A novel monoclonal antibody generated by immunization with granular tau oligomers binds to tau aggregates at 423-430 amino acid sequence.

Prior to the formation of amyloid fibrils, the pathological hallmark in tau-related neurodegenerative disease, tau monomers aggregate into a diverse range of oligomers. Granular tau oligomers, consisting of approximately 40 tau protein molecules, are present in the prefrontal cortex of patients at Braak stages I-II, preclinical stages of Alzheimer's disease (AD). Antibodies to granular tau oligomers as antigens have not been reported. Therefore, we generated new rat monoclonal antibodies by immunization with granular tau oligomers. Three antibodies from different hybridoma clones showed stronger immunoreactivity to granular tau oligomers and tau fibrils compared with monomeric tau. Of the three antibodies, 2D6-2C6 showed 3000-fold greater immunoreactivity in P301L-tau transgenic (rTg4510) mice than in non-transgenic mice, while MC1 antibody, which detects pathological conformations of tau, showed a 5.5-fold increase. These results suggest that 2D6-2C6 recognizes aggregates more specifically than MC1. In AD subjects, 2D6-2C6 recognized neurofibrillary tangles and pretangles, and co-localized within AT8-positive cells containing phosphorylated tau aggregates. The epitope of 2D6-2C6 is the 423-430 amino acid (AA) sequence of C-terminal regions. Taken together, a novel monoclonal antibody, 2D6-2C6, generated by immunization with granular tau oligomers binds to tau aggregates at the 423-430 AA sequence.

Distinct Expression Profiles of Neuroblastoma-Associated mRNAs in Peripheral Blood and Bone Marrow of Non-High-Risk and High-Risk Neuroblastoma Patients.

Non-high-risk (non-HR) neuroblastoma (NB) patients have excellent outcomes, with more than a 90% survival rate, whereas HR NB patients expect less than a 50% survival rate. Metastatic disease is the principal cause of death among both non-HR and HR NB patients. Previous studies have reported the significant but limited prognostic value of quantitative PCR (qPCR)-based assays, measuring overlapping but different sets of neuroblastoma-associated mRNAs (NB-mRNAs), to detect metastatic disease in both non-HR and HR patient samples. A droplet digital PCR (ddPCR)-based assay measuring seven NB-mRNAs (CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs) was recently developed and exhibited a better prognostic value for HR patient samples than qPCR-based assays. However, it remained to be tested on non-HR patient samples. In the present study, we employed the ddPCR-based assay to study peripheral blood (PB) and bone marrow (BM) samples collected at diagnosis from eight non-HR and eleven HR cases and characterized the expression profiles of NB-mRNAs. The most highly expressed NB-mRNAs in PB and BM differed between non-HR and HR cases, with the CRMP1 mRNA being predominant in non-HR cases and the GAP43 mRNA in HR cases. The levels of NB-mRNAs in PB and BM were 5 to 1000 times lower in non-HR cases than in HR cases. The PB to BM ratio of NB-mRNAs was 10 to 100 times higher in non-HR cases compared to HR cases. The present case series suggests that non-HR and HR NB patients have the distinct expression profiles of NB-mRNAs in their PB and BM.

A novel NFKB1 variant in a Japanese pedigree with common variable immunodeficiency.

Recently, heterozygous loss-of-function NFKB1 variants were identified as the primary cause of common variable immunodeficiency (CVID) in the European population. However, pathogenic NFKB1 variants have never been reported in the Japanese population. We present a 29-year-old Japanese woman with CVID. A novel variant, c.136 C > T, p.(Gln46*), was identified in NFKB1. Her mother and daughter carried the same variant, demonstrating the first Japanese pedigree with an NFKB1 pathogenic variant.

Higher levels of minimal residual disease in peripheral blood than bone marrow before 1st and 2nd relapse/regrowth in a patient with high­risk neuroblastoma: A case report.

More than half of patients with high-risk neuroblastoma (HR-NB) experience relapse/regrowth due to the activation of chemoresistant minimal residual disease (MRD). MRD in patients with HR-NB can be evaluated by quantitating neuroblastoma-associated mRNAs (NB-mRNAs) in bone marrow (BM) and peripheral blood (PB) samples. Although several sets of NB-mRNAs have been shown to possess a prognostic value for MRD in BM samples (BM-MRD), MRD in PB samples (PB-MRD) is considered to be low and difficult to evaluate. The present report describes an HR-NB case presenting higher PB-MRD than BM-MRD before 1st and 2nd relapse/regrowth. A 3-year-old female presented with an abdominal mass, was diagnosed with HR-NB, and treated according to the nationwide standard protocol for HR-NB. Following systemic induction and consolidation therapy with local therapy, the patient achieved complete remission but experienced a 1st relapse/regrowth 6 months after maintenance therapy. The patient partially responded to salvage chemotherapy and anti-GD2 immunotherapy but had a 2nd relapse/regrowth 14 months after the 1st relapse/regrowth. Consecutive PB-MRD and BM-MRD monitoring revealed that PB-MRD was lower than BM-MRD at diagnosis (100 times) and 1st and 2nd relapse/regrowth (1,000 and 3 times) but became higher than BM-MRD before 1st and 2nd relapse/regrowth. The present case highlights that PB-MRD can become higher than BM-MRD before relapse/regrowth of patients with HR-NB.

Minimal residual disease detected by droplet digital PCR in peripheral blood stem cell grafts has a prognostic impact on high-risk neuroblastoma patients.

More than half of high-risk neuroblastoma (NB) patients have experienced relapse due to the activation of chemoresistant minimal residual disease (MRD) even though they are treated by high-dose chemotherapy with autologous peripheral blood stem cell (PBSC) transplantation. Although MRD in high-risk NB patients can be evaluated by quantitative PCR with several sets of neuroblastoma-associated mRNAs (NB-mRNAs), the prognostic significance of MRD in PBSC grafts (PBSC-MRD) is unclear. In the present study, we collected 20 PBSC grafts from 20 high-risk NB patients and evaluated PBSC-MRD detected by droplet digital PCR (ddPCR) with 7NB-mRNAs (CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNA). PBSC-MRD in 11 relapsed patients was significantly higher than that in 9 non-relapsed patients. Patients with a higher PBSC-MRD had a lower 3-year event-free survival (P = 0.0148). The present study suggests that PBSC-MRD detected by ddPCR with 7NB-mRNAs has a prognostic impact on high-risk NB patients.

Limited correlation between tumor markers and minimal residual disease detected by seven neuroblastoma-associated mRNAs in high-risk neuroblastoma patients.

Vanillylmandelic acid (VMA), homovanillic acid (HVA), neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) are classical tumor markers and are used as standard clinical evaluations for patients with neuroblastoma (NB). Minimal residual disease (MRD) can be monitored by quantifying several sets of NB-associated mRNAs in the bone marrow (BM) and peripheral blood (PB) of patients with NB. Although MRD in BM and PB has been revealed to be a strong prognostic factor that is independent of standard clinical evaluations, its interrelation with tumor markers remains uncharacterized. The present study determined the levels of tumor markers (VMA, HVA, NSE and LDH) and MRD (BM-MRD and PB-MRD) in 133 pairs of concurrently collected BM, PB and urine samples from 19 patients with high-risk NB. The patients were evaluated during the entire course of treatment, which included 10 diagnoses, 32 treatments, 36 post-treatment, 9 relapses and 46 post-relapse sample pairs. The level of BM-MRD and PB-MRD was determined by quantifying 7 NB-mRNAs (collapsin response mediator protein 1, dopamine beta-hydroxylase, dopa decarboxylase, growth-associated protein 43, ISL LIM homeobox 1, pairedlike homeobox 2b and tyrosine hydroxylase) using droplet digital PCR. In overall sample pairs, tumor markers (VMA, HVA, NSE and LDH) demonstrated weak but significant correlations (P<0.011) with BM-MRD and PB-MRD. In subgroups according to each patient evaluation, the degree of correlation between tumor markers and MRD became stronger in patients with adrenal gland tumors, BM metastasis at diagnosis and relapse/regrowth compared with overall sample pairs. In contrast, tumor markers demonstrated variable correlations with MRD in subgroups according to each sample evaluation (BM infiltration at sampling, collection time point and disease status). The results suggested that tumor markers may demonstrate limited correlation with MRD in patients with high-risk NB.

Minimal residual disease in high-risk neuroblastoma shows a dynamic and disease burden-dependent correlation between bone marrow and peripheral blood.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and originates from sympathoadrenal or Schwann cell precursors derived from neural crest. These neural crest derivatives also constitute the hematopoietic and mesenchymal stem cells in bone marrow (BM) that is the most frequent site of NB metastasis and relapse. In NB patients, NB cells have been pathologically detected in BM and peripheral blood (PB), and minimal residual disease (MRD) in BM and PB (BM-MRD and PB-MRD) can be monitored by quantitating several sets of NB-associated mRNAs (NB-mRNAs). Although previous studies have shown varying degrees of correlation between BM-MRD and PB-MRD, the underlying factors and/or mechanisms remains unknown. In the present study, we determined the levels of BM-MRD and PB-MRD by quantitating seven NB-mRNAs in 133 pairs of concurrently collected BM and PB samples from 19 high-risk NB patients with clinical disease evaluation, and examined their correlation in overall and subgroups of sample pairs. The levels of BM-MRD and PB-MRD were moderately (r = 0.418, p < 0.001) correlated with each other in overall sample pairs. The correlation became strong (r = 0.725, p < 0.001), weak (r = 0.284, p = 0.008), and insignificant (p = 0.194) in progression, stable, and remission subgroups of sample pairs, respectively. It also became stronger in subgroups of sample pairs with poor treatment responses and poor prognostic factors. Present study suggests that MRD in high-risk NB shows a dynamic and disease burden-dependent correlation between BM and PB.

Catheter-related blood stream infection caused by Mycobacterium chelonae in a child with myeloid leukemia associated with Down syndrome.

Rapidly growing nontuberculous mycobacteria should be considered if GPRs gram-positive rods are detected in blood cultures 2-3 days after the blood sample collection.

Pazopanib maintenance therapy after tandem high-dose chemotherapy for disseminated Ewing sarcoma.

The dismal prognosis of patients with disseminated Ewing sarcoma necessitates the development of novel treatment strategies. Pazopanib is an oral multi-targeted tyrosine kinase inhibitor that is active against advanced soft tissue sarcoma. However, the clinical activity and feasibility of pazopanib for treating Ewing sarcoma remain poorly understood. Moreover, clinical information on the use of tandem high-dose chemotherapy for Ewing sarcoma is limited. A 14-year-old boy with Ewing sarcoma was transferred to our hospital for treatment. Magnetic resonance imaging, computed tomography scans, and bone scintigraphy revealed multiple lesions in the pubis, ilium, ischium, femur, rib, cranial bone, thoracic vertebrae, sacrum, obturator muscle, adductor magnus muscle, testicular cord, and lungs. Bone scintigraphy after intensive chemotherapies confirmed that multiple abnormal accumulations were still present in the cranial bone and pubis. Subsequently, the patient received tandem high-dose chemotherapy including topotecan, and radiotherapy. Abnormal accumulations have disappeared in bone scintigraphy. Subsequently, pazopanib maintenance therapy was initiated. Despite the presence of innumerable lesions at diagnosis, the patient has been in near-complete remission for the past 1 year with pazopanib administration. This confirms that adding pazopanib maintenance therapy after tandem high-dose chemotherapy is a therapeutic option for cases with disseminated Ewing sarcoma.

Generation of a monoclonal antibody against the extracellular domain of IGF-1R.

Type I insulin-like growth factor receptor (IGF-1R) is a receptor tyrosine kinase that is involved in the transformation of cells, cancer proliferation, and metastatic events in various types of cancer. The present study reports on the generation of a mouse monoclonal antibody (MAb) to IGF-1R using the mouse lymph node method. MAb 1B1, which reacts specifically with the extracellular domain of IGF-1R, was obtained using flow cytometry (FCM) screening using MCF-7 cells. Using immunostaining, MAb 1B1 detected IGF-1R mainly on the plasma membrane of MCF-7 cells. MAb 1B1 would be useful in FCM and immunofluorescence assays to detect IGF-1R-expressing cells for basic and clinical research.

Adenosine and ATP affect LPS-induced cytokine production in canine macrophage cell line DH82 cells.

Macrophages are essential for controlling the majority of infections, and are mediators of natural immunity. During infection, lipopolysaccharide (LPS) stimulates macrophages to produce pro-inflammatory cytokines. Adenosine and ATP released into the extracellular space by immunological stimuli have been shown to regulate various immune functions. More recently, it has been shown adenosine and ATP have a critical role on the physiological negative feedback mechanism for limitation and termination of tissue-specific and systemic inflammatory responses. It was useful and meaningful to gain information about interaction between LPS, which generates the inflammation, and adenosine and ATP, which terminate the inflammation. We evaluate effects of adenosine and ATP on the production of cytokines related to inflammation in canine macrophage cell line DH82 cells. Adenosine and ATP respectively increased the production of IL-10 without affecting the production of IL-6, TNF-α and IL-12 in DH82 cells. In addition, adenosine and ATP prevented the production of LPS-induced IL-6, TNF-α and IL-12 in DH82 cells. In contrast, adenosine and ATP potentiated LPS-induced IL-10 production in DH82 cells. Moreover, adenosine, but not ATP inhibited LPS-induced expression of TLR4 in DH82 cells. These results suggest that conditions related to increased adenosine and/or ATP may play an important role in the inflammatory reactions.