Risk factors affecting the failure to complete treatment for patients with latent tuberculosis infection in Tokyo, Japan.

OBJECTIVE: The treatment completion rate for all latent tuberculosis infection (LTBI) patients in Tokyo was 83.6% in 2014: somewhat lower than the targeted goal of 85%. This study examines the association between risk factors and LTBI patients' failure to complete treatment. METHODS: We collected data related to the treatment of LTBI patients who were reported to public health centers in Tokyo from January 2014 through December 2014. Data included potential risk factors affecting treatment, treatment results, and patient characteristics. We used Fisher's exact test to evaluate risk factors affecting failure to complete treatment. The failure rate was defined as the incompletion rate of treatment. RESULTS: Of 1060 notified cases, 877 had completed treatment; 116 had not completed treatment. Of these 116 cases, failure to complete treatment in 52 cases was attributable to side effects of anti-tuberculosis drugs. Reasons other than side effects were given for 64 cases. Another 67 cases could not be followed up. In all, 941 cases were analyzed, excluding cases lost to follow-up and cases for which patients had not completed treatment because of anti-tuberculosis drug side effects. Failure rates among foreign-born patients (26.9%) were significantly much higher than those among Japan-born patients (3.9%). Statistical tests indicated no presumed potential risk factor as significant. However, "no supporter for foreign-born LTBI patients" was marginally significant. CONCLUSION: Only "no supporter for foreign-born LTBI patients" was found to be marginally significant. More data must be accumulated to assess the risk factors affecting LTBI treatment.

Matrix Gla protein maintains normal and malignant hematopoietic progenitor cells by interacting with bone morphogenetic protein-4.

Matrix Gla protein (MGP), a modulator of the BMP-SMAD signals, inhibits arterial calcification in a Glu γ-carboxylation dependent manner but the role of MGP highly expressed in a subset of bone marrow (BM) mesenchymal stem/stromal cells is unknown. Here we provide evidence that MGP might be a niche factor for both normal and malignant myelopoiesis. When mouse BM hematopoietic cells were cocultured with mitomycin C-treated BM stromal cells in the presence of anti-MGP antibody, growth of hematopoietic cells was reduced by half, and maintenance of long-term culture-initiating cells (LTC-ICs) was profoundly attenuated. Antibody-mediated blockage of MGP also inhibited growth (by a fifth) and cobblestone formation (by half) of stroma-dependent MB-1 myeloblastoma cells. MGP was undetectable in normal hematopoietic cells but was expressed in various mesenchymal cells and was aberrantly high in MB-1 cells. MGP and bone morphogenetic protein (BMP)-4 were co-induced in stromal cells cocultured with both normal hematopoietic cells and MB-1 myeloblastoma cells in an oscillating several days-periodic manner. BMP-2 was also induced in stromal cells cocultured with normal hematopoietic cells but was barely expressed when cocultured with MB-1 cells. GST-pulldown and luciferase reporter assays showed that uncarboxylated MGP interacted with BMP-4 and that anti-MGP antibody abolished this interaction. LDN-193189, a selective BMP signaling inhibitor, inhibited growth and cobblestone formation of MB-1 cells. The addition of warfarin, a selective inhibitor of vitamin K-dependent Glu γ-carboxylation, did not affect MB-1 cell growth, suggesting that uncarboxylated MGP has a biological effect in niche. These results indicate that MGP may maintain normal and malignant hematopoietic progenitor cells, possibly by modulating BMP signals independently of Glu γ-carboxylation. Aberrant MGP by leukemic cells and selective induction of BMP-4 relative to BMP-2 in stromal cells might specify malignant niche.

Translin modulates mesenchymal cell proliferation and differentiation in mice.

Translin, a highly conserved DNA/RNA binding protein that forms a hetero-octamer together with Translin-associated factor X (TRAX), possesses a broad variety of functions, including RNA processing and DNA repair. Recent studies have reported that Translin is involved in mesenchymal cell physiology. Thus, here we analyzed the intrinsic role of Translin in mesenchymal cell proliferation and differentiation. Translin-deficient E11.5 mouse embryonic fibroblasts showed enhanced growth. Translin-deficient bone marrow-derived mesenchymal stem cells showed substantial expansion in vivo and enhanced proliferation in vitro. These cells also showed enhanced osteogenic and adipocytic differentiation. Histological analyses showed adipocytic hypertrophy in various adipose tissues. Translin knockout did not affect the growth of subcutaneous white adipose tissue-derived stem cells, but enhanced adipocytic differentiation was observed in vitro. Contrary to previous reports, in vitro-fertilized Translin-null mice were not runted and exhibited normal metabolic homeostasis, indicating the fragility of these mice to environmental conditions. Together, these data suggest that Translin plays an intrinsic role in restricting mesenchymal cell proliferation and differentiation.

Two year field study to evaluate the efficacy of Mamestra brassicae nucleopolyhedrovirus combined with proteins derived from Xestia c-nigrum granulovirus.

Japan has only three registered baculovirus biopesticides despite its long history of studies on insect viruses. High production cost is one of the main hindrances for practical use of baculoviruses. Enhancement of insecticidal effect is one possible way to overcome this problem, so there have been many attempts to develop additives for baculoviruses. We found that alkaline soluble proteins of capsules (GVPs) of Xestia c-nigrum granulovirus can increase infectivity of some viruses including Mamestra brassicae nucleopolyhedrovirus (MabrNPV), and previously reported that MabrNPV mixed with GVPs was highly infectious to three important noctuid pests of vegetables in the following order, Helicoverpa armigera, M. brassicae, and Autographa nigrisigna. In this study, small-plot experiments were performed to assess concentrations of MabrNPV and GVPs at three cabbage fields and a broccoli field for the control of M. brassicae. In the first experiment, addition of GVPs (10 µg/mL) to MabrNPV at 106 OBs/mL resulted in a significant increase in NPV infection (from 53% to 66%). In the second experiment, the enhancing effect of GVP on NPV infection was confirmed at 10-times lower concentrations of MabrNPV. In the third and fourth experiments, a 50% reduction in GVPs (from 10 µg/mL to 5 µg/mL) did not result in a lowering of infectivity of the formulations containing MabrNPV at 105 OBs/mL. These results indicate that GVPs are promising additives for virus insecticides.

Re-Evaluation of the PBAN Receptor Molecule: Characterization of PBANR Variants Expressed in the Pheromone Glands of Moths.

Sex pheromone production in most moths is initiated following pheromone biosynthesis activating neuropeptide receptor (PBANR) activation. PBANR was initially cloned from pheromone glands (PGs) of Helicoverpa zea and Bombyx mori. The B. mori PBANR is characterized by a relatively long C-terminus that is essential for ligand-induced internalization, whereas the H. zea PBANR has a shorter C-terminus that lacks features present in the B. mori PBANR critical for internalization. Multiple PBANRs have been reported to be concurrently expressed in the larval CNS of Heliothis virescens. In the current study, we sought to examine the prevalence of multiple PBANRs in the PGs of three moths and to ascertain their potential functional relevance. Multiple PBANR variants (As, A, B, and C) were cloned from the PGs of all species examined with PBANR-C the most highly expressed. Alternative splicing of the C-terminal coding sequence of the PBAN gene gives rise to the variants, which are distinguishable only by the length and composition of their respective C-terminal tails. Transient expression of fluorescent PBANR chimeras in insect cells revealed that PBANR-B and PBANR-C localized exclusively to the cell surface while PBANR-As and PBANR-A exhibited varying degrees of cytosolic localization. Similarly, only the PBANR-B and PBANR-C variants underwent ligand-induced internalization. Taken together, our results suggest that PBANR-C is the principal receptor molecule involved in PBAN signaling regardless of moth species. The high GC content of the C-terminal coding sequence in the B and C variants, which makes amplification using conventional polymerases difficult, likely accounts for previous "preferential" amplification of PBANR-A like receptors from other species.

Mamestra brassicae nucleopolyhedrovirus infection and enhancing effect of proteins derived from Xestia c-nigrum granulovirus in larvae of Mamestra brassicae and Helicoverpa armigera (Lepidoptera: Noctuidae) on cabbage.

The insecticidal effect of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) T5 against Mamestra brassicae (L.) and Helicoverpa armnigera (Hiibner) (Lepidoptera: Noctuidae), important pests of various vegetables and ornamental crops in Japan as well as many other countries, and the enhancing activity of proteins derived from occlusion bodies of Xestia c-nigrum granulovirus (XecnGV) a-4, which was named GVPs, on the infectivity of MabrNPV were evaluated in a bioassay with second-instar larvae fed on virus-applied cabbage, Brassica oleracea L. var. capitata, plants. The lethal concentrations of MabrNPV achieving 95% mortality (LC95) were estimated to be 7.7 x 10(5) and 1.8 x 10(5) OBs per ml for M. brassicae and H. armigera, respectively, with MabrNPV-alone treatment. Because the mean areas of cabbage leaf consumed by one larva in 60 h were not significantly different between M. brassicae and H. armigera, we conclude that MabrNPV is more infectious to H. armigera than to M. brassicae. When larvae were fed on cabbage plants treated with 10(4) OBs per ml MabrNPV and various concentrations of the GVPs, the mortality of the two noctuid larvae increased in relation to GVP concentration. The requisite concentrations of the GVPs achieving 95% mortality with the MabrNPV for M. brassicae and H. armigera were estimated to be 5.93-8.30 and 1.94-3.48 microg/ml, respectively. In a comparison of the MabrNPV-alone treatment with equivalent 95% mortality, addition of GVPs increased the rate of larval death at younger instars, especially in M. brassicae. Our results indicate that GVPs are a potentially useful additive for improving the insecticidal efficacy of MabrNPV.

Comparative studies of lepidopteran baculovirus-specific protein FP25K: development of a novel Bombyx mori nucleopolyhedrovirus-based vector with a modified fp25K gene.

Lepidopteran baculovirus-specific protein FP25K performs many roles during the infection cycle, including functions in the production of occlusion bodies (OBs) and budded viruses (BVs), oral infection, and postmortem host degradation. To explore the common and specific functions of FP25K proteins among lepidopteran baculoviruses, we performed comparative analyses of FP25K proteins from group I and group II nucleopolyhedroviruses (NPVs) and granulovirus (GV). Using recombinant Bombyx mori NPVs (BmNPVs), we showed that the FP25Ks from NPVs were able to eliminate all the phenotypic defects observed in an infection with a BmNPV mutant lacking functional fp25K but that FP25K from GV did not show abilities to recover oral infectivity and postmortem host degradation. We also observed that introduction of Autographa californica multiple NPV (AcMNPV) fp25K into the BmNPV genome enhanced OB and BV production. According to these results, we generated a novel BmNPV-based expression vector with AcMNPV fp25K and examined its potential in BmN cells and B. mori larvae. Our results showed that the introduction of AcMNPV fp25K significantly increases the expression of foreign gene products in cultured cells and shortens the time for obtaining the secreted recombinant proteins from larval hemolymph.

In vivo characterization of two granuloviruses in larvae of Mythimna separata (Lepidoptera: Noctuidae).

The pathogenicity of two granuloviruses (GVs), Xestia c-nigrum GV (XecnGV) and Pseudaletia unipuncta GV (PsunGV), was examined in Mythimna separata. Partial sequencing of the genome of PsunGV indicated that it is related closely to XecnGV, but considered to be a different species. PsunGV and XecnGV showed similar pathogenicity in terms of dose-mortality response and pattern of host mass changes following infection. Both GVs killed infected larvae in 2-3 weeks. Temporal changes in the concentrations of GV-specific DNA in the larval haemolymph were measured by using a real-time quantitative PCR. Viral DNA concentration increased quickly and reached a plateau at 60-72 h post-inoculation. Rates of budded virus (BV) production of each GV were estimated on the basis of viral DNA concentrations by a modified Gompertz model. The slopes of the estimated BV growth curves of both XecnGV and PsunGV in M. separata larvae were equivalent to that of Mamestra brassicae nucleopolyhedrovirus (NPV) in its original host, reported in our previous study. This suggested that BV production is not a major factor in the slower killing speed of GVs in comparison to NPVs. The GV-infected larvae survived for an additional 10 days or more after reaching a maximum level of BV concentration, and kept growing without pupation. These findings also suggested that the GVs have a unique mechanism to regulate the growth of host larvae.

Cryptic fragment alpha4 LG4-5 derived from laminin alpha4 chain inhibits de novo adipogenesis by modulating the effect of fibroblast growth factor-2.

Cleavage of the extracellular matrix (ECM) by proteolysis unmasks cryptic sites and generates novel fragments with biological activities functionally distinct from those of the intact ECM molecule. The laminin G-like (LG)4-5 fragment has been shown to be excised from the laminin alpha4 chain in various tissues. However, the functional role of this fragment has remained unknown to date. To investigate this, we prepared alpha4 LG1-3 and alpha4 LG4-5 fragments by elastase digestion of recombinant alpha4 LG1-5, and examined their effects on de novo adipogenesis in mice at the site of injection of basement membrane extract (Matrigel) and fibroblast growth factor (FGF)-2. Although the addition of whole alpha4 LG1-5 suppressed adipogenesis to some extent, the alpha4 LG4-5 fragment could strongly suppress adipogenesis at a concentration of less than 20 nm. Addition of the alpha4 LG4 module, which contains a heparin-binding region, had a suppressive effect, but this was lost in mutants with reduced heparin-binding activity. In addition, antibodies against the extracellular domain of syndecan-2 and -4, which are known receptors for the alpha4 LG4 module, suppressed adipogenesis. Thus, these results suggest that the cryptic alpha4 LG4-5 fragment derived from the laminin alpha4 chain inhibits de novo adipogenesis by modulating the effect of FGF-2 through syndecans.

Enhancement of nucleopolyhedrovirus infectivity against Mamestra brassicae (Lepidoptera: Noctuidae) by proteins derived from granulovirus and a fluorescent brightener.

The synergistic enhancement of nucleopolyhedrovirus (NPV) infection by granuloviruses (GVs) is well documented; and a GV granule protein, named viral enhancin, has been identified as an active contributor to this effect. We detected the presence of two proteins with molecular mass of 93 and 108 kDa in granules of a GV isolated from Xestia c-nigrum (L.) (XecnGV) as candidates for enhancin, and we confirmed that at least the 108-kDa protein enhances the infectivity of Mamestra brassicae nucleopolyhedrovirus (MabrNPV). We tested the effect of virion-free proteins obtained from XecnGV granules (GVPs) on MabrNPV infection, and we made a comparison with an enhancing chemical, the stilbene-derived fluorescent brightener Tinopal. Bioassay was performed employing the diet contamination method, by using second instars of Mamestra brassicae (L.) (Lepidoptera: Noctuidae). The enhancing effects of GVPs (0.1 mg/g diet) and Tinopal (1 mg/g diet) were estimated to be 70.7-81.5-fold and 26.9-33.7-fold, respectively, as calculated from the LC50 values of MabrNPV with or without the additives. The additives reduced the lethal time of MabrNPV-infected larvae and they caused death at a younger instar. These results suggest that GVPs can enhance MabrNPV infection as effectively as Tinopal.

In vivo characterization of a group II nucleopolyhedrovirus isolated from Mamestra brassicae (Lepidoptera: Noctuidae) in Japan.

A Japanese isolate of Mamestra brassicae nucleopolyhedrovirus (MabrNPV) was identified phylogenetically as a group II nucleopolyhedrovirus (NPV) that is related closely to other NPVs isolated from Mamestra spp. based on nucleotide sequence data of its polh, egt and lef-3 genes. The multiplication of MabrNPV in M. brassicae larvae was characterized following inoculation at various doses and in combination with the fluorescent brightener Tinopal by measuring temporal changes in the concentrations of its viral DNA using real-time quantitative PCR. The growth curves of budded-virus replication were analysed by fitting the data of viral DNA concentration in the host haemolymph to a modified Gompertz model. When fifth-instar larvae were inoculated with an LD95 equivalent dose of MabrNPV and Tinopal, the time lag between the onset of primary and secondary infection was estimated to be 25 h. Another 65 h was required to reach a plateau titre equivalent to a level of 10(9) virions ml(-1) in the haemolymph. All larvae died during the sixth instar following this inoculation regime. In contrast, following inoculation with a 1000-fold higher dose of MabrNPV and Tinopal, the time lag between the onset of primary and secondary infection was only 20 h. Subsequently, the same plateau titre was reached after a further 20 h. Following this inoculation regime, most larvae died during the fifth instar. Quantification of viral DNA by real-time quantitative PCR and application of the Gompertz model are valuable for the characterization of baculovirus replication in vivo.

Genome sequence and organization of a nucleopolyhedrovirus isolated from the smaller tea tortrix, Adoxophyes honmai.

Adoxophyes honmai nucleopolyhedrovirus (AdhoNPV) has a distinctive pathology in A. honmai larvae, killing the host more slowly than other NPVs. To further understand the pathology of AdhoNPV, its genome was completely sequenced and compared with those of other baculoviruses. The AdhoNPV genome is 113,220 bp, with a G + C content of 35.6%. It contains 125 putative open reading frames (ORFs), of which 8 are unique to AdhoNPV, and 4 homologous regions. The other 117 ORFs display similarity to previously characterized baculovirus genes involved in early and late gene expression, DNA replication, and structural and auxiliary functions. The phylogenetic position of AdhoNPV, in relation to 15 other baculoviruses whose genomes have been completely sequenced, was assessed by three different analyses: gene sequence, gene order, and gene content. Although gene content analysis failed to support the group II NPVs, phylogenetic trees based on gene sequence and gene order showed AdhoNPV to be closely related to the group II NPVs.