Modified ESR-based photosafety test (ESR-PT) detecting singlet oxygen and free radical formation.

The electron spin resonance-based photosafety test (ESR-PT) was modified using a new parameter, photoreactivity index (PRI), to detect singlet oxygen and free radical photoproducts simultaneously. With this modification, the modified ESR-PT is expected to reduce the number of false negative results due to chemicals producing free radical photoproducts other than singlet oxygen. The assay performance of the modified ESR-PT was evaluated using 56 chemicals, including hydrophobic chemicals. When using the PRI cutoff value of 2.0 in the modified ESR-PT, the accuracy relative to photosafety reference data was 91.1%, and the applicability (100%) was better than the other non-animal photosafety test. Among the chemicals producing positive results, bithionol, fenticlor, and doxycycline HCl were considered positive based on the detection of free radical photoproducts, suggesting that these three chemicals may have phototoxic or photoallergic potential via radical reactions. Additionally, this finding demonstrated the fundamental advantage of the modified ESR-PT using ESR spectroscopy, which can detect radicals selectively and quantitatively. Accordingly, the new parameter PRI is effective for photosafety evaluations based on not only singlet oxygen but also free radical photoproducts generated from chemicals. Therefore, the modified ESR-PT has a great potential for a photosafety test method applicable to various chemicals.

Epididymitis with Pasteurella multocida isolated from two male Japanese Black beef calves.

Two male Japanese Black calves developed an enlarged scrotum and testis. Orchiectomy was performed and pus was collected during surgery. After removal of the testis, bacteriological and histopathological examinations were conducted to investigate the cause and confirm the diagnosis. Based on the results obtained, both cases were diagnosed with epididymitis caused by an infection with Pasteurella multocida. This is the first study to show that P. multocida causes epididymitis in male calves. Further studies are required to clarify the details underlying the infection of calves with P. multocida.

Differential Ability of Spike Protein of SARS-CoV-2 Variants to Downregulate ACE2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 19 (COVID-19) and employs angiotensin-converting enzyme 2 (ACE2) as the receptor. Although the expression of ACE2 is crucial for cellular entry, we found that the interaction between ACE2 and the Spike (S) protein in the same cells led to its downregulation through degradation in the lysosomal compartment via the endocytic pathway. Interestingly, the ability of the S protein from previous variants of concern (VOCs) to downregulate ACE2 was variant-dependent and correlated with disease severity. The S protein from the Omicron variant, associated with milder disease, exhibited a lower capacity to downregulate ACE2 than that of the Delta variant, which is linked to a higher risk of hospitalization. Chimeric studies between the S proteins from the Delta and Omicron variants revealed that both the receptor-binding domain (RBD) and the S2 subunit played crucial roles in the reduced ACE2 downregulation activity observed in the Omicron variant. In contrast, three mutations (L452R/P681R/D950N) located in the RBD, S1/S2 cleavage site, and HR1 domain were identified as essential for the higher ACE2 downregulation activity observed in the Delta variant compared to that in the other VOCs. Our results suggested that dysregulation of the renin-angiotensin system due to the ACE2 downregulation activity of the S protein of SARS-CoV-2 may play a key role in the pathogenesis of COVID-19.

Movements of Ancient Human Endogenous Retroviruses Detected in SOX2-Expressing Cells.

Human endogenous retroviruses (HERVs) occupy approximately 8% of the human genome. HERVs, transcribed in early embryos, are epigenetically silenced in somatic cells, except under pathological conditions. HERV-K is thought to protect embryos from exogenous viral infection. However, uncontrolled HERV-K expression in somatic cells has been implicated in several diseases. Here, we show that SOX2, which plays a key role in maintaining the pluripotency of stem cells, is critical for HERV-K LTR5Hs. HERV-K undergoes retrotransposition within producer cells in the absence of Env expression. Furthermore, we identified new HERV-K integration sites in long-term culture of induced pluripotent stem cells that express SOX2. These results suggest that the strict dependence of HERV-K on SOX2 has allowed HERV-K to protect early embryos during evolution while limiting the potentially harmful effects of HERV-K retrotransposition on host genome integrity in these early embryos. IMPORTANCE Human endogenous retroviruses (HERVs) account for approximately 8% of the human genome; however, the physiological role of HERV-K remains unknown. This study found that HERV-K LTR5Hs and LTR5B were transactivated by SOX2, which is essential for maintaining and reestablishing pluripotency. HERV-K can undergo retrotransposition within producer cells without env expression, and new integration sites may affect cell proliferation. In induced pluripotent stem cells (iPSCs), genomic impairment due to HERV-K retrotransposition has been identified, but it is a rare event. Considering the retention of SOX2-responsive elements in the HERV-K long terminal repeat (LTR) for over 20 million years, we conclude that HERV-K may play important physiological roles in SOX2-expressing cells.

Control of HIV-1 Replication by CD8+ T Cells Specific for Two Novel Pol Protective Epitopes in HIV-1 Subtype A/E Infection.

Although many HIV-1-specific CD8+ T cell epitopes have been identified and used in various HIV-1 studies, most of these epitopes were derived from HIV-1 subtypes B and C. Only 17 well-defined epitopes, none of which were protective, have been identified for subtype A/E infection. The roles of HIV-1-specific T cells have been rarely analyzed for subtype A/E infection. In this study, we identified six novel HLA-B*15:02-restricted optimal HIV-1 subtype A/E epitopes and then analyzed the presentation of these epitopes by HIV-1 subtype A/E virus-infected cells and the T cell responses to these epitopes in treatment-naive HIV-1 subtype A/E-infected HLA-B*15:02+ Vietnamese individuals. Responders to the PolTY9 or PolLF10 epitope had a significantly lower plasma viral load (pVL) than nonresponders among HLA-B*15:02+ individuals, whereas no significant difference in pVL was found between responders to four other epitopes and nonresponders. The breadth of T cell responses to these two Pol epitopes correlated inversely with pVL. These findings suggest that HLA-B*15:02-restricted T cells specific for PolTY9 and PolLF10 contribute to the suppression of HIV-1 replication in HLA-B*15:02+ individuals. The HLA-B*15:02-associated mutation Pol266I reduced the recognition of PolTY9-specific T cells in vitro but did not affect HIV-1 replication by PolTY9-specific T cells in Pol266I mutant virus-infected individuals. These findings indicate that PolTY9-specific T cells suppress replication of the Pol266I mutant virus even though the T cells selected this mutant. This study demonstrates the effective role of T cells specific for these Pol epitopes to control circulating viruses in HIV-1 subtype A/E infection. IMPORTANCE It is expected that HIV-1-specific CD8+ T cells that effectively suppress HIV-1 replication will contribute to HIV-1 vaccine development and therapy to achieve an HIV cure. T cells specific for protective epitopes were identified in HIV-1 subtype B and C infections but not in subtype A/E infection, which is epidemic in Southeast Asia. In the present study, we identified six T cell epitopes derived from the subtype A/E virus and demonstrated that T cells specific for two Pol epitopes effectively suppressed HIV-1 replication in treatment-naive Vietnamese individuals infected with HIV-1 subtype A/E. One of these Pol protective epitopes was conserved among circulating viruses, and one escape mutation was accumulated in the other epitope. This mutation did not critically affect HIV-1 control by specific T cells in HIV-1 subtype A/E-infected individuals. This study identified two protective Pol epitopes and characterized them in cases of HIV-1 subtype A/E infection.

Establishment of Cell-Based Assay System for Evaluating Cytotoxic Activity Modulated by the Blockade of PD-1 and PD-L1 Interactions with a Therapeutic Antibody.

BACKGROUND: Therapeutic antibodies targeting the PD-1/PD-L1 immune checkpoint are widely used in cancer therapy and are under active further development. Historically, the antitumor activity of PD-1/PD-L1 immune checkpoint inhibitors has been evaluated using in vivo and ex vivo test methods; however, a simple in vitro assay method to evaluate antitumor activity accurately is needed for the efficient development of new therapeutic agents. In the present study, we attempted to establish a simple cell-based assay system to evaluate the modulating effect of PD-1/PD-L1 immune checkpoint inhibitors on cytotoxic activity. METHODS: We established a new natural killer (NK) cell line stably transfected with the PD-1 and IL-2 genes and a new NK-sensitive target cell line stably transfected with the PD-L1 gene. Then, the assay system was established by co-cultivation of the established cell lines and measurement of the cytotoxic activities using the europium release assay. To confirm the performance of the established assay system, model therapeutic antibodies to block the PD-1/PD-L1 signal, nivolumab and atezolizumab were added to the co-culture system and the modulating effect on the cytotoxic activities were evaluated. RESULTS: Nivolumab and atezolizumab clearly showed a modulating effect on cytotoxic activity in a dose-dependent manner in our assay system, whereas a human IgG isotype control antibody did not show any modulating effect on the assay system. CONCLUSION: The newly established cell-based assay system can quantitatively evaluate the modulating effect of PD-1/PD-L1 immune checkpoint inhibitors by measuring cytotoxic activity, playing an important role in antitumor effects as innate immunity.

Turkish Plants, Including Quercetin and Oenothein B, Inhibit the HIV-1 Release and Accelerate Cell Apoptosis.

The introduction of combined anti-retroviral therapy (cART) in 1996, along with a continual breakthrough in anti-human immunodeficiency virus-1 (HIV-1) drugs, has improved the life expectancies of HIV-1-infected individuals. However, the incidence of drug-resistant viruses between individuals undergoing cART and treatment-naïve individuals is a common challenge. Therefore, there is a requirement to explore potential drug targets by considering various stages of the viral life cycle. For instance, the late stage, or viral release stage, remains uninvestigated extensively in antiviral drug discovery. In this study, we prepared a natural plant library and selected candidate plant extracts that inhibited HIV-1 release based on our laboratory-established screening system. The plant extracts from Epilobium hirsutum L. and Chamerion angustifolium (L.) Holub, belonging to the family Onagraceae, decreased HIV-1 release and accelerated the apoptosis in HIV-1-infected T cells but not uninfected T cells. A flavonol glycoside quercetin with oenothein B in Onagraceae reduced HIV-1 release in HIV-1-infected T cells. Moreover, extracts from Chamerion angustifolium (L.) Holub and Senna alexandrina Mill. inhibited the infectivity of progeny viruses. Together, these results suggest that C. angustifolium (L.) Holub contains quercetin with oenothein B that synergistically blocks viral replication and kills infected cells via an apoptotic pathway. Consequently, the plant extracts from the plant library of Turkey might be suitable candidates for developing novel anti-retroviral drugs that target the late phase of the HIV-1 life cycle.

Interaction of TSG101 with the PTAP Motif in Distinct Locations of Gag Determines the Incorporation of HTLV-1 Env into the Retroviral Virion.

Human T-cell tropic virus type 1 (HTLV-1) is known to be mainly transmitted by cell-to-cell contact due to the lower infectivity of the cell-free virion. However, the reasons why cell-free HTLV-1 infection is poor remain unknown. In this study, we found that the retrovirus pseudotyped with HTLV-1 viral envelope glycoprotein (Env) was infectious when human immunodeficiency virus type 1 (HIV-1) was used to produce the virus. We found that the incorporation of HTLV-1 Env into virus-like particles (VLPs) was low when HTLV-1 Gag was used to produce VLPs, whereas VLPs produced using HIV-1 Gag efficiently incorporated HTLV-1 Env. The production of VLPs using Gag chimeras between HTLV-1 and HIV-1 Gag and deletion mutants of HIV-1 Gag showed that the p6 domain of HIV-1 Gag was responsible for the efficient incorporation of HTLV-1 Env into the VLPs. Further mutagenic analyses of the p6 domain of HIV-1 Gag revealed that the PTAP motif in the p6 domain of HIV-1 Gag facilitates the incorporation of HTLV-1 Env into VLPs. Since the PTAP motif is known to interact with tumor susceptibility gene 101 (TSG101) during the budding process, we evaluated the effect of TSG101 knockdown on the incorporation of HTLV-1 Env into VLPs. We found that TSG101 knockdown suppressed the incorporation of HTLV-1 Env into VLPs and decreased the infectivity of cell-free HIV-1 pseudotyped with HTLV-1 Env. Our results suggest that the interaction of TSG101 with the PTAP motif of the retroviral L domain is involved not only in the budding process but also in the efficient incorporation of HTLV-1 Env into the cell-free virus.

Plasma N-Cleaved Galectin-9 Is a Surrogate Marker for Determining the Severity of COVID-19 and Monitoring the Therapeutic Effects of Tocilizumab.

Galectin-9 (Gal-9) is known to contribute to antiviral responses in coronavirus disease 2019 (COVID-19). Increased circulating Gal-9 in COVID-19 is associated with COVID-19 severity. In a while, the linker-peptide of Gal-9 is susceptible to proteolysis that can cause the change or loss of Gal-9 activity. Here, we measured plasma levels of N-cleaved-Gal9, which is Gal9 carbohydrate-recognition domain at the N-terminus (NCRD) with attached truncated linker peptide that differs in length depending on the type of proteases, in COVID-19. We also investigated the time course of plasma N-cleaved-Gal9 levels in severe COVID-19 treated with tocilizumab (TCZ). As a result, we observed an increase in plasma N-cleaved-Gal9 levels in COVID-19 and its higher levels in COVID-19 with pneumonia compared to the mild cases (healthy: 326.1 pg/mL, mild: 698.0 pg/mL, and with pneumonia: 1570 pg/mL). N-cleaved-Gal9 levels were associated with lymphocyte counts, C-reactive protein (CRP), soluble interleukin-2 receptor (sIL-2R), D-dimer, and ferritin levels, and ratio of percutaneous oxygen saturation to fraction of inspiratory oxygen (S/F ratio) in COVID-19 with pneumonia and discriminated different severity groups with high accuracy (area under the curve (AUC): 0.9076). Both N-cleaved-Gal9 and sIL-2R levels were associated with plasma matrix metalloprotease (MMP)-9 levels in COVID-19 with pneumonia. Furthermore, a decrease in N-cleaved-Gal9 levels was associated with a decrease of sIL-2R levels during TCZ treatment. N-cleaved-Gal9 levels showed a moderate accuracy (AUC: 0.8438) for discriminating the period before TCZ from the recovery phase. These data illustrate that plasma N-cleaved-Gal9 is a potential surrogate marker for assessing COVID-19 severity and the therapeutic effects of TCZ.

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

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.IMPORTANCE 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.

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

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.

High Levels of the Cleaved Form of Galectin-9 and Osteopontin in the Plasma Are Associated with Inflammatory Markers That Reflect the Severity of COVID-19 Pneumonia.

Numbers of patients with coronavirus disease 2019 (COVID-19) have increased rapidly worldwide. Plasma levels of full-length galectin-9 (FL-Gal9) and osteopontin (FL-OPN) as well as their truncated forms (Tr-Gal9, Ud-OPN, respectively), are representative inflammatory biomarkers. Here, we measured FL-Gal9, FL-OPN, Tr-Gal9, and Ud-OPN in 94 plasma samples obtained from 23 COVID-19-infected patients with mild clinical symptoms (CV), 25 COVID-19 patients associated with pneumonia (CP), and 14 patients with bacterial infection (ID). The four proteins were significantly elevated in the CP group when compared with healthy individuals. ROC analysis between the CV and CP groups showed that C-reactive protein had the highest ability to differentiate, followed by Tr-Gal9 and ferritin. Spearman's correlation analysis showed that Tr-Gal9 and Ud-OPN but not FL-Gal9 and FL-OPN, had a significant association with laboratory markers for lung function, inflammation, coagulopathy, and kidney function in CP patients. CP patients treated with tocilizumab had reduced levels of FL-Gal9, Tr-Gal9, and Ud-OPN. It was suggested that OPN is cleaved by interleukin-6-dependent proteases. These findings suggest that the cleaved forms of OPN and galectin-9 can be used to monitor the severity of pathological inflammation and the therapeutic effects of tocilizumab in CP patients.

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

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.

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

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.

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

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.

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

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 in vitro 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.

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

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, p110ß, 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.

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

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.

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

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.

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

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.