition from conversion disorders. Severe truncal ataxia was the initial manifestation in this case. The patient had no abnormalities in objective tests but began to show extraordinary circadian variations in certain parameters. Her cerebellar symptoms were thus considered to possibly be associated with conversion disorders. Later, she tested positive for cerebrospinal fluid anti-glutamic acid receptor (GluR) delta2 antibody. The lymphocyte stimulation test yielded a positive reaction to GluRdelta2 antigen. In addition, in the chronic stage SPECT revealed reduced cerebellar blood flow. She was thus diagnosed as having persistent cerebellar ataxia due to autoimmune mechanisms and modification of cerebellar symptoms due to secondary conversion disorders. Our experience with this case suggests that checking cerebrospinal fluid for anti-GluRdelta2 antibody is possibly useful for distinguishing between conversion disorders and cerebellar ataxia due to autoimmune mechanisms.

Neutrophil infiltration and oxidant-production in human atherosclerotic carotid plaques.

To clarify the clinical implications of neutrophils in vulnerable plaques we evaluated the function and activity of infiltrated neutrophils in an atherosclerotic plaque, focusing on oxidant production. A histopathological investigation was performed using carotid arterial samples obtained from seven patients. The atherosclerotic plaques were examined cytochemically for naphthol-ASD-chloroacetate esterase activity and oxidant-production, and immunohistochemically using N-formyl peptide receptor-like 1 (fPRL1)-, CD66b-, CD68- or p22phox-specific antibodies. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque was estimated using an activity index. Naphthol-ASD-chloroacetate esterase activity was found in cells located in the atherosclerotic plaque, indicating that the cells were neutrophils. The cytoplasmic fPRL1 intensity value of the neutrophils in the plaque decreased to approximately 60% of the intensity observed in the capillary vessels. Oxidant-production was also detected in the plaques, and both neutrophils and macrophages were observed at the corresponding oxidant-production sites. p22phox expression was also located in the same areas in which oxidant-production was observed in these plaques. We could not directly evaluate how much ROS generated from the infiltrated neutrophils contributed the plaque vulnerability followed by its rupture. However, the infiltrated neutrophils in the atherosclerotic plaques morphologically appeared activated and were actively generating oxidant, implying that neutrophils, together with macrophages, infiltrate into atherosclerotic plaques and contribute to plaque vulnerability.

Effect of pravastatin on cisplatin-induced nephrotoxicity in rats.

We investigated whether pravastatin ameliorates renal damage induced by cisplatin (CP). Forty-three male Wistar rats were divided into four groups: rats treated with a control diet for 19 days and saline injection on day 14 (group1), group 1 with pravastatin treatment with 19 days (group 2), group 1 with CP injection on day 14 (group 3), and group 2 with CP injection (group 4). Renal function and serum lipids, renal malondialdehyde (MDA) and glutathione (GSH) levels, glutathione peroxidase (GPx) mRNA expression and activity, and kidney triglyceride (TG) concentrations were measured. Histology was evaluated by light microscopy with immunohistochemistry for p53, p53-upregulated modulation of apoptosis (PUMA), and terminal deoxynucleotide transferase dUTP nick end-labeling (TUNEL) staining. CP induced renal tubular damage with a higher MDA level, increased PUMA expression, p53- and TUNEL-positive cells counts, elevation of serum lipids, and decreased GSH level, GPx mRNA expression, and activity. Pravastatin partially ameliorated CP-induced renal injury, based on suppression of the renal MDA and TG levels, decreased p53 expression, and apoptosis in CP-treated rats. These findings suggest that pravastatin has a partial protective effect against CP nephrotoxicity via antioxidant activity as well as attenuation of the p53 response, and lipid-lowering effects.

Pediatric Hirayama disease.

We report on a 16-year-old girl with muscular atrophy of a distal upper extremity (Hirayama disease). The disease progressed insidiously, and during our first examination, she exhibited weakness and wasting in the right hand, accompanied by cold paresis. No sensory disturbance was evident. A nerve conduction test revealed reduced compound muscle action potential of the ulnar and median nerves of the affected hand. However, conduction velocities were normal. An F-wave test of the right ulnar nerve indicated reduced frequency and prolonged minimum latency. Magnetic resonance imaging of the cervical spinal cord demonstrated abnormal signal intensity in the anterior horn, atrophy in the lower cervical cord, a forward shift of the lower cervical cord upon neck flexion, and flow voids in the epidural space. These clinical findings are typical of Hirayama disease. The use of a cervical collar did improve the patient's hand strength after 2 years. Pediatric neurologists should be aware of this disease, particularly in its early course, to diagnose it early, and to introduce a neck cervical collar as soon as possible after diagnosis.