Hirotake Kasai

Microglia release ATP by exocytosis.

Microglia survey the brain environment by sensing several types of diffusible molecules, among which extracellular nucleotides released/leaked from damaged cells have central roles. Microglia sense ATP or other nucleotides by multiple P2 receptors, after which they change into several different phenotypes. However, so far, it is largely unknown whether microglia themselves release ATP and, if so, by what mechanism. Here we show that exocytosis is the mechanism by which microglia release ATP. When we stimulated microglia with ionomycin, they released ATP and the release was dependent on Ca2+, vesicular H+‐ATPase, or SNAREs but independent of connexin/pannexin hemichannels. VNUT was found to be expressed in microglia and exhibited no colocalization with lysosome. We also visualized the exocytosis of ATP by a quinacrine‐based fluorescent time‐lapse imaging. Moreover, we found that lipopolysaccharide increased the ionomycin‐induced release of ATP, which was dependent on the increase in VNUT. Taken together, our data suggested that exocytosis is the mechanism of ATP release from microglia. When activated, they would release ATP by increasing VNUT‐dependent exocytotic mechanisms. GLIA 2013;61:1320–1330

Glycyrrhizin enhances interleukin-12 production in peritoneal macrophages

Interleukin‐12 (IL‐12) is a monocyte/macrophage‐derived cytokine that plays a prominent role in the development of T helper type 1 (Th1) cell‐mediated immune responses. Glycyrrhizin (GL), an aqueous extract of liquorice root, used as Chinese medicine, is known to have various immunomodulating activities. In this study, GL showed a dose‐dependent priming effect on lipopolysaccharide (LPS)‐induced IL‐12 p40 and IL‐12 p70 (heterodimer of p40 and p35) protein production by peritoneal macrophages (PM). The maximal effect was observed when GL was intraperitoneally administered 12 hr before the PM were harvested and stimulated in vitro with LPS. The increases in IL‐12 p70 and p40 protein production were primarily due to up‐regulated transcription of IL‐12 p35 and p40 messenger RNAs (mRNAs), as demonstrated by RNase protection assay. The augmentation of IL‐12 p40 mRNA expression induced by GL pretreatment was associated with increased NF‐κB activation. Moreover, GL exhibited the same priming effect on IL‐12 production in interferon‐γ knockout (IFN‐γ–/–) mice. The production of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was not induced at any time point after GL pretreatment. These findings demonstrated the ability of GL to enhance LPS‐induced IL‐12 production by peritoneal macrophages, and indicated that the priming effect of GL on IL‐12 production was independent of both IFN‐γ and GM‐CSF.

IL-12 Production Induced by Agaricus blazei Fraction H (ABH) Involves Toll-like Receptor (TLR)

Agaricus blazei Murill is an edible fungus used in traditional medicine, which has various well-documented medicinal properties. In the present study, we investigated the effects of hemicellulase-derived mycelia extract (Agaricus blazei fraction H: ABH) on the immune system. First, we examined the cytokine-inducing activity of ABH on human peripheral mononuclear cells (PBMC). The results indicated that ABH induced expression of IL-12, a cytokine known to be a critical regulator of cellular immune responses. Flow cytometric analysis demonstrated the induction of IL-12 production by the CD14-positive cell population, consisting of monocytes/macrophages (Mo/Mφ). Furthermore, the elimination of Mo/Mφ attenuated IL-12 production in PBMC. ABH-induced IL-12 production was inhibited by anti-CD14 and anti-TLR4 antibodies but not by anti-TLR2 antibody. The activity of ABH was not inhibited by polymyxin B, while the activity of lipopolysaccharide used as a reference was inhibited. Oral administration of ABH enhanced natural killer (NK) activity in the spleen. These findings suggest that ABH activated Mo/Mφ in a manner dependent on CD14/TLR4 and NK activity.

Prevalence of drug-resistance-associated mutations in antiretroviral drug-naive Zambians infected with subtype C HIV-1.

The ability of HIV-1 to evolve resistance to antiretroviral drugs leads to treatment failure. By nucleotide sequencing of HIV-1 subtype B isolates, amino acids responsible for drug resistance have been identified. Less information is available, however, on the extent and distribution of these amino acids in HIV-1 nonsubtype B viruses circulating mainly in developing countries. More HIV-infected patients in the developing world are now using antiretroviral drugs, and hence there is a need to monitor drug resistance mutations in HIV-1 non-subtype B viruses. This study examines the prevalence of drug resistance mutations in 28 antiretroviral drug-naive HIV-1-infected Zambians. HIV-1 proviral DNA was extracted from peripheral blood mononuclear cells. The region encompassing gag p17 to env C2-V3-C3 was amplified by the polymerase chain reaction followed by direct sequencing. Sequence analyses for drug resistance-associated mutations in th e protease and reverse transcriptase genes, and HIV-1 subtyping, were done. Overall, 92.8% of the generated sequences were HIV-1 subtype C. The generated sequences revealed only secondary associated, but no primary, drug-resistance mutations The most frequent secondary mutations in the protease and RT genes were, respectively, I93L(91.7%), L89M (79.2%), M3611V (79%, 4.2%), and R211K (70.8%), S48T (62.5%). The atypical residues M41N (3.6%) and D67A (3.6%) were detected in the RT gene. This study reveals many naturally occurring polymorphisms in HIV-1 subtype C isolates from antiretroviral drug-naive individuals. Such polymorphisms could lead to rapid treatment failure and development of drug-resistant HIV-1 mutants in individuals undergoing antiretroviral therapy.

Hallmarks of Hepatitis C Virus in Equine Hepacivirus

ABSTRACT Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3′ untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3′ UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile190 and Phe191 of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV. IMPORTANCE EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.

Antithetical effects of hemicellulase-treated Agaricus blazei on the maturation of murine bone-marrow-derived dendritic cells

We report the effects of hemicellulase‐treated Agaricus blazei (ABH) on the maturation of bone‐marrow‐derived dendritic cells (BMDCs). ABH activated immature BMDCs, inducing up‐regulation of surface molecules, such as CD40, CD80 and major histocompatibility complex class I antigens, as well as inducing allogeneic T‐cell proliferation and T helper type 1 cell development. However, unlike lipopolysaccharide (LPS), ABH did not stimulate the BMDCs to produce proinflammatory cytokines, such as interleukin‐12 (IL‐12) p40, tumour necrosis factor‐α, or IL‐1β. In addition, ABH suppressed LPS‐induced DC responses. Pretreatment of DCs with ABH markedly reduced the levels of LPS‐induced cytokine secretion, while only slightly decreasing up‐regulation of the surface molecules involved in maturation. ABH also had a significant impact on peptidoglycan‐induced or CpG oligodeoxynucleotide‐induced IL‐12p40 production in DCs. The inhibition of LPS‐induced responses was not associated with a cytotoxic effect of ABH nor with an anti‐inflammatory effect of IL‐10. However, ABH decreased NF‐κB‐induced reporter gene expression in LPS‐stimulated J774.1 cells. Interestingly, DCs preincubated with ABH and then stimulated with LPS augmented T helper type 1 responses in culture with allogeneic T cells as compared to LPS‐stimulated but non‐ABH‐pretreated DCs. These observations suggest that ABH regulates DC‐mediated responses.

Increased expression of macrophage colony-stimulating factor in ankylosing spondylitis and rheumatoid arthritis

Increasing evidence suggest that macrophage colony-stimulating factor (M-CSF) plays a part in rheumatoid arthritis, but few studies have focused on its role in ankylosing spondylitis. This study aimed at investigating M-CSF expression and its clinical significance in active ankylosing spondylitis and rheumatoid arthritis. Sandwich ELISA was used to measure serum M-CSF levels in 12 active patients with ankylosing spondylitis (Bath Ankylosing Spondylitis Disease Activity Index of ⩾3)1 and 12 active patients with rheumatoid arthritis.2 Eleven serum samples of healthy volunteers were examined as controls. We also examined M-CSF mRNA expression in peripheral blood mononuclear cells (PBMCs) using real-time reverse transcription-polymerase chain reaction. The relative fold increase in M-CSF mRNA expression in patient groups was determined by 2−ΔΔC.3 Considering that tumour necrosis factor (TNF) α and interleukin (IL) 1α have the ability to induce M-CSF,4 we also investigated their levels and correlations with M-CSF. …

Delayed Cell Cycle Progression and Apoptosis Induced by Hemicellulase-Treated Agaricus blazei

We examined the effects of hemicellulase-treated Agaricus blazei (AB fraction H, ABH) on growth of several tumor cell lines. ABH inhibited the proliferation of some cell lines without cytotoxic effects. It markedly prolonged the S phase of the cell cycle. ABH also induced mitochondria-mediated apoptosis in different cell lines. However, it had no impact on the growth of other cell lines. ABH induced strong activation of p38 mitogen-activated protein kinase (MAPK) in the cells in which it evoked apoptosis. On the other hand, ABH showed only a weak p38 activation effect in those cell lines in which it delayed cell cycle progression with little induction of apoptosis. However, p38 MAPK-specific inhibitor inhibited both ABH-induced effects, and ABH also caused apoptosis in the latter cells under conditions of high p38 MAPK activity induced by combined treatment with TNF-α. These results indicate that the responsiveness of p38 MAPK to ABH, which differs between cell lines, determines subsequent cellular responses on cell growth.

Long-term imipramine treatment increases N-methyl-D-aspartate receptor activity and expression via epigenetic mechanisms

Imipramine, a major antidepressant, is known to inhibit reuptake of serotonin and norepinephrine, which contributes to recovery from major depressive disorder. It has recently been reported that acute imipramine treatment inhibits N-methyl-d-aspartate (NMDA) receptor activity. However, the mechanisms underlying long-term effects of imipramine have not been identified. We tested these distinct effects in mouse cortical neurons and found that acute (30s) imipramine treatment decreased Ca(2+) influx through NMDA receptors, whereas long-term treatment (48h) increased Ca(2+) influx via the same receptors. Furthermore, long-term treatment increased NMDA receptor 2B (NR2B) subunit expression via epigenetic changes, including increased acetylation of histones H3K9 and H3K27 in the NR2B promoter and decreased activity of histone deacetylase 3 (HDAC3) and HDAC4. These results suggest that the long-term effects of imipramine on NMDA receptors are quite different from its acute effects. Furthermore, increased NR2B expression via epigenetic alterations might be a part of the mechanism responsible for this long-term effect.

Emergence of new HIV-1 subtypes other than Subtype C among antenatal women in Lusaka, Zambia.

Molecular epidemiology of HIV-1 in Zambia was investigated by direct sequencing of PCR products from samples collected from antenatal attendees in Lusaka, Zambia. One hundred and forty samples were initially screened for HIV, using antibody assays. Thirty-three (23.6%) samples were HIV-1 positive. Sequences of the HIV-1 env gp120 region were obtained from 28 of 33 (85%) HIV-1-positive samples. Twenty-six of the 28 sequences were HIV-1 env subtype C-like as previously reported. However, one HIV-1 env subtype D-like virus and one HIV-1 env subtype G-like virus were identified. This is the first time that these two HIV-1 env subtype viruses have been identified in Zambia, suggesting that more subtypes could be in existence.