Liver-specific Mettl14 deletion induces nuclear heterotypia and dysregulates RNA export machinery.

Modification of RNA with N6-methyladenosine (m6A) has gained attention in recent years as a general mechanism of gene regulation. In the liver, m6A, along with its associated machinery, has been studied as a potential biomarker of disease and cancer, with impacts on metabolism, cell cycle regulation, and pro-cancer state signaling. However these observational data have yet to be causally examined in vivo. For example, neither perturbation of the key m6A writers Mettl3 and Mettl14, nor the m6A readers Ythdf1 and Ythdf2 have been thoroughly mechanistically characterized in vivo as they have been in vitro. To understand the functions of these machineries, we developed mouse models and found that deleting Mettl14 led to progressive liver injury characterized by nuclear heterotypia, with changes in mRNA splicing, processing and export leading to increases in mRNA surveillance and recycling.

Virological characteristics of a SARS-CoV-2-related bat coronavirus, BANAL-20-236.

BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).

Cancer therapeutic trispecific antibodies recruiting both T and natural killer cells to cancer cells.

T cells and natural killer (NK) cells are major effector cells recruited by cancer therapeutic bispecific antibodies; however, differences in the populations of these cells in individual tumors limit the general use of these antibodies. In the present study, trispecific antibodies were created, namely T cell and NK cell engagers (TaKEs), that recruit both T cells and NK cells. Notably, three Fc­fused TaKEs were designed, TaKE1­Fc, TaKE2­Fc and TaKE3­Fc, using variable fragments targeting the epidermal growth factor receptor on tumor cells, CD3 on T cells, and CD16 on NK cells. Among them, TaKE1­Fc was predicted to form a circular tetrabody­like configuration and exhibited the highest production and greatest cancer growth inhibitory effects. TaKE1 was prepared from TaKE1­Fc by digesting the Fc region for further functional evaluation. The resulting TaKE1 exhibited trispecificity via its ability to bind cancer cells, T cells and NK cells, as well as comparable or greater cancer growth inhibitory effects to those of two bispecific antibodies that recruit T cells and NK cells, respectively. A functional trispecific antibody with the potential to exert strong therapeutic effects independent of T cell and NK cell populations was developed.

Incorporation of a repeated polypeptide sequence in therapeutic antibodies as a universal masking procedure: A case study of T cell-engaging bispecific antibodies.

Prodrug design is a promising approach for reducing the off-target effects of therapeutic antibodies, particularly bispecific antibodies (bsAbs) that recruit T cells for activation; this design uses masking sequences that inhibit antibody binding until they reach the tumor microenvironment, where they are removed. In this study, we propose PAS, a polypeptide sequence composed of repeated Pro, Ala, and Ser residues, as a universal masking sequence. PAS has no specificity, but can inhibit antibody binding through steric hindrance caused by its large fluid dynamic radius and disordered structure; additionally, its length can be adjusted. We fused PAS to the N-terminus of an anti-CD3 single-chain variable fragment (scFv) and a bsAb, that targets both the epidermal growth factor receptor and CD3, via a recognition sequence cleaved by cancer-related proteases. PAS integration inhibited anti-CD3 scFv binding with higher efficacy than the epitope sequence, and the extent of inhibition was proportional to the length of the PAS sequence. For masked bsAbs, T cell-binding ability, cancer growth inhibition effects, and T cell activation effects were also reduced depending on the length of PAS and were fully restored upon removing PAS sequences using protease. The masking procedure using PAS was successfully applied to another scFv. The provision to adjust the masking effects of PAS by tuning its length, makes PAS fusion a valuable tool for the universal design of prodrug antibodies.

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate.

SignificanceHepatitis C virus chronically infects approximately 1% of the world's population, making an effective vaccine for hepatitis C virus a major unmet public health need. The membrane-associated E1E2 envelope glycoprotein has been used in clinical studies as a vaccine candidate. However, limited neutralization breadth and difficulty in producing large amounts of homogeneous membrane-associated E1E2 have hampered efforts to develop an E1E2-based vaccine. Our previous work described the design and biochemical validation of a native-like soluble secreted form of E1E2 (sE1E2). Here, we describe the immunogenic characterization of the sE1E2 complex. sE1E2 elicited broadly neutralizing antibodies in immunized mice, with increased neutralization breadth relative to the membrane-associated E1E2, thereby validating this platform as a promising model system for vaccine development.

Efficacy of a Cap-Dependent Endonuclease Inhibitor and Neuraminidase Inhibitors against H7N9 Highly Pathogenic Avian Influenza Virus Causing Severe Viral Pneumonia in Cynomolgus Macaques.

H7N9 highly pathogenic avian influenza virus (HPAIV) infection in a human was first reported in 2017. A/duck/Japan/AQ-HE29-22/2017 (H7N9) (Dk/HE29-22), found in imported duck meat at an airport in Japan, possesses a hemagglutinin with a multibasic cleavage site, indicating high pathogenicity in chickens, as in the case of other H7 HPAIVs. In the present study, we examined the pathogenicity of Dk/HE29-22 and the effectiveness of a cap-dependent endonuclease inhibitor (baloxavir) and neuraminidase inhibitors (oseltamivir and zanamivir) against infection with this strain in a macaque model (n = 3 for each group). All of the macaques infected with Dk/HE29-22 showed severe signs of disease and pneumonia even after the virus had disappeared from lung samples. Virus titers in macaques treated with baloxavir were significantly lower than those in the other treated groups. After infection, levels of interferon alpha and beta (IFN-α and IFN-ß) in the blood of macaques in the baloxavir group were the highest among the groups, whereas levels of tumor necrosis factor alpha (TNF-α) and interleukin 13 (IL-13) were slightly increased in the untreated group. In addition, immune checkpoint proteins, including programmed death 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT), were expressed at high levels in the untreated group, especially in one macaque that showed severe signs of disease, indicating that negative feedback responses against vigorous inflammation may contribute to disease progression. In the group treated with baloxavir, the percentages of PD-1-, CTLA-4-, and TIGIT-positive T lymphocytes were lower than those in the untreated group, indicating that reduction in virus titers may prevent expression of immune checkpoint molecules from downregulation of T cell responses.

Inactivated whole virus particle vaccine with potent immunogenicity and limited IL-6 induction is ideal for influenza.

In contrast to current ether- or detergent-disrupted "split" vaccines (SVs) for influenza, inactivated whole influenza virus particle vaccines (WPVs) retain the original virus structure and components and as such may confer similar immunity to natural infection. In a collaboration between academia and industry, the potential of WPV as a new seasonal influenza vaccine was investigated. Each of the four seasonal influenza vaccine manufacturers in Japan prepared WPVs and SVs from the same batches of purified influenza virus. Both mice and monkeys vaccinated with the WPVs exhibited superior immune responses to those vaccinated with the corresponding SVs. Vaccination with A/California/07/2009 (H1N1) WPV enabled mice to survive a lethal challenge dose of homologous virus whereas those vaccinated with SV succumbed to infection within 6 days. Furthermore, mice vaccinated with WPV induced substantial numbers of multifunctional CD8+ T cells, important for control of antigenically drifted influenza virus strains. In addition, cytokines and chemokines were detected at early time points in the sera of mice vaccinated with WPV but not in those animals vaccinated with SV. These results indicate that WPVs induce enhanced innate and adaptive immune responses compared to equivalent doses of SVs. Notably, WPV at one fifth of the dose of SV was able to induce potent immunity with limited production of IL-6, one of the pyrogenic cytokines. We thus propose that WPVs with balanced immunogenicity and safety may set a new global standard for seasonal influenza vaccines.

Engineering the hinge region of human IgG1 Fc-fused bispecific antibodies to improve fragmentation resistance.

Fc domain fusion can improve the therapeutic effects of relatively small biological molecules such as peptides, cytokines, and antibody fragments. Fc fusion proteins can also be used to enhance the cytotoxic effects of small bispecific antibodies (bsAbs). However, fragmentation of Fc fusion proteins, which mainly occurs around the hinge regions during production, storage, and circulation in the blood, is a major issue. In this study, we first investigated the mechanisms of fragmentation around the hinge region during storage using Fc-fused bsAbs with specificity for epidermal growth factor receptor and CD3 as a model. The fragmentation peaks generated by gel filtration analysis indicated that both contaminating proteases and dissolved active oxygen should be considered causes of fragmentation. We designed and constructed variants by introducing a point mutation into the upper hinge region, which reduced the cleavage caused by dissolved active oxygen, and shortened the hinge region to restrict access of proteases. These hinge modifications improved fragmentation resistance and did not affect the biological activity of the bsAbs in vitro. We confirmed the versatility of the hinge modifications using another Fc-fused bsAb. Our results show that hinge modifications to the Fc fusion protein, especially the introduction of a point mutation into the upper hinge region, can reduce fragmentation substantially, and these modifications can be used to improve the fragmentation resistance of other recombinant Fc fusion proteins.

Induction of humoural and cellular immunity by immunisation with HCV particle vaccine in a non-human primate model.

OBJECTIVE: Although HCV is a major cause of chronic liver disease worldwide, there is currently no prophylactic vaccine for this virus. Thus, the development of an HCV vaccine that can induce both humoural and cellular immunity is urgently needed. To create an effective HCV vaccine, we evaluated neutralising antibody induction and cellular immune responses following the immunisation of a non-human primate model with cell culture-generated HCV (HCVcc). DESIGN: To accomplish this, 10 common marmosets were immunised with purified, inactivated HCVcc in combination with two different adjuvants: the classically used aluminum hydroxide (Alum) and the recently established adjuvant: CpG oligodeoxynucleotide (ODN) wrapped by schizophyllan (K3-SPG). RESULTS: The coadministration of HCVcc with K3-SPG efficiently induced immune responses against HCV, as demonstrated by the production of antibodies with specific neutralising activity against chimaeric HCVcc with structural proteins from multiple HCV genotypes (1a, 1b, 2a and 3a). The induction of cellular immunity was also demonstrated by the production of interferon-γ mRNA in spleen cells following stimulation with the HCV core protein. These changes were not observed following immunisation with HCVcc/Alum preparation. No vaccination-related abnormalities were detected in any of the immunised animals. CONCLUSIONS: The current preclinical study demonstrated that a vaccine included both HCVcc and K3-SPG induced humoural and cellular immunity in marmosets. Vaccination with this combination resulted in the production of antibodies exhibiting cross-neutralising activity against multiple HCV genotypes. Based on these findings, the vaccine created in this study represents a promising, potent and safe prophylactic option against HCV.

Glycyrrhizin inhibits human parainfluenza virus type 2 replication by the inhibition of genome RNA, mRNA and protein syntheses.

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.

Increased expression of the regulatory T cell-associated marker CTLA-4 in bovine leukemia virus infection.

Regulatory T cells (Tregs) play a critical role in the maintenance of the host's immune system. Tregs, particularly CD4(+)CD25(+)Foxp3(+) T cells, have been reported to be involved in the immune evasion mechanism of tumors and several pathogens that cause chronic infections. Recent studies showed that a Treg-associated marker, cytotoxic T-lymphocyte antigen 4 (CTLA-4), is closely associated with the progression of several diseases. We recently reported that the proportion of Foxp3(+)CD4(+) cells was positively correlated with the number of lymphocytes, virus titer, and virus load but inversely correlated with IFN-γ expression in cattle infected with bovine leukemia virus (BLV), which causes chronic infection and lymphoma in its host. Here the kinetics of CTLA-4(+) cells were analyzed in BLV-infected cattle. CTLA-4 mRNA was predominantly expressed in CD4(+) T cells in BLV-infected cattle, and the expression was positively correlated with Foxp3 mRNA expression. To test for differences in the protein expression level of CTLA-4, we measured the proportion of CTLA-4-expressing cells by flow cytometry. In cattle with persistent lymphocytosis (PL), mean fluorescence intensities (MFIs) of CTLA-4 on CD4(+) and CD25(+) T cells were significantly increased compared with that in control and aleukemic (AL) cattle. The percentage of CTLA-4(+) cells in the CD4(+) T cell subpopulation was positively correlated with TGF-ß mRNA expression, suggesting that CD4(+)CTLA-4(+) T cells have a potentially immunosuppressive function in BLV infection. In the limited number of cattle that were tested, the anti-CTLA-4 antibody enhanced the expression of CD69, IL-2, and IFN-γ mRNA in anti-programmed death ligand 1 (PD-L1) antibody-treated peripheral blood mononuclear cells from BLV-infected cattle. Together with previous findings, the present results indicate that Tregs may be involved in the inhibition of T cell function during BLV infection.

Expression analysis of Foxp3 in T cells from bovine leukemia virus infected cattle.

In the present study, we monitored Foxp3(+) T cells in bovine leukemia virus (BLV)-infected cattle. By flow cytometric analysis, the proportion of Foxp3(+) CD4(+) cells from persistent lymphocytotic cattle was significantly increased compared to control and AL cattle. Interestingly, the proportion of Foxp3(+) CD4(+) cells correlated positively with the increased number of lymphocytes, virus titer and virus load, whereas it inversely correlated with IFN-γ mRNA expression, suggesting that Foxp3(+) CD4(+) T cells in cattle have a potentially immunosuppressive function. Further studies are necessary to elucidate the detailed mechanism behind the increased Treg during BLV infection.

Enhanced expression of LAG-3 on lymphocyte subpopulations from persistently lymphocytotic cattle infected with bovine leukemia virus.

An immunoinhibitory receptor, lymphocyte activation gene-3 (LAG-3), which is mainly expressed in T-cells, is involved in the immune evasion of several pathogens causing chronic infections and tumors. However, unlike human or mouse LAG-3, no functional analysis of LAG-3 has been reported in domestic animals. Thus, in this study, bovine LAG-3 expression was analyzed in bovine leukemia virus (BLV)-infected cattle. In persistent lymphocytotic (PL) cattle, the numbers of LAG-3(+)CD4(+) cells and LAG-3(+)CD8(+) cells were conserved whilst the number of MHC class II(+) cells was remarkably higher than in the control animals. In contrast, the mean fluorescence intensity (MFI) for LAG-3 on PBMCs from PL cattle was significantly increased compared to control and asymptomatic (AL) cattle. Specifically, the LAG-3 expression level was significantly increased in both CD4(+) and CD8(+) T cells from PL cattle. LAG-3 expression correlated positively with increased numbers of lymphocytes and MHC class II(+) cells in infected animals. Preliminary results from PD-L1 and LAG-3 blockade assay revealed that IFN-γ and IL-2 expressions were significantly up-regulated by addition of anti- PD-L1 and LAG-3 antibodies in PBMCs from PL cattle. These findings suggest that LAG-3 might be involved in the inhibition of T-cell function through its binding and signaling on MHC class II molecule during BLV infection.

Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva.

Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus caffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo.

Increased bovine Tim-3 and its ligand expressions during bovine leukemia virus infection.

The immunoinhibitory receptor T cell immunoglobulin domain and mucin domain-3 (Tim-3) and its ligand, galectin-9 (Gal-9), are involved in the immune evasion mechanisms for several pathogens causing chronic infections. However, there is no report concerning the role of Tim-3 in diseases of domestic animals. In this study, cDNA encoding for bovine Tim-3 and Gal-9 were cloned and sequenced, and their expression and role in immune reactivation were analyzed in bovine leukemia virus (BLV)-infected cattle. Predicted amino acid sequences of Tim-3 and Gal-9 shared high homologies with human and mouse homologues. Functional domains, including tyrosine kinase phosphorylation motif in the intracellular domain of Tim-3 were highly conserved among cattle and other species. Quantitative real-time PCR analysis showed that bovine Tim-3 mRNA is mainly expressed in T cells such as CD4+ and CD8+ cells, while Gal-9 mRNA is mainly expressed in monocyte and T cells. Tim-3 mRNA expression in CD4+ and CD8+ cells was upregulated during disease progression of BLV infection. Interestingly, expression levels for Tim-3 and Gal-9 correlated positively with viral load in infected cattle. Furthermore, Tim-3 expression level closely correlated with up-regulation of IL-10 in infected cattle. The expression of IFN-γ and IL-2 mRNA was upregulated when PBMC from BLV-infected cattle were cultured with Cos-7 cells expressing Tim-3 to inhibit the Tim-3/Gal-9 pathway. Moreover, combined blockade of the Tim-3/Gal-9 and PD-1/PD-L1 pathways significantly promoted IFN-γ mRNA expression compared with blockade of the PD-1/PD-L1 pathway alone. These results suggest that Tim-3 is involved in the suppression of T cell function during BLV infection.

Molecular cloning of bovine lymphocyte activation gene-3 and its expression characteristics in bovine leukemia virus-infected cattle.

Lymphocyte activation gene-3 (LAG-3), a major histocompatibility complex (MHC) class II binding CD4 homologue has recently been shown as one of the mechanisms for down-regulating immune responses during chronic disease progression. For the first time, we cloned LAG-3 from two breeds of cattle (Holstein and Japanese Black), and analyzed its expression levels in cattle infected with bovine leukemia virus (BLV), a chronic viral infection that leads to immuno-suppression. The cloned cDNA of bovine LAG-3 have an open reading frame of 1551 nucleotides, encoding a polypeptide of 515 amino acids in length. Similar to the swine LAG-3, the bovine LAG-3 protein sequence consisted of four extracellular domains, a transmembrane domain and an inhibitory motif, KTGELE. We found that the bovine LAG-3 mRNA transcripts were expressed predominantly on T-cells such as CD4(+) and CD8(+) cells, among peripheral blood mononuclear cells. In subsequent expression analysis, LAG-3 mRNA expression on CD4(+) T-cells from BLV-infected cattle was upregulated compared to that in normal cattle. Comparable results were obtained with CD8(+) T-cells from cattle infected with BLV. We further observed strong upregualtion of MHC class II molecule, the ligand for LAG-3 in BLV-infected cattle. These findings indicate an important role for inhibitory receptor molecules such as LAG-3 in chronic bovine infections and future studies will elucidate the specific role of LAG-3 in bovine diseases.

Development of a quantitative bioassay to assess preventive compounds against inflammation-based carcinogenesis.

Reducing cancer incidence and mortality by use of cancer-chemopreventive agents is an important goal. We have established an in vitro bioassay that is able to screen large numbers of candidate chemicals that are positive for prevention of inflammation-related carcinogenesis. To accomplish this we have added candidate chemicals or vehicles and freshly isolated, fluorescent dye-labeled inflammatory cells that were overlaid on TNF-alpha-stimulated mouse endothelial cells in a 96-well plate. Inhibition of inflammatory cell attachment to the endothelial cells by the chemicals was quantified by the intensity of fluorescence from the adherent inflammatory cells after removing unattached cells. Using this assay, we selected two chemicals, auraptene and turmerones, for further study. As an in vivo test, diets containing these test chemicals were administered to mice with a piece of foreign body, gelatin sponge, that had been implanted to cause inflammation, and we found that the number of inflammatory cells that infiltrated into the subcutaneously implanted gelatin sponge was reduced compared to that found in the mice fed with a control diet. Moreover, diets containing either of the two chemicals prevented inflammation-based carcinogenesis in a mouse model. We found that the compounds reduced not only the number of infiltrating cells but also the expression of inducible nitric oxide synthase (iNOS) or formation of 8-hydroxy-2'-deoxyguanine (8-OHdG) in the infiltrated cells. Moreover, both compounds but not controls sustained the reducing activity in the inflammatory lesion, and this finding was confirmed by using non-invasive in vivo electron spin resonance. The newly established in vitro screening assay will be useful for finding biologically effective chemopreventive agents against inflammation-related carcinogenesis.

Effects of pregnancy and lactation on warfarin-induced changes in blood coagulation-related parameters in rats.

Effects of pregnancy and lactation on warfarin-induced changes in blood coagulation-related parameters were examined in rats. Warfarin (0.5 mg/kg/day) was given orally to pregnant and non-pregnant rats for 3 days from gestation day (GD) 17 to 19 or to lactating and non-pregnant rats for 3 days from post partum day (PPD) 10 to 12. Blood samples were collected from the rats on the day following the last administration (GD 20 or PPD 13) to measure prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombotest (TBT), factor VII and X activities and anti-thrombin III concentration (ATIII). Administration of warfarin to non-pregnant rats resulted in significant prolongation of APTT and TBT and significant decreases in factor VII and X activities. On the other hand, similar but not significant changes were observed in pregnant rats and similar significant but less prominent changes were observed in lactating rats. The reduction of the anticoagulant effects of warfarin may partially be related to high plasma 17beta-estradiol concentration in pregnant rats and to high plasma prolactin concentration in lactating rats, respectively.

Therapeutic potential of proapoptotic molecule Noxa in the selective elimination of tumor cells.

The selective elimination of tumor cells by inducing apoptosis is one of the most important issues in cancer therapy. In this context, artificial expression of the p53 tumor-suppressor gene has been an attractive approach and numerous studies have shown its efficacy in combination with other therapies such as radiation or chemotherapy. One of the critical issues for current cancer gene therapy is how to induce apoptosis in cancer cells without affecting normal cells. In the present study, we examined the potential of Noxa, a BH3-only protein with proapoptotic activity that functions downstream of the p53-mediated apoptotic pathway, to selectively induce apoptosis in tumor cells. We found that upon infection of a recombinant adenovirus contrived to express the Noxa gene, apoptosis was induced in vitro in several human breast cancer cell lines, but not in normal mammary epithelial cell lines. Furthermore, intratumoral injection of the Noxa-expressing adenovirus resulted in marked shrinkage of the transplanted tumor derived from breast cancer cells without any notable adverse effect on the surrounding normal tissue. In contrast, the expression of Puma, another BH3-only protein that also functions downstream of the p53 pathway, induced apoptosis in both cancer and normal cells. Thus, our results suggest a mechanism wherein Noxa, but not Puma, selectively induces apoptosis in human tumor cells. These data provide a new prospect for cancer therapy by the Noxa-mediated selective elimination of malignant cells.

7Alpha-hydroxypregnenolone mediates melatonin action underlying diurnal locomotor rhythms.

Melatonin regulates diurnal changes in locomotor activity in vertebrates, but the molecular mechanism for this neurohormonal regulation of behavior is poorly understood. Here we show that 7alpha-hydroxypregnenolone, a previously undescribed avian neurosteroid, mediates melatonin action on diurnal locomotor rhythms in quail. In this study, we first identified 7alpha-hydroxypregnenolone and its stereoisomer 7beta-hydroxypregnenolone in quail brain. These neurosteroids have not been described previously in avian brain. We then demonstrated that 7alpha-hydroxypregnenolone acutely increased quail locomotor activity. To analyze the production of 7alpha-hydroxypregnenolone, cytochrome P450(7alpha), a steroidogenic enzyme of this neurosteroid, was also identified. Subsequently, we demonstrated diurnal changes in 7alpha-hydroxypregnenolone synthesis in quail. 7Alpha-Hydroxypregnenolone synthesis and locomotor activity in males were much higher than in females. This is the first demonstration in any vertebrate of a clear sex difference in neurosteroid synthesis. This sex difference in 7alpha-hydroxypregnenolone synthesis corresponded to the sex difference in locomotion. We show that only males exhibited marked diurnal changes in 7alpha-hydroxypregnenolone synthesis, and these changes occurred in parallel with changes in locomotor activity. Finally, we identified melatonin as a key component of the mechanism regulating 7alpha-hydroxypregnenolone synthesis. Increased synthesis of 7alpha-hydroxypregnenolone occurred in males in vivo after melatonin removal via pinealectomy and orbital enucleation (Px plus Ex). Conversely, decreased synthesis of this neurosteroid occurred after melatonin administration to Px plus Ex males. This study demonstrates that melatonin regulates synthesis of 7alpha-hydroxypregnenolone, a key factor for induction of locomotor activity, thus inducing diurnal locomotor changes in male birds. This is a previously undescribed role for melatonin.