Publications by authors named "Yosuke Maeda"

43 Publications

Influence of Tooth Mold Materials in Composite Resin CAD/CAM Crown Destructive Test.

Bull Tokyo Dent Coll 2021 Mar 15;62(1):15-26. Epub 2021 Feb 15.

Department of Fixed Prosthodontics, Tokyo Dental College.

A range of experimental designs have been used in destructive testing of composite resin CAD/CAM crowns. Various materials have been adopted for the abutment in such tests, including human or bovine dentin, stainless steel, PMMA, and composite resin, the selection of which is made in accordance with study objective or preference of the researcher. The purpose of this study was to determine how the material selected for the abutment material affected fracture load and maximum displacement. Destructive tests were conducted on composite resin crowns of the same design. Three types of material were used for the abutments together with 2 types of adhesive material. Images of each sample were acquired before destruction using a microfocus X-ray CT scanner to confirm the feasibility of a non-destructive test.The load required to fracture the composite CAD/CAM resin crowns depended on the abutment material used, with a decrease being observed in the order of composite resin, stainless steel, and PMMA. Maximum displacement decreased in the order of PMMA, composite resin, and stainless steel. Differences in the material used for setting (adhesive resin or polycarboxylate cement) showed no effect on fracture load. These results indicate that the load required to achieve destruction of resin CAD/CAM crowns varies according to the abutment material used.
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http://dx.doi.org/10.2209/tdcpublication.2020-0028DOI Listing
March 2021

Plasma Levels of a Cleaved Form of Galectin-9 Are the Most Sensitive Biomarkers of Acquired Immune Deficiency Syndrome and Tuberculosis Coinfection.

Biomolecules 2020 10 30;10(11). Epub 2020 Oct 30.

Department of Health Science and Social Welfare, Kibi International University, Takahashi 716-8508, Japan.

Acquired immunodeficiency syndrome (AIDS) complicated with tuberculosis (TB) is a global public issue. Due to the paucity of bacteria in AIDS/TB, blood-based biomarkers that reflect disease severity are desired. Plasma levels of matricellular proteins, such as osteopontin (OPN) and galectin-9 (Gal-9), are known to be elevated in AIDS and TB. Therefore, full-length (FL)-Gal9 and FL-OPN, and their truncated forms (Tr-Gal9, Ud-OPN), and 38 cytokines/chemokines were measured in the plasma of 24 AIDS (other than TB), 49 TB, and 33 AIDS/TB patients. Receiver-operating characteristic analysis was used to screen molecules that could distinguish either between disease and normal group, among each disease group, or between deceased patients and survivors. Selected molecules were further analyzed for significant differences. Tr-Gal9 had the highest ability to differentiate TB from AIDS or AIDS/TB, while Ud-OPN distinguished multidrug resistance (MDR)-TB from non-MDR TB, and extra-pulmonary TB from pulmonary TB. Molecules significantly elevated in deceased patients included; FL-Gal9, Tr-Gal9, interleukin (IL)-1 receptor antagonist, IL-17A and transforming growth factor-α in AIDS; IL-6, granulocyte colony-stimulating factor and monocyte chemotactic protein-1 in TB; and macrophage inflammatory protein-1β in AIDS/TB. From the sensitivity, specificity, and significant elevation, Tr-Gal9 is the best biomarker of inflammation and severity in AIDS and AIDS/TB.
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http://dx.doi.org/10.3390/biom10111495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7693693PMC
October 2020

Development of a new photosafety test method based on singlet oxygen generation detected using electron spin resonance.

J Appl Toxicol 2021 Feb 15;41(2):247-255. Epub 2020 Jul 15.

Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Saitama, Japan.

Photosafety evaluations of chemicals used in consumer products, such as pharmaceuticals and cosmetics, are very important. Currently, two non-animal tests for photosafety evaluations, the in vitro 3T3 neutral red uptake phototoxicity test (NRU PT) and the reactive oxygen species (ROS) assay, are used to detect photoreactive chemicals. However, these two tests are difficult to apply to hydrophobic chemicals. In the present study, we attempted to develop a new photosafety test method, named the electron spin resonance-based photosafety test (ESR-PT), that would be applicable even to hydrophobic chemicals based on the detection of singlet oxygen generation after irradiation using ESR spectroscopy with 4-hydroxy-2,2,6,6-tetramethyl-piperidine as a spin trap reagent. To achieve a quantitative evaluation, the singlet oxygen formation (SOF) value, which can be calculated as the increment in relative intensity after irradiation of the test mixture normalized by the increment in relative intensity after irradiation of the vehicle control solution, was calculated. The performance of the ESR-PT was evaluated by testing all the proficiency chemicals of the ROS assay plus additional chemicals, including hydrophobic chemicals and chemicals that tested false negative in the 3T3-NRU PT and ROS assay. SOF values were successfully calculated for all the chemicals tested including the hydrophobic chemicals, and the accuracy of the ESR-PT using a tentative cutoff value of 2.8 against the photosafety information was 100%. Therefore, the SOF value could be an effective parameter for photosafety evaluations, suggesting that the newly developed ESR-PT is a promising non-animal test applicable even to hydrophobic chemicals.
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http://dx.doi.org/10.1002/jat.4040DOI Listing
February 2021

Applicability of the proposed GHS subcategorization criterion for LLNA:BrdU-ELISA (OECD TG442B) to the CBA/J strain mouse.

J Appl Toxicol 2020 10 5;40(10):1435-1439. Epub 2020 May 5.

Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Shimotakano, Sugito-machi, Kitakatsushika-gun, Saitama, Japan.

The Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is a hazard classification and communication system for providing information on the safe handling of chemicals worldwide. In this study, we evaluated the applicability of the newly proposed GHS subcategorization criterion for murine local lymph node assay:2-bromodeoxyuridine enzyme-linked immunosorbent assay (LLNA:BrdU-ELISA), Category 1A:EC1.6 ≤6%, Category 1B:EC1.6 >6%, to data derived from LLNA:BrdU-ELISA performed in the CBA/J strain mouse. Fifteen chemicals categorized in GHS hazard Category 1 sensitizers listed in the LLNA performance standard were tested by LLNA:BrdU-ELISA in the CBA/J strain mouse and were classified according to the new criterion. The results revealed that all of the GHS 1A or 1B category chemicals classified according to the EC3 values derived from radioisotopic LLNA (LLNA-RI) could be correctly assigned into the respective 1A and 1B categories using the newly proposed GHS subclassification criterion. In addition, analysis of the correlation between the reported EC3 values and EC1.6 values derived from the LLNA:BrdU-ELISA performed in the CBA/J strain mouse confirmed the existence of a strong correlation (r = 0.9076, P < .0001). These findings suggest that the newly proposed GHS subcategorization criterion for LLNA:BrdU-ELISA is potentially applicable for practical use in GHS subcategorization.
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http://dx.doi.org/10.1002/jat.3996DOI Listing
October 2020

α-Sens: The improved ARE-Nrf2-based sensitization screening assay using normalized transcriptional activity.

Toxicology 2020 06 23;439:152476. Epub 2020 Apr 23.

The United Graduate School of Veterinary Science, Yamaguchi University, Japan.

Two non-animal test methods, KeratinoSens™ and LuSens, have been approved by the Organization of Economic Cooperation and Development (OECD) test guidelines for evaluating the sensitization potential of chemicals, and been positioned as a method for appraising key event (KE)-2, namely, the keratinocyte response component of the Adverse Outcome Pathway (AOP) in sensitization process. However, these two methods require separate cytotoxicity tests to determine the concentrations to be tested in the main test. Therefore, we developed a simple and highly accurate KE-2 test method named α-Sens that uses the dual luciferase assay system and attempted a further application of luciferase-based determination of cell viability to calculate the normalized Antioxidant response element (ARE)-mediated transcriptional activity, named normalized ARE Activity (nAA), to evaluate the sensitizing potential of chemicals. A cell line carrying the ARE-inducible Firefly luciferase reporter gene and Thymidine kinase (TK) promoter-driven Renilla luciferase gene was established and used for the α-Sens. A total of 28 chemicals, consisting of 19 skin sensitizers and nine non-skin sensitizers were tested by this assay system. The α-Sens yielded an accuracy (%), sensitivity (%), and specificity (%) against corresponding values for local lymph node assay of 96.4 %, 95.0 %, and 100 %, respectively, and for human data of 100 % for all. The α-Sens gave clear positive results for phenyl benzoate and eugenol, chemicals for which KeratinoSens™ or LuSens yielded false-negative results, using a new parameter. Our results suggest that better prediction capacity could be achieved by using nAA as a classifier compared to other existing KE-2 test methods. In conclusion, the α-Sens is promising as a simple and highly accurate in vitro skin sensitization test method for evaluation of KE-2.
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http://dx.doi.org/10.1016/j.tox.2020.152476DOI Listing
June 2020

Serotonin Syndrome Developing Immediately after the Initiation of Low-Dose Methadone Therapy: A Case Report.

Case Rep Oncol 2020 Jan-Apr;13(1):281-284. Epub 2020 Mar 24.

Department of Pharmacy, Hokkaido University Hospital, Sapporo, Japan.

We present a case in which serotonin syndrome developed immediately after the initiation of low-dose methadone following an increase in oxycodone dose and the initiation of duloxetine. The symptoms of serotonin syndrome were alleviated and later disappeared upon cessation of methadone alone. The case was a 47-year-old woman with a desmoid tumor. The administration of duloxetine (20 mg/day) was initiated while the patient took oxycodone sustained-release tablets (40 mg/day). The following day, excessive perspiration, chills, and tremors appeared after the initiation of 15 mg/day methadone. Discontinuation of methadone led to an alleviation of the symptoms which completely disappeared 3 days later. The results suggest that low-dose methadone can trigger serotonin syndrome as early as after the first dose. Thus, it is important to be aware of the risks and to immediately take action if symptoms appear.
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http://dx.doi.org/10.1159/000506443DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7154242PMC
March 2020

Existence of Replication-Competent Minor Variants with Different Coreceptor Usage in Plasma from HIV-1-Infected Individuals.

J Virol 2020 06 1;94(12). Epub 2020 Jun 1.

Center for AIDS Research, Kumamoto University, Kumamoto, Japan.

Cell entry by HIV-1 is mediated by its principal receptor, CD4, and a coreceptor, either CCR5 or CXCR4, with viral envelope glycoprotein gp120. Generally, CCR5-using HIV-1 variants, called R5, predominate over most of the course of infection, while CXCR4-using HIV-1 variants (variants that utilize both CCR5 and CXCR4 [R5X4, or dual] or CXCR4 alone [X4]) emerge at late-stage infection in half of HIV-1-infected individuals and are associated with disease progression. Although X4 variants also appear during acute-phase infection in some cases, these variants apparently fall to undetectable levels thereafter. In this study, replication-competent X4 variants were isolated from plasma of drug treatment-naive individuals infected with HIV-1 strain CRF01_AE, which dominantly carries viral RNA (vRNA) of R5 variants. Next-generation sequencing (NGS) confirmed that sequences of X4 variants were indeed present in plasma vRNA from these individuals as a minor population. On the other hand, in one individual with a mixed infection in which X4 variants were dominant, only R5 replication-competent variants were isolated from plasma. These results indicate the existence of replication-competent variants with different coreceptor usage as minor populations. The coreceptor switch of HIV-1 from R5 to CXCR4-using variants (R5X4 or X4) has been observed in about half of HIV-1-infected individuals at late-stage infection with loss of CD4 cell count and disease progression. However, the mechanisms that underlie the emergence of CXCR4-using variants at this stage are unclear. In the present study, CXCR4-using X4 variants were isolated from plasma samples of HIV-1-infected individuals that dominantly carried vRNA of R5 variants. The sequences of the X4 variants were detected as a minor population using next-generation sequencing. Taken together, CXCR4-using variants at late-stage infection are likely to emerge when replication-competent CXCR4-using variants are maintained as a minor population during the course of infection. The present study may support the hypothesis that R5-to-X4 switching is mediated by the expansion of preexisting X4 variants in some cases.
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http://dx.doi.org/10.1128/JVI.00193-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7307085PMC
June 2020

Incidence of postoperative nausea and vomiting is not increased by combination of low concentration sevoflurane and propofol compared with propofol alone in patients undergoing laparoscopic gynecological surgery.

JA Clin Rep 2019 Nov 2;5(1):70. Epub 2019 Nov 2.

Department of Anesthesiology, Hakodate Central General Hospital, 3-2, Honcho3, Hakodate, 040-8585, Japan.

Background: The incidence of postoperative nausea and vomiting (PONV) is higher in patients receiving volatile anesthetics than those receiving total intravenous anesthesia (TIVA) with propofol. However, it is unclear whether its incidence is increased when a low concentration of sevoflurane is used in combination with propofol.

Methods: This prospective, randomized, controlled trial enrolled women undergoing laparoscopic gynecological surgery. Patients were randomly assigned to receive general anesthesia either with propofol alone (group P) or with 0.8% sevoflurane and propofol (group SP, n = 36, each group) for maintenance of anesthesia. The incidence of PONV and the number of patients who required antiemetics were compared.

Results: There were no differences in the incidence of PONV and the number of patients who required antiemetics between the P and SP groups.

Conclusions: A combination of 0.8% sevoflurane and propofol to maintain anesthesia does not increase the incidence of PONV compared with TIVA with propofol.

Trial Registration: UMIN-CTR UMIN000023647 , registered 14 August 2016.
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http://dx.doi.org/10.1186/s40981-019-0292-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6966744PMC
November 2019

Proposal of GHS sub-categorization criteria for LLNA: BrdU-ELISA (OECD TG442B).

Regul Toxicol Pharmacol 2019 Oct 18;107:104409. Epub 2019 Jun 18.

Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute (CERI), 1600, Shimotakano, Sugito-machi, Kitakatsushika-gun, Saitama, 3450043, Japan.

The Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is a hazard classification and communication system for providing information on the safe handling of chemicals worldwide. While the GHS provides sub-categorization criteria for sensitizers when using the guinea pig maximization test/Buehler test (OECD TG406) and the standard radioisotopic LLNA (OECD TG429), the sub-categorization criteria for LLNA: BrdU-ELISA (OECD TG442B) are not currently provided. In this study, we re-analyzed the existing data of 32 sensitizers classified in the 1A or 1B categories of the GHS, and attempted to determine optimal criteria for GHS sub-categorization using LLNA: BrdU-ELISA. Consequently, the optimal criterion for the GHS sub-categorization was determined to be 6% when using EC1.6, showing the correct outcomes (%) for GHS 1A and GHS 1B category chemicals were 92.3 and 84.2 for all 32 chemicals, respectively. When excluding 2-mercaptobenzothiazole which may cause strain specific low response in this assay system, the correct outcomes (%) for GHS 1A chemicals was 100. Further work would be necessary, but the GHS sub-categorization criteria proposed in this study might be promising when using LLNA: BrdU-ELISA to provide information on the skin sensitization potency category of chemicals.
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http://dx.doi.org/10.1016/j.yrtph.2019.104409DOI Listing
October 2019

The Phosphatidylinositol 3-Kinase p110α/PTEN Signaling Pathway Is Crucial for HIV-1 Entry.

Biol Pharm Bull 2019 ;42(1):130-138

Division of Hematopoiesis, Center for AIDS Research, Kumamoto University.

Human immunodeficiency virus type 1 (HIV-1) drives multiple signaling pathways to facilitate its cellular entry and replication. The interaction between HIV-1 envelope (env) protein and target cell surface CD4 first activates the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway, and the subsequent interaction between HIV-1 env glycoprotein and CCR5/CXCR4 coreceptors establishes viral fusion and entry. Four isoforms of the class-I PI3K catalytic subunits (p110α, p110β, p110γ, and p110δ) have been identified so far, but the isoform(s) involved in the HIV-1 entry is still unknown. This study aimed to identify the PI3K isoform(s) using recently developed isoform-specific inhibitors and the roles of their negative regulators, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1), in HIV-1 infection. We found that the PI3K p110α isoform-specific inhibitor PIK-75 suppressed HIV-1 entry in HIV-1 permissive T cells, PM1 cells, and TZM-bl cells (HeLa cell-derived indicator cells that coexpress CD4, CCR5, and CXCR4) and decreased the HIV-1-induced phosphorylation of Akt. Moreover, wild-type PTEN (but neither phosphatase-deficient PTEN nor wild-type SHIP1) was a key regulator of HIV-1 entry. Cell-to-cell fusion by HIV-1 env-CD4 interaction was suppressed in the presence of PI3K p110α-specific inhibitor. These data suggest that the PI3K p110α/PTEN signaling pathway is indispensable for HIV-1 entry, including HIV-1 env-mediated cell-to-cell fusion.
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http://dx.doi.org/10.1248/bpb.b18-00801DOI Listing
April 2019

Effects of Soluble Tumor Necrosis Factor (TNF) on Antibody-Dependent Cellular Cytotoxicity of Therapeutic anti-TNF-α Antibody.

Immunol Invest 2019 Jul 20;48(5):441-450. Epub 2018 Dec 20.

a Chemicals Assessment and Research Center , Chemicals Evaluation and Research Institute , Saitama , Japan.

Anti-TNF antibodies are major therapeutics for rheumatoid arthritis and have been approved for marketing in many countries. Antibody-dependent cellular cytotoxicity (ADCC) is considered to be a potential mechanism of action of anti-TNF antibodies, since some anti-TNF antibodies have been confirmed to induce cytotoxic effects on TNF-producing cells via ADCC and complement-dependent cytotoxicity (CDC) in experiments. In this study, we established a new stable effector cell line expressing human FcγRIIIa, CD16:KHYG-1, and compared the performance of this cell line with that of peripheral blood mononuclear cells (PBMCs) in ADCC assays against CHO-derived target cells expressing protease-sensitive pro-TNF. Although an inhibitory effect of soluble TNF released from pro-TNF expressing cells on ADCC activity was seen, clear dose-responsive ADCC activities were observed even in the presence or absence of TNF-α converting enzyme (TACE) inhibitor. However, significant differences in the ADCC activities in the presence or absence of TACE inhibitor were only noted when CD16:KHYG-1 cells were used as the effector cells. Our findings indicate that soluble TNF may influence ADCC activity of anti-TNF antibody. Moreover, the fact that the influence was able to be detected only in the case using stable effector cell also suggests that the stable effector cell established this time enable highly accurate ADCC measurement.
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http://dx.doi.org/10.1080/08820139.2018.1549067DOI Listing
July 2019

The role of conventional antibodies targeting the CD4 binding site and CD4-induced epitopes in the control of HIV-1 CRF01_AE viruses.

Biochem Biophys Res Commun 2019 01 20;508(1):46-51. Epub 2018 Nov 20.

Center for AIDS Research, Kumamoto University, Kumamoto, Japan.

HIV-1 CRF01_AE viruses are highly prevalent in Southeast Asia. However, vulnerability sites in Env of CRF01_AE viruses have not been investigated sufficiently. We examined the sensitivity of CRF01_AE viruses from Japan and Vietnam, together with subtype B viruses from Japan, to neutralization and Fc-mediated signaling. Neutralization coverage of broadly neutralizing antibodies (bnAbs), 2G12 and b12, was significantly low against CRF01_AE viruses, compared with subtype B viruses. In contrast, the conventional antibody targeting the CD4 binding site (CD4bs), 49G2, showed better neutralization and Fc-mediated signaling activities against CRF01_AE viruses than subtype B viruses. Fc-mediated signaling activity of anti-CD4 induced (CD4i) antibody, 4E9C, was also detected against CRF01_AE viruses more than subtype B viruses. These results suggest that conventional antibodies against CD4bs and CD4i may play an important role in the control of CRF01_AE viruses.
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http://dx.doi.org/10.1016/j.bbrc.2018.11.063DOI Listing
January 2019

Variations in the viral genome and biological properties of bovine leukemia virus wild-type strains.

Virus Res 2018 07 18;253:103-111. Epub 2018 Jun 18.

Laboratory of Animal Health II, 1-17-71 Fuchinobe, Chuo-ku, Sagamihara, Kanagawa, 252-5201, Japan.

Bovine leukemia virus (BLV) is the etiological agent of enzootic bovine leukosis (EBL), which causes enormous economic losses in the livestock industry worldwide. To reduce the economic loss caused by BLV infection, it is important to clarify the characters associated with BLV transmissibility and pathogenesis in cattle. In this study, we focused on viral characters and examined spontaneous mutations in the virus and viral properties by analyses of whole genome sequences and BLV molecular clones derived from cows with and without EBL. Genomic analysis indicated that all 28 strains harbored limited genetic variations but no deletion mutations that allowed classification into three groups (A, B, and C), except for one strain. Some nucleotide/amino acid substitutions were specific to a particular group. On the other hand, these genetic variations were not associated with the host bovine leukocyte antigen-DRB3 allele, which is known to be related to BLV pathogenesis. The viral replication activity in vitro was high, moderate, and low in groups A, B, and C, respectively. In addition, the proviral load, which is related to BLV transmissibility and pathogenesis, was high in cows infected with group A strains and low in those infected with group B/C strains. Therefore, these results suggest that limited genetic variations could affect viral properties relating to BLV transmissibility and pathogenesis.
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http://dx.doi.org/10.1016/j.virusres.2018.06.005DOI Listing
July 2018

New approach to predict photoallergic potentials of chemicals based on murine local lymph node assay.

J Appl Toxicol 2018 10 23;38(10):1316-1322. Epub 2018 May 23.

Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute, Saitama, Japan.

Photoallergic dermatitis, caused by pharmaceuticals and other consumer products, is a very important issue in human health. However, S10 guidelines of the International Conference on Harmonization do not recommend the existing prediction methods for photoallergy because of their low predictability in human cases. We applied local lymph node assay (LLNA), a reliable, quantitative skin sensitization prediction test, to develop a new photoallergy prediction method. This method involves a three-step approach: (1) ultraviolet (UV) absorption analysis; (2) determination of no observed adverse effect level for skin phototoxicity based on LLNA; and (3) photoallergy evaluation based on LLNA. Photoallergic potential of chemicals was evaluated by comparing lymph node cell proliferation among groups treated with chemicals with minimal effect levels of skin sensitization and skin phototoxicity under UV irradiation (UV+) or non-UV irradiation (UV-). A case showing significant difference (P < .05) in lymph node cell proliferation rates between UV- and UV+ groups was considered positive for photoallergic reaction. After testing 13 chemicals, seven human photoallergens tested positive and the other six, with no evidence of causing photoallergic dermatitis or UV absorption, tested negative. Among these chemicals, both doxycycline hydrochloride and minocycline hydrochloride were tetracycline antibiotics with different photoallergic properties, and the new method clearly distinguished between the photoallergic properties of these chemicals. These findings suggested high predictability of our method; therefore, it is promising and effective in predicting human photoallergens.
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http://dx.doi.org/10.1002/jat.3643DOI Listing
October 2018

Applicability of an Integrated Testing Strategy consisting of in silico, in chemico and in vitro assays for evaluating the skin sensitization potencies of isocyanates.

Toxicology 2018 01 1;393:9-14. Epub 2017 Nov 1.

Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute (CERI), 1-4-25 Koraku, Bunkyo-ku, Tokyo 112-0004, Japan.

The skin sensitization potential of chemicals has been traditionally assessed using regulatory accepted in vivo methods, such as guinea pig maximization test or mouse local lymph node assays (LLNAs). A huge effort to reduce and replace the use of animals for safety assessments of chemicals because of regulatory requirements and ethical issues is presently underway, and alternative non-animal methods have been greatly developed. So far, a few studies have investigated the sensitization potencies of isocyanates which is a group of highly reactive chemicals that are known to be occupational allergens. The present study evaluated nine commonly used isocyanates using an in vivo LLNA and assessed the applicability of an Integrated Testing Strategy (ITS) consisting of an in silico Derek Nexus prediction, an in chemico direct peptide reactivity assay (DPRA), and an in vitro human Cell Line Activation Test (h-CLAT) to isocyanates. All nine isocyanates were evaluated as positive using the LLNA, Derek Nexus and DPRA, whereas seven chemicals tested positive using the h-CLAT: hexamethylene diisocyanate tested negative, and 1,5-diisocyanatonaphthalene could not be examined because of a solubility issue. When assessed using the ITS, the positive/negative evaluations of skin sensitization hazard were consistent with those assessed using the LLNA for all nine chemicals. However, the potency prediction results of the ITS tended to be underestimated, compared with those of the LLNA. The data presented in this work provide insights into the performance of non-animal testing approaches for evaluating the skin sensitization potencies of isocyanates.
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http://dx.doi.org/10.1016/j.tox.2017.10.015DOI Listing
January 2018

Molecular mechanisms by which HERV-K Gag interferes with HIV-1 Gag assembly and particle infectivity.

Retrovirology 2017 04 26;14(1):27. Epub 2017 Apr 26.

Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA.

Background: Human endogenous retroviruses (HERVs), the remnants of ancient retroviral infections, constitute approximately 8% of human genomic DNA. Since HERV-K Gag expression is induced by HIV-1 Tat in T cells, induced HERV-K proteins could affect HIV-1 replication. Indeed, previously we showed that HERV-K Gag and HIV-1 Gag coassemble and that this appears to correlate with the effect of HERV-K Gag expression on HIV-1 particle release and its infectivity. We further showed that coassembly requires both MA and NC domains, which presumably serve as scaffolding for Gag via their abilities to bind membrane and RNA, respectively. Notably, however, despite possessing these abilities, MLV Gag failed to coassemble with HIV-1 Gag and did not affect assembly and infectivity of HIV-1 particles. It is unclear how the specificity of coassembly is determined.

Results: Here, we showed that coexpression of HERV-K Gag with HIV-1 Gag changed size and morphology of progeny HIV-1 particles and severely diminished infectivity of such progeny viruses. We further compared HERV-K-MLV chimeric constructs to identify molecular determinants for coassembly specificity and for inhibition of HIV-1 release efficiency and infectivity. We found that the CA N-terminal domain (NTD) of HERV-K Gag is important for the reduction of the HIV-1 release efficiency, whereas both CA-NTD and major homology region of HERV-K Gag contribute to colocalization with HIV-1 Gag. Interestingly, these regions of HERV-K Gag were not required for reduction of progeny HIV-1 infectivity.

Conclusions: Our results showed that HERV-K Gag CA is important for reduction of HIV-1 release and infectivity but the different regions within CA are involved in the effects on the HIV-1 release and infectivity. Altogether, these findings revealed that HERV-K Gag interferes the HIV-1 replication by two distinct molecular mechanisms.
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http://dx.doi.org/10.1186/s12977-017-0351-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5406883PMC
April 2017

Increased HIV-1 sensitivity to neutralizing antibodies by mutations in the Env V3-coding region for resistance to CXCR4 antagonists.

J Gen Virol 2016 09 1;97(9):2427-2440. Epub 2016 Jul 1.

AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, 162-8640 Tokyo, Japan.

HIV-1 passage in cell culture in the presence of chemokine receptor antagonists can result in selection of viruses with env mutations that confer resistance to these inhibitors. In the present study, we examined the effect of HIV-1env mutations that confer resistance to CXCR4 antagonists on envelope (Env) sensitivity to neutralizing antibodies (NAbs). Serial passage of CXCR4-tropic HIV-1 NL4-3 in PM1/CCR5 cells under CXCR4 antagonists KRH-3955, AMD3100 and AMD070 yielded two KRH-3955-resistant, one AMD3100-resistant and one AMD070-resistant viruses. These viruses had multiple env mutations including the Env gp120 V3 region. The majority of viruses having these CXCR4 antagonist-resistant Envs showed higher sensitivity to NAbs 447-52D, b12 and 2F5 targeting the V3 region, the gp120 CD4-binding site and the gp41 membrane proximal region, respectively, compared to NL4-3 WT virus. Recombinant NL4-3 viruses with the V3-coding region replaced with those derived from the CXCR4 antagonist-resistant viruses showed increased sensitivity to NAbs b12, 2F5 and 447-52D. Molecular dynamics simulations of Env gp120 outer domains predicted that the V3 mutations increased levels of fluctuations at the tip and stem of the V3 loop. These results indicate that mutations in the V3-coding region that result in loss of viral sensitivity to CXCR4 antagonists increase viral sensitivity to NAbs, providing insights into our understanding of the interplay of viral Env accessibility to chemokine receptors and sensitivity to NAbs.
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http://dx.doi.org/10.1099/jgv.0.000536DOI Listing
September 2016

Comparison of outcomes obtained in murine local lymph node assays using CBA/J or CBA/Ca mice.

J Appl Toxicol 2016 08 12;36(8):1011-4. Epub 2015 Oct 12.

Chemicals Assessment and Research Center, Chemicals Evaluation and Research Institute (CERI), 1600, Shimotakano, Sugito-machi, Kitakatsushika-gun, Saitama, 345-0043, Japan.

CBA/J and CBA/Ca mice are the recommended strains for local lymph node assays (LLNAs). Here, we report quantitative and qualitative comparisons between both mouse strains to provide useful information for the strain selection of sensitization testing. LLNA was conducted, in accordance with Organisation for Economic Co-operation and Development Test Guideline No. 429, with CBA/J and CBA/Ca mice using five chemicals including typical contact sensitizers and non-sensitizers: 2,4-dinitrochlorobenzene (DNCB), isoeugenol, α-hexylcinnamic aldehyde (HCA), propylene glycol (PG), and hexane; then outcomes were compared based on the raw data (disintegrations per minute, DPM), stimulation index (SI) values, EC3 values and positive/negative decisions. Although a significant difference was noted between DPM values derived from each strain of mice, SI values exhibited no considerable difference. The EC3 values for DNCB in CBA/J and CBA/Ca mice were 0.04 and 0.03, those for isoeugenol were 1.4 and 0.9, and those for HCA were 7.7 and 6.0, respectively. All EC values derived from each test system were almost equivalent and were within the range of acceptance criteria of the ICCVAM performance standard for LLNA. Positive/negative outcomes for all test chemicals were consistent. In conclusion, no considerable differences were observed in the final outcomes derived from CBA/J and CBA/Ca mice in LLNA. Copyright © 2015 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/jat.3250DOI Listing
August 2016

Fibrocytes Differ from Macrophages but Can Be Infected with HIV-1.

J Immunol 2015 Nov 28;195(9):4341-50. Epub 2015 Sep 28.

Center for AIDS Research, Kumamoto University, Kumamoto 860-0811, Japan; International Research Center for Medical Sciences, Kumamoto University, Kumamoto 860-0811, Japan;

Fibrocytes (fibroblastic leukocytes) are recently identified as unique hematopoietic cells with features of both macrophages and fibroblasts. Fibrocytes are known to contribute to the remodeling or fibrosis of various injured tissues. However, their role in viral infection is not fully understood. In this study, we show that differentiated fibrocytes are phenotypically distinguishable from macrophages but can be infected with HIV-1. Importantly, fibrocytes exhibited persistently infected cell-like phenotypes, the degree of which was more apparent than macrophages. The infected fibrocytes produced replication-competent HIV-1, but expressed HIV-1 mRNA at low levels and strongly resisted HIV-1-induced cell death, which enabled them to support an extremely long-term HIV-1 production at low but steady levels. More importantly, our results suggested that fibrocytes were susceptible to HIV-1 regardless of their differentiation state, in contrast to the fact that monocytes become susceptible to HIV-1 after the differentiation into macrophages. Our findings indicate that fibrocytes are the previously unreported HIV-1 host cells, and they suggest the importance of considering fibrocytes as one of the long-lived persistently infected cells for curing HIV-1.
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http://dx.doi.org/10.4049/jimmunol.1500955DOI Listing
November 2015

Differential Ability of Primary HIV-1 Nef Isolates To Downregulate HIV-1 Entry Receptors.

J Virol 2015 Sep 15;89(18):9639-52. Epub 2015 Jul 15.

Center for AIDS Research, Kumamoto University, Kumamoto, Japan International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan

Unlabelled: HIV-1 Nef downregulates the viral entry receptor CD4 as well as the coreceptors CCR5 and CXCR4 from the surface of HIV-infected cells, and this leads to promotion of viral replication through superinfection resistance and other mechanisms. Nef sequence motifs that modulate these functions have been identified via in vitro mutagenesis with laboratory HIV-1 strains. However, it remains unclear whether the same motifs contribute to Nef activity in patient-derived sequences and whether these motifs may differ in Nef sequences isolated at different infection stages and/or from patients with different disease phenotypes. Here, nef clones from 45 elite controllers (EC), 46 chronic progressors (CP), and 43 acute progressors (AP) were examined for their CD4, CCR5, and CXCR4 downregulation functions. Nef clones from EC exhibited statistically significantly impaired CD4 and CCR5 downregulation ability and modestly impaired CXCR4 downregulation activity compared to those from CP and AP. Nef's ability to downregulate CD4 and CCR5 correlated positively in all cohorts, suggesting that they are functionally linked in vivo. Moreover, impairments in Nef's receptor downregulation functions increased the susceptibility of Nef-expressing cells to HIV-1 infection. Mutagenesis studies on three functionally impaired EC Nef clones revealed that multiple residues, including those at novel sites, were involved in the alteration of Nef functions and steady-state protein levels. Specifically, polymorphisms at highly conserved tryptophan residues (e.g., Trp-57 and Trp-183) and immune escape-associated sites were responsible for reduced Nef functions in these clones. Our results suggest that the functional modulation of primary Nef sequences is mediated by complex polymorphism networks.

Importance: HIV-1 Nef, a key factor for viral pathogenesis, downregulates functionally important molecules from the surface of infected cells, including the viral entry receptor CD4 and coreceptors CCR5 and CXCR4. This activity enhances viral replication by protecting infected cells from cytotoxicity associated with superinfection and may also serve as an immune evasion strategy. However, how these activities are maintained under selective pressure in vivo remains elusive. We addressed this question by analyzing functions of primary Nef clones isolated from patients at various infection stages and with different disease phenotypes, including elite controllers, who spontaneously control HIV-1 viremia to undetectable levels. The results indicated that downregulation of HIV-1 entry receptors, particularly CCR5, is impaired in Nef clones from elite controllers. These functional impairments were driven by rare Nef polymorphisms and adaptations associated with cellular immune responses, underscoring the complex molecular pathways responsible for maintaining and attenuating viral protein function in vivo.
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http://dx.doi.org/10.1128/JVI.01548-15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4542390PMC
September 2015

Separate cellular localizations of human T-lymphotropic virus 1 (HTLV-1) Env and glucose transporter type 1 (GLUT1) are required for HTLV-1 Env-mediated fusion and infection.

J Virol 2015 Jan 22;89(1):502-11. Epub 2014 Oct 22.

Department of Medical Virology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.

Unlabelled: Interaction of the envelope glycoprotein (Env) of human T-lymphotropic virus 1 (HTLV-1) with the glucose transporter type 1 (GLUT1) expressed in target cells is essential for viral entry. This study found that the expression level of GLUT1 in virus-producing 293T cells was inversely correlated with HTLV-1 Env-mediated fusion activity and infectivity. Chimeric studies between GLUT1 and GLUT3 indicated that the extracellular loop 6 (ECL6) of GLUT1 is important for the inhibition of cell-cell fusion mediated by Env. When GLUT1 was translocated into the plasma membrane from intracellular storage sites by bafilomycin A1 (BFLA1) treatment in 293T cells, HTLV-1 Env-mediated cell fusion and infection also were inhibited without the overexpression of GLUT1, indicating that the localization of GLUT1 in intracellular compartments rather than in the plasma membrane is crucial for the fusion activity of HTLV-1 Env. Immunoprecipitation and laser scanning confocal microscopic analyses indicated that under normal conditions, HTLV-1 Env and GLUT1 do not colocalize or interact. BFLA1 treatment induced this colocalization and interaction, indicating that GLUT1 normally accumulates in intracellular compartments separate from that of Env. Western blot analyses of FLAG-tagged HTLV-1 Env in virus-producing cells and the incorporation of HTLV-1 Env in virus-like particles (VLPs) indicate that the processing of Env is inhibited by either overexpression of GLUT1 or BFLA1 treatment in virus-producing 293T cells. This inhibition probably is due to the interaction of the Env with GLUT1 in intracellular compartments. Taken together, separate intracellular localizations of GLUT1 and HTLV-1 Env are required for the fusion activity and infectivity of HTLV-1 Env.

Importance: The deltaretrovirus HTLV-1 is a causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Although HTLV-1 is a complex retrovirus that has accessory genes, no HTLV-1 gene product has yet been shown to regulate its receptor GLUT1 in virus-producing cells. In this study, we found that a large amount of GLUT1 or translocation of GLUT1 to the plasma membrane from intracellular compartments in virus-producing cells enhances the colocalization and interaction of GLUT1 with HTLV-1 Env, leading to the inhibition of cell fusion activity and infectivity. The results of our study suggest that GLUT1 normally accumulates in separate intracellular compartments from Env, which is indeed required for the proper processing of Env.
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http://dx.doi.org/10.1128/JVI.02686-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301130PMC
January 2015

Preferential recognition of monomeric CCR5 expressed in cultured cells by the HIV-1 envelope glycoprotein gp120 for the entry of R5 HIV-1.

Virology 2014 Mar 31;452-453:117-24. Epub 2014 Jan 31.

Department of Medical Virology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Electronic address:

Bimolecular fluorescence complementation (BiFC) and western blot analysis demonstrated that CCR5 exists as constitutive homo-oligomers, which was further enhanced by its antagonists such as maraviroc (MVC) and TAK-779. Staining by monoclonal antibodies recognizing different epitopes of CCR5 revealed that CCR5 oligomer was structurally different from the monomer. To determine which forms of CCR5 are well recognized by CCR5-using HIV-1 for the entry, BiFC-positive and -negative cell fractions in CD4-positive 293T cells were collected by fluorescent-activated cell sorter, and infected with luciferase-reporter HIV-1 pseudotyped with CCR5-using Envs including R5 and R5X4. R5 and dual-R5 HIV-1 substantially infected BiFC-negative fraction rather than BiFC-positive fraction, indicating the preferential recognition of monomeric CCR5 by R5 and dual-R5 Envs. Although CCR5 antagonists enhanced oligomerization of CCR5, MVC-resistant HIV-1 was found to still recognize both MVC-bound and -unbound forms of monomeric CCR5, suggesting the constrained use of monomeric CCR5 by R5 HIV-1.
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http://dx.doi.org/10.1016/j.virol.2013.12.034DOI Listing
March 2014

V3-independent competitive resistance of a dual-X4 HIV-1 to the CXCR4 inhibitor AMD3100.

PLoS One 2014 19;9(2):e89515. Epub 2014 Feb 19.

Department of Medical Virology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.

A CXCR4 inhibitor-resistant HIV-1 was isolated from a dual-X4 HIV-1 in vitro. The resistant variant displayed competitive resistance to the CXCR4 inhibitor AMD3100, indicating that the resistant variant had a higher affinity for CXCR4 than that of the wild-type HIV-1. Amino acid sequence analyses revealed that the resistant variant harbored amino acid substitutions in the V2, C2, and C4 regions, but no remarkable changes in the V3 loop. Site-directed mutagenesis confirmed that the changes in the C2 and C4 regions were principally involved in the reduced sensitivity to AMD3100. Furthermore, the change in the C4 region was associated with increased sensitivity to soluble CD4, and profoundly enhanced the entry efficiency of the virus. Therefore, it is likely that the resistant variant acquired the higher affinity for CD4/CXCR4 by the changes in non-V3 regions. Taken together, a CXCR4 inhibitor-resistant HIV-1 can evolve using a non-V3 pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089515PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3929750PMC
October 2014

Promoter Targeting shRNA Suppresses HIV-1 Infection In vivo Through Transcriptional Gene Silencing.

Mol Ther Nucleic Acids 2013 Dec 3;2:e137. Epub 2013 Dec 3.

St. Vincent's Centre for Applied Medical Research, Darlinghurst, New South Wales, Australia.

Despite prolonged and intensive application, combined antiretroviral therapy cannot eradicate human immunodeficiency virus (HIV)-1 because it is harbored as a latent infection, surviving for long periods of time. Alternative approaches are required to overcome the limitations of current therapy. We have been developing a short interfering RNA (siRNA) gene silencing approach. Certain siRNAs targeting promoter regions of genes induce transcriptional gene silencing. We previously reported substantial transcriptional gene silencing of HIV-1 replication by an siRNA targeting the HIV-1 promoter in vitro. In this study, we show that this siRNA, expressed as a short hairpin RNA (shRNA) (shPromA-JRFL) delivered by lentiviral transduction of human peripheral blood mononuclear cells (PBMCs), which are then used to reconstitute NOJ mice, is able to inhibit HIV-1 replication in vivo, whereas a three-base mismatched variant (shPromA-M2) does not. In shPromA-JRFL-treated mice, HIV-1 RNA in serum is significantly reduced, and the ratio of CD4(+)/CD8(+) T cells is significantly elevated. Expression levels of the antisense RNA strand inversely correlates with HIV-1 RNA in serum. The silenced HIV-1 can be reactivated by T-cell activation in ex vivo cultures. HIV-1 suppression is not due to offtarget effects of shPromA-JRFL. These data provide "proof-of principle" that an shRNA targeting the HIV-1 promoter is able to suppress HIV-1 replication in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e137; doi:10.1038/mtna.2013.64; published online 3 December 2013.
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http://dx.doi.org/10.1038/mtna.2013.64DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3894581PMC
December 2013

Structure and dynamics of the gp120 V3 loop that confers noncompetitive resistance in R5 HIV-1(JR-FL) to maraviroc.

PLoS One 2013 28;8(6):e65115. Epub 2013 Jun 28.

Transfusion Transmitted Diseases Center, Institute of Blood Transfusion, Chinese Academy of Medical Science, Chenghua District, Chengdu, Sichuan Province, PR China.

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4(+) cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1(V3-M5) derived from HIV-1(JR-FLan) is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1(JR-FLan) to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0065115PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3695986PMC
January 2014

Dynamic appearance of antigenic epitopes effective for viral neutralization during membrane fusion initiated by interactions between HIV-1 envelope proteins and CD4/CXCR4.

Immunobiology 2012 Sep 23;217(9):864-72. Epub 2011 Dec 23.

Department of Immunology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.

HIV-1 entry into cells is mediated by interactions between the envelope (Env) gp120 and gp41 proteins with CD4 and chemokine receptors via an intermediate called the viral fusion complex (vFC). Here, mAbs were used to find the dynamic changes in expression of antigenic epitopes during vFC formation. A CD4-specific mAb (R275) and anti-vFC mAbs, designated F12-1, F13-6 and F18-4 that recognize the epitopes only appeared by the co-culture of env-transfected 293FT and CD4-transfected 293 cells, were developed by immunizing ganp-gene transgenic mice with an vFC-like structure formed by the same co-culture. The epitopes recognized by the mAbs appeared at different time points during vFC formation: F18-4 appeared first, followed by F13-6, and finally F12-1. The anti-vFC mAbs had little effect on vFC formation or virus neutralization; however, interestingly F12-1 and F18-4 increased exposure of the OKT4-epitope on the domain 3 in the extracellular region of CD4. R275, which recognizes the epitope closely associated with the OKT4-determinant on the domain 3, showed the marked inhibition of vFC formation and viral neutralization activity. The Ab binding to the epitopes appeared during viral membrane fusion might reinforce the appearance of the target epitopes for effective neutralization activity.
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http://dx.doi.org/10.1016/j.imbio.2011.12.007DOI Listing
September 2012

A combination of polymorphic mutations in V3 loop of HIV-1 gp120 can confer noncompetitive resistance to maraviroc.

Virology 2011 May 26;413(2):293-9. Epub 2011 Mar 26.

Department of Medical Virology, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8665, Japan.

Maraviroc binds to the pocket of extracellular loops of the cell surface CCR5 and prevents R5 HIV-1 from using CCR5 as a coreceptor for entry into CD4-positive cells. To evaluate the contribution of the V3 loop structure in gp120 to maraviroc resistance, we isolated maraviroc-resistant variants from the V3 loop library virus (HIV-1(V3Lib)) containing a set of random combinations of 0-10 polymorphic mutations in vitro. HIV-1(V3Lib) at passage 17 could not be suppressed even at 10 μM (>1400-fold resistance), while HIV-1(JR-FL) at passage 17 revealed an 8-fold resistance to maraviroc. HIV-1(V3Lib-P17) contained T199K and T275M plus 5 mutations in the V3 loop, I304V/F312W/T314A/E317D/I318V. The profile of pseudotyped virus containing I304V/F312W/T314A/E317D/I318V in V3 loop alone revealed a typical noncompetitive resistance, although T199K and/or T275M could not confer noncompetitive resistance. This type of library virus is useful for isolation of escape viruses from effective entry inhibitors.
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http://dx.doi.org/10.1016/j.virol.2011.02.019DOI Listing
May 2011

Involvement of inhibitory factors in the inefficient entry of HIV-1 into the human CD4 positive HUT78 cell line.

Virus Res 2011 Jan 20;155(1):368-71. Epub 2010 Oct 20.

Department of Medical Virology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan.

Little is known about whether human CD4 positive T cells, the principal natural target of HIV-1, have intrinsic factors, other than the receptor/coreceptor molecules, which modulate the entry efficiency of HIV-1. In the present study, we found that human T cell lines, HUT78 and PM1, were less permissive to VSV-G-mediated HIV-1 infection compared with the Jurkat cell line. Furthermore, HUT78 cells were also less sensitive to HIV-1 Env-mediated infection, while PM1 cells became susceptible to HIV-1. Real-time PCR analyses showed that less susceptibility of the cells to HIV-1 was due to block at, or prior to, reverse transcription of viral RNA. To clarify the entry efficiency of HIV-1 into these cell lines, we analyzed the internalization of p24 Ag into the cytosolic and vesicular fractions of post-nuclear extracts at 4h post-infection. When the cells were infected with HIV-1 pseudotyped with VSV-G, the amount of p24 Ag in the cytosolic fractions in both HUT78 and PM1 cells was lower than that observed in Jurkat cells. In the case of HIV-1 Env-mediated infection, however, PM1 cells exhibited comparable amounts of p24 Ag in the cytosolic fraction compared with Jurkat cells, while the amount of p24 Ag in HUT78 cells remained low. Heterokaryon experiments between susceptible and less susceptible cell lines suggested that some inhibitory factors counteracted VSV-G-mediated viral entry in PM1 and HUT78 cells, and HIV-1 Env-mediated viral entry in HUT78 cells.
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http://dx.doi.org/10.1016/j.virusres.2010.10.010DOI Listing
January 2011

The antiretroviral potency of APOBEC1 deaminase from small animal species.

Nucleic Acids Res 2008 Dec 29;36(21):6859-71. Epub 2008 Oct 29.

Department of Retrovirology and Self-Defense, Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.

Although the role of the APOBEC3-dependent retroelement restriction system as an intrinsic immune defense against human immunodeficiency virus type1 (HIV-1) infection is becoming clear, only the rat ortholog of mammalian APOBEC1s (A1) thus far has been shown to possess antiviral activity. Here, we cloned A1 cDNAs from small animal species, and showed that similar to rat A1, both wild-type and Deltavif HIV-1 infection was inhibited by mouse and hamster A1 (4- to 10-fold), whereas human A1 had negligible effects. Moreover, rabbit A1 significantly reduced the infectivity of both HIV-1 virions (>300-fold), as well as that of SIVmac, SIVagm, FIV and murine leukemia virus. Immunoblot analysis showed that A1s were efficiently incorporated into the HIV-1 virion, and their packaging is mediated through an interaction with the nucleocapsid Gag domain. Interestingly, there was a clear accumulation of particular C-T changes in the genomic RNAs of HIV-1 produced in their presence, with few G-A changes in the proviral DNA. Together, these data reveal that A1 may function as a defense mechanism, regulating retroelements in a wide range of mammalian species.
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http://dx.doi.org/10.1093/nar/gkn802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2588513PMC
December 2008

Wheezing illness caused by respiratory syncytial virus and other agents.

Pediatr Int 2008 Aug;50(4):500-5

Department of Pediatrics, Kyoto City Hospital, Kyoto, Japan.

Background: Respiratory syncytial virus (RSV) and other pathogenic agents cause lower respiratory infection with wheezing in infants (wheezing illness in infancy). Wheezing illness in infancy due to RSV can be life-threatening and can induce recurrent wheezing; but these events can also be produced by infection by other pathogenic agents. Thus, whether RSV induces more severe wheezing illness in infancy remains poorly understood.

Methods: Infants with an initial wheezing illness were divided into an RSV-positive group and an RSV-negative group and the differences in disease severity, intensity of acute symptoms, and susceptibility to recurrent wheezing, between these two groups, were investigated.

Results: The RSV-positive group accounted for 57.4% of the infants. The infants in the RSV-positive group were significantly younger than those in the RSV-negative group. Other background parameters, gender and family history, were not significantly different. There were no significant differences in indicators of severity (hospitalization periods, periods of persistent wheezing and requirement of oxygen supplementation) between the two groups.

Conclusion: Wheezing illness in infancy caused by RSV is more common in younger infants, but it is not more severe than that caused by other pathogenic agents.
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http://dx.doi.org/10.1111/j.1442-200X.2008.02702.xDOI Listing
August 2008