One-year follow-up of transgene expression by integrase-defective lentiviral vectors and their therapeutic potential in spinocerebellar ataxia model mice.

We examined integrase-defective lentiviral vectors (IDLVs) with a mutant (D64V) integrase in terms of their residual integration capability, the levels and duration of transgene expression and their therapeutic potential in comparison to wild-type lentiviral vectors (WTLVs) with a wild-type integrase gene. Compared with WTLVs, the IDLV-mediated proviral integration into host-cell chromosomes was approximately 1/3850 in HeLa cells and approximately 1/111 in mouse cerebellar neurons in vivo. At 2 months, transgene expression by IDLVs in the mouse cerebellum was comparable to that by WTLVs, but then significantly decreased. The mRNA levels at 6 and 12 months after injection in IDLV-infected cerebella were approximately 26% and 5%, respectively, of the mRNA levels in WTLV-injected cerebella. To examine the therapeutic potential, IDLVs or WTLVs expressing a molecule that enhances the ubiquitin-proteasome pathway were injected into the cerebella of spinocerebellar ataxia type 3 model mice (SCA3 mice). IDLV-injected SCA3 mice showed a significantly improved rotarod performance even at 1 year after-injection. Immunohistochemistry at 1 year after injection showed a drastic reduction of mutant aggregates in Purkinje cellsfrom IDLV-injected, as well as WTLV-injected, SCA3 mice. Our results suggest that because of the substantially reduced risk of insertional mutagenesis, IDLVs are safer and potentially effective as gene therapy vectors.

Human metapneumovirus infection among family members.

The transmission of human metapneumovirus (hMPV) among family members is not well understood. We identified 15 families in which multiple members were diagnosed with hMPV infection by real-time PCR in 2008 and 2010. Index patients ranged in age from 2 years to 11 years (median 5 years), and all 15 index cases were children who attended primary school, kindergarten, or nursery school. Contact patients ranged in age from 2 months to 46 years (median 6 years). Excluding five adult cases, contact patients were significantly younger than index patients (P = 0·0389). Of the 12 contact children, seven (58%) were infants who were taken care of at home. The serial interval between the onset of symptoms in an index patient and the onset of symptoms in a contact patient was estimated to be 5 days. These results suggest that the control of school-based outbreaks is important for preventing hMPV infection in infants.

Diffuse and focal palmoplantar keratoderma can be caused by a keratin 6c mutation.

The palmoplantar keratodermas (PPKs) are a large group of genodermatoses comprising nearly 60 genetically distinct diseases. They are characterized by hyperkeratosis on the palms and soles with or without extrapalmoplantar hyperkeratotic lesions. Focal PPK is one of the hallmarks of pachyonychia congenita, a rare autosomal dominant disorder resulting from mutations in the keratin genes KRT6A, KRT6B, KRT16 or KRT17. Recently, in-frame deletion mutations of KRT6C have been identified in three families with focal PPK with slight or no nail changes. We report here a novel KRT6C mutation identified in a Japanese family with PPK with phenotypic heterogeneity, presenting with not only focal but also diffuse hyperkeratosis. The proband had diffuse hyperkeratosis on the soles and small focal hyperkeratoses on the palms, while the two other affected individuals showed focal hyperkeratoses on the soles. All three patients were heterozygotes for c.1414G>A in KRT6C, predicted to result in p.Glu472Lys. These findings strongly suggest that screening of patients with nonepidermolytic diffuse PPK, in whom the pathogenic mutations are yet to be determined, might identify mutations in KRT6C.

Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation.

We found that c-kit-positive, lineage marker-negative, Thy-1lo cells are present in both bone marrow and thymus ("BM c-kit" and "thymus c-kit" cells). Although the two cell types are phenotypically similar, only BM c-kit cells showed the potential to form colonies in vitro as well as in vivo. However, both of them revealed extensive growth and differentiation potential to T cells after direct transfer into an irradiated adult thymus, or a deoxyguanosine-treated fetal thymus. Time course analysis showed that thymus c-kit cells differentiated into CD4CD8 double-positive cells approximately 4 d earlier than BM c-kit cells did. In addition, anti-c-kit antibody blocked T cell generation of BM c-kit cells but not of thymus c-kit cells. Intravenous injection of thymus c-kit resulted in the generation of not only T cells, but B as well as NK1.1+ cells. These data provide evidence that thymus c-kit cells represent common lymphoid progenitors with the differentiation potential to T, B, and possibly NK cells. The c-kit-mediated signaling appears to be essential in the transition from BM c-kit to thymus c-kit cells.

Expression and function of c-kit in hemopoietic progenitor cells.

The expression and function of a receptor tyrosine kinase, c-kit, in the adult bone marrow of the mouse were investigated by using monoclonal antibodies (mAbs) against the extracellular domain of murine c-kit. In adult C57BL/6 mouse, 7.8% of total bone marrow cells express c-kit on their surface. Half of the c-kit+ cells do not express lineage markers including Mac-1, Gr-1, TER-119, and B220, while the remainder coexpress myeloid lineage markers such as Mac-1 and Gr-1. After c-kit+ cells were removed from the bone marrow cell preparation, hemopoietic progenitor cells reactive to IL-3, GM-CSF, or M-CSF and also those which give rise to spleen colonies in irradiated recipients disappeared almost completely. Thus, most hemopoietic progenitors in the adult bone marrow express c-kit. To investigate whether or not c-kit has any role in the hemopoiesis of adult bone marrow, we took the advantage of one of the anti-c-kit mAbs that can antagonize the function of c-kit. As early as two days after the injection of 1 milligram of an antagonistic antibody, ACK2, almost all hemopoietic progenitor cells disappeared from the bone marrow, which eventually resulted in the absence of mature myeloid and erythroid cells in the bone marrow. These results provide direct evidence that c-kit is an essential molecule for constitutive intramarrow hemopoiesis, especially for the self-renewal of hemopoietic progenitor cells at various stages of differentiation.

In vivo imaging in humanized mice.

The radiological modalities that are currently utilized as critical components in clinical medicine have also been adapted to small-animal imaging, among which are ultrasound imaging, X-ray computerized tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), and single-photon emission computed tomography (SPECT). Optical imaging techniques such as bioluminescence imaging (BLI) and fluorescence imaging (FLI) are approaches that are commonly used in small animals. Longitudinal surveys of living (i.e., nonsacrificed) animal models with these modalities provide some clues for the development of clinical applications. The techniques are absolutely essential for translational research. However, there are currently few tools available with sufficient spatial or temporal resolution ideal for all experimental studies. In this chapter, we provide a rationale and techniques for visualizing target cells in living small animals and an overview of the advantages and limitations of current imaging technology. Finally, we introduce a humanized mouse and a novel in vivo imaging system that we have developed. We also discuss real-time observations of reconstructs and clinical manifestations.

Vasoactive intestinal peptide: a possible transmitter of nonadrenergic relaxation of guinea pig airways.

Vasoactive intestinal peptide, a smooth-muscle relaxant neuropeptide with neurotransmitter properties, was relaxed during electrical field stimulation of guinea pig trachea. The amount released correlated with the degree of relaxation, and the release was blocked by tetrodotoxin. Prior incubation of the trachea with antiserum to vasoactive intestinal peptide reduced the relaxation. Thus vasoactive intestinal peptide may mediate the nonadrenergic relaxation of tracheal smooth muscle.

Characteristics of light intensity enhancement of a silver nanoprism with rounded corners.

We have fabricated silver nanoprisms of 100-600 nm side length by focussed ion beam lithography and measured the light intensity scattering spectra using dark-field microscopy. Two resonance peaks due to localized surface plasmon excitation were observed in the spectra and their central frequency shown to depend on the prism size. The near-field electromagnetic intensity distribution with TE-polarized light at the vacuum wavelength of 632.8 nm was measured. We have obtained a much lower light intensity enhancement than previously numerically predicated. However, scattering spectra obtained numerically, taking into account roundness of the prism corners, agree well with experimental ones. At the same time, the numerically determined field distribution was different to the near-field intensity obtained experimentally. Our results suggest the particular shape of the corner region of the prism is a key factor for obtaining a large light intensity enhancement and shaping the local field distribution.

Frequent immune response to a melanocyte specific protein KU-MEL-1 in patients with Vogt-Koyanagi-Harada disease.

AIM: To isolate autoantigens possibly involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease. METHODS: Autoantigens recognised by immunoglobulin G antibodies (IgG Ab) in sera from VKH patients were isolated by screening the lambda phage cDNA libraries made from melanocytes and a highly pigmented melanoma cell line with the patients' sera. Presence of IgG specific for the autoantigens in sera from patients with various panuveitis and healthy individuals was evaluated. Relation between the specific IgG and various clinicopathological features was examined. RESULTS: KU-MEL-1 was found to be one of the 81 isolated positive clones representing 35 distinct genes, which is a previously isolated melanoma antigen preferentially expressed in melanocytes. The IgG Ab specific for KU-MEL-1 was detected in sera from patients with VKH in significantly higher amounts than in sera from patients with Behçet's disease, sarcoidosis, and from healthy individuals. Positive serum KU-MEL-1 Ab was significantly associated with HLA-DRB1*0405 and male VKH patients. CONCLUSION: KU-MEL-1 was identified as a new autoantigen for VKH. The highly frequent induction of IgG Ab for KU-MEL-1 in HLA-DRB1*0405 positive VKH patients may suggest the possible involvement of KU-MEL-1 specific CD4(+) T cells in the pathogenesis of VKH, suggesting the possible use in the development of diagnostic and therapeutic treatments for VKH patients.

[Surgical treatment for traumatic tracheobronchial injuries].

Tracheobronchial injury is a relatively rare but often fatal condition due to the injury from the neck to the chest. Different clinical features depend on the site of injury. We experienced 5 cases of tracheobronchial injury; cervical trachea in 2, thoracic trachea in 1, tracheal carina in 1, left main bronchus in 1. Three cases were caused by blunt trauma by traffic accident and 2 cases were due to penetrating injury (stab wound and gunshot wound). Thoracotomy with primary repair for 3 (simple repair, bronchoplasty, pneumonectomy) and cervicotomy for 2 (end-to-end anastomosis) were performed. One patient with severe associated injury died of multiple organ failure after surgery. Accurate diagnosis and the appropriate treatment in the early stage is essential in the treatment of tracheobronchial injury.

[pN0 stage IA lung cancer downstaged from pN2 IIIA by induction therapy; report of a case].

A 75-year-old male of adenocarcinoma in non-small cell lung cancer (NSCLC) was diagnosed to be p-staged IIIA by a preoperative mediastinoscopy. After the induction chemotherapy of cisplatin (80 mg/m2, day 1, 4 weeks, 2 cycles) plus docetaxel hydrate (60 mg/m2, day 1, 4 weeks, 2 cycles), we performed lobectomy and lymph nodes dissection. The postoperative pathological results showed no metastasis of the resected lymph nodes. The postoperative stage has been down to be pN0 IA from pN2 IIIA. After the adjuvant therapy of docetaxel hydrate (60 mg/m2, day 1, 4 weeks, 2 cycles), the postoperative serum carcinoembryonic antigen level has been normalized without a recurrence for more than 2 years. A pathological staging with a mediastinoscopic diagnosis for the suspected clinical IIIA-staged NSCLC should bring us a good assessment of the induction therapy.

[New method for localization of the small ground-glass opacity lesion in resected lung].

A small lesion showing ground-glass opacity (GGO) by preoperative computed tomography (CT) is sometimes difficult to detect after lobectomy when it locates in the central part of the lobe. In order to facilitate to identify the lesion for marking pathological specimen, we developed a new method using CT. After surgery, the resected pulmonary lobe was expanded with airflow through the bronchial stump and the target lesion was examined with CT. The laser beam of the CT on the surface of the lung is used as a guiding line for cutting. Through the application of this method for 2 clinical cases, it was found to be possible to exactly identify the GGO lesion from the surface of the resected lung enabling to visualize a fresh surface of the lesion like a CT image with minimal destruction of the structure.

Optically detected magnetic resonance of high-density ensemble of NV- centers in diamond.

Optically detected magnetic resonance (ODMR) is a way to characterize the ensemble of NV-centers. Recently, a remarkably sharp dip was observed in the ODMR with a high-density ensemble of NV centers. The model (Zhu et al 2014 Nat. Commun. 5 3424) indicated that such a dip was due to the spin-1 properties of the NV- centers. Here, we present many more details of the analysis to show how this model can be applied to investigate the properties of the NV- centers. By using our model, we have reproduced the ODMR with and without applied external magnetic fields. Additionally, we investigate how the ODMR is affected by the typical parameters of the ensemble NV- centers such as strain distributions, inhomogeneous magnetic fields, and homogeneous broadening width. Our model provides a way to characterize the NV- center from the ODMR, which would be crucial to realize diamond-based quantum information processing.

Purified Human Dental Pulp Stem Cells Promote Osteogenic Regeneration.

Human dental pulp stem/progenitor cells (hDPSCs) are attractive candidates for regenerative therapy because they can be easily expanded to generate colony-forming unit-fibroblasts (CFU-Fs) on plastic and the large cell numbers required for transplantation. However, isolation based on adherence to plastic inevitably changes the surface marker expression and biological properties of the cells. Consequently, little is currently known about the original phenotypes of tissue precursor cells that give rise to plastic-adherent CFU-Fs. To better understand the in vivo functions and translational therapeutic potential of hDPSCs and other stem cells, selective cell markers must be identified in the progenitor cells. Here, we identified a dental pulp tissue-specific cell population based on the expression profiles of 2 cell-surface markers LNGFR (CD271) and THY-1 (CD90). Prospectively isolated, dental pulp-derived LNGFR(Low+)THY-1(High+) cells represent a highly enriched population of clonogenic cells--notably, the isolated cells exhibited long-term proliferation and multilineage differentiation potential in vitro. The cells also expressed known mesenchymal cell markers and promoted new bone formation to heal critical-size calvarial defects in vivo. These findings suggest that LNGFR(Low+)THY-1(High+) dental pulp-derived cells provide an excellent source of material for bone regenerative strategies.

Effect of bile acids on the uptake of irinotecan and its active metabolite, SN-38, by intestinal cells.

The intestinal transport of irinotecan (CPT-11) and its active metabolite, SN-38, has been previously reported (K. Kobayashi et al., Int. J. Cancer, 83 (1999) 491-496). In the present study, the effect of the two major primary bile acids, cholic acid (CA) and taurocholic acid (TCA), on the uptake of CPT-11 and SN-38 by hamster intestinal epithelial cells was investigated. These two bile acids at concentrations up to 200 microM did not directly alter the cellular uptake of CPT-11 and SN-38. However, under physiologically acidic intestinal pH conditions, micelle formation induced by 20 mM TCA significantly reduced the cellular uptake of CPT-11 and SN-38 by 60% and 80%, respectively.