Down-regulation of macrophage Ia mRNA expression by interferon (IFN)-alpha and IFN-beta mediated by de novo synthesized protein

We investigated the regulation of class I and class II major histocompatibility complex (MHC) antigen expression of murine peritoneal macrophages (M phi) by interferons (IFNs) at the mRNA level. Enhancement of class I antigen expression by IFNs (IFN-alpha, beta, and gamma), induction of class II antigen expression by IFN-gamma, and inhibition of class II antigen expression by IFN-alpha or IFN-beta all corresponded to steady-state levels of these MHC-specific mRNAs. Cycloheximide (CHX), a protein synthesis inhibitor, was used to elucidate whether IFN regulation of MHC mRNA expression depends on the newly synthesized proteins. CHX concentration was carefully chosen so that M phi viability was not decreased, total protein synthesis was considerably but not completely inhibited, and suppression of surface class II expression was virtually perfect. Under these conditions CHX did not affect the levels of either class I or class II mRNA, but it prevented IFN-beta from interfering with class II mRNA induction by IFN-gamma. These results indicate that the augmentation of induction and/or accumulation of MHC mRNA by IFNs is not dependent on the de novo synthesis of protein, but the down-regulation of class II mRNA level by IFN-beta is mediated by some newly synthesized protein(s).     

Three-year survey of the epidemiology of rotavirus, enteric adenovirus, and some small spherical viruses including "Osaka-agent" associated with infantile diarrhea

Studies were made by electron microscopy (EM) on the viruses associated with diarrhea of outpatients at a pediatric clinic in Osaka Prefecture during the three year period from 1980 through 1982. The viruses detected by EM by negative staining with phosphotungstic acid (PTA) were classified morphologically into 6 groups: rotavirus, adenovirus and four kinds of small spherical viruses, calicivirus, astrovirus, picornavirus/parvovirus (P/P)-like agent and Osaka-agent. Osaka-agent seems to be a newly identified small virus. It is 35-40 nm in diameter with a fringe of spike-like structures on its surface. Viruses were detected in 181 of the 395 cases of diarrhea (45.8%). Rotavirus was detected in 122 (30.9%) of the total cases and in 67.4% of the virus-positive cases, while other viruses were detected in 15% of the total cases; adenovirus in 23 (6%) and small agents in 36 (9%). Rotavirus infection showed a distinctive seasonal variation, being mainly restricted to cooler months, but infections with other viruses did not show any seasonal variation. The age distribution of patients suggested that infants of 0 to 2 years old are very susceptible to all viruses. Attempts to cultivate these viruses in vitro were successful with only two isolates of adenovirus type 5.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

Vitamin D receptor contains multiple dimerization interfaces that are functionally different.

The vitamin D receptor mediates the signal of 1 alpha, 25-dihydroxyvitamin D3 by binding to vitamin D responsive elements in DNA as a homodimer or as a heterodimer composed of one vitamin D receptor subunit and one retinoid X receptor subunit. We have mapped the dimerization interfaces of the vitamin D receptor that is involved in homo- or heterodimer formation in the absence of DNA. While deletion of the first zinc finger region of vitamin D receptor diminished homodimerization activity, it did not affect heterodimerization. In contrast, a deletion just beyond the zinc finger region affected heterodimerization with retinoid X receptor, but not homodimerization. The zinc finger region alone could form a homodimer with full-length vitamin D receptor, but not a heterodimer with retinoid X receptor. The carboxy-terminal region was also necessary for heterodimer formation. This region showed only a weak dimerization activity in the absence of ligand, but this was dramatically increased in the presence of ligand for both homo- and heterodimerization. These results suggest that the vitamin D receptor has at least three dimerization interfaces whose functions are apparently distinguishable. These are located in the first zinc finger region, the region just beyond this zinc finger and in the carboxy-terminal region.     

Induced pluripotent stem cell‐derived myeloid cells expressing OX40 ligand amplify antigen‐specific T cells in advanced melanoma

Immune checkpoint inhibitors improved the survival rate of patients with unresectable melanoma. However, some patients do not respond, and variable immune‐related adverse events have been reported. Therefore, more effective and antigen‐specific immune therapies are urgently needed. We previously reported the efficacy of an immune cell therapy with immortalized myeloid cells derived from induced pluripotent stem cells (iPS‐ML). In this study, we generated OX40L‐overexpressing iPS‐ML (iPS‐ML‐Zsgreen‐OX40L) and investigated their characteristics and in vivo efficacy against mouse melanoma. We found that iPS‐ML‐Zsgreen‐OX40L suppressed the progression of B16‐BL6 melanoma, and prolonged survival of mice with ovalbumin (OVA)‐expressing B16 melanoma (MO4). The number of antigen‐specific CD8⁺ T cells was higher in spleen cells treated with OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L than in those without OX40L. The OVA peptide‐pulsed iPS‐ML‐Zsgreen‐OX40L significantly increased the number of tumor‐infiltrating T lymphocytes (TILs) in MO4 tumor. Flow cytometry showed decreased regulatory T cells but increased effector and effector memory T cells among the TILs. Although we plan to use allogeneic iPS‐ML in the clinical applications, iPS‐ML showed the tumorgenicity in the syngeneic mice model. Incorporating the suicide gene is necessary to ensure the safety in the future study. Collectively, these results indicate that iPS‐ML‐Zsgreen‐OX40L therapy might be a new method for antigen‐specific cancer immunotherapy.     

Partial silencing of fucosyltransferase 8 gene expression inhibits proliferation of Ishikawa cells,a cell line of endometrial cancer

Endometrial cancer is the most common gynecologic malignancy and is associated with increased morbidity each year, including young people. However, its mechanisms of proliferation and progression are not fully elucidated. It is well known that abnormal glycosylation is involved in oncogenesis, and fucosylation is one of the most important types of glycosylation. In particular, fucosyltransferase 8 (FUT8) is the only FUT responsible for α1, 6-linked fucosylation (core fucosylation), and it is involved in various physiological as well as pathophysiological processes, including cancer biology. Therefore, we aimed to identify the expression of FUT8 in endometrial endometrioid carcinoma and investigate the effect of the partial silencing of the FUT8 gene on the cell proliferation of Ishikawa cells, an epithelial-like endometrial cancer cell line. Quantitative real-time PCR analysis showed that FUT8 gene expression was significantly elevated in the endometrial endometrioid carcinoma, compared to the normal endometrium. The immunostaining of FUT8 and Ulex europaeus Agglutinin 1 (UEA-1), a kind of lectin family specifically binding to fucose, was detected endometrial endometrioid carcinoma. The proliferation assay showed FUT8 partial knockdown by transfection of siRNA significantly suppressed the proliferation of Ishikawa cells, concomitant with the upregulation in the gene expressions associated with the interesting pathways associated with de-ubiquitination, aspirin trigger, mesenchymal-epithelial transition (MET) et al. It was suggested that the core fucosylation brought about by FUT8 might be involved in the proliferation of endometrial endometrioid carcinoma cells.     

Polymorphic mutations in mouse mitochondrial DNA regulate a tumor phenotype

To examine whether polymorphic mtDNA mutations that do not induce significant respiration defects regulate phenotypes of tumor cells, we used mouse transmitochondrial tumor cells (cybrids) with nuclear DNA from C57BL/6 (B6) strain and mtDNA from allogenic C3H strain. The results showed that polymorphic mutations of C3H mtDNA in the cybrids induced hypoxia sensitivity, resulting in a delay of tumor formation on their subcutaneous inoculation into B6 mice. Therefore, the effects of polymorphic mutations in normal mtDNA have to be carefully considered, particularly when we apply the gene therapy to the embryos to replace their pathogenic mtDNA by normal mtDNA.     

TNF-α Acts as an Immunoregulator in the Mouse Brain by Reducing the Incidence of Severe Disease Following Japanese Encephalitis Virus Infection

Japanese encephalitis virus (JEV) causes acute central nervous system (CNS) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. However, the mechanism of severe encephalitis has not been fully elucidated. In this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal JEV infection. Following extraneural infection with the JaOArS982 strain of JEV, infected mice exhibited clinical signs ranging from mild to fatal outcome. Comparison of the pathogenetic response between severe and mild cases of JaOArS982-infected mice revealed increased levels of TNF-α in the brains of severe cases. However, unexpectedly, the mortality rate of TNF-α KO mice was significantly increased compared with that of WT mice, indicating that TNF-α plays a protective role against fatal infection. Interestingly, there were no significant differences of viral load in the CNS between WT and TNF-α KO mice. However, exaggerated inflammatory responses were observed in the CNS of TNF-α KO mice. Although these observations were also obtained in IL-10 KO mice, the mortality and enhanced inflammatory responses were more pronounced in TNF-α KO mice. Our findings therefore provide the first evidence that TNF-α has an immunoregulatory effect on pro-inflammatory cytokines in the CNS during JEV infection and consequently protects the animals from fatal disease. Thus, we propose that the increased level of TNF-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. In future, further elucidation of the immunoregulatory mechanism of TNF-α will be an important priority to enable the development of effective treatment strategies for Japanese encephalitis.     

Expression of Pituitary Adenylate Cyclase-Activating Peptide (PACAP) and PAC1 in the Periodontal Ligament After Tooth Luxation

Pituitary adenylate cyclase-activating peptide (PACAP) is widely distributed throughout the nervous system. PACAP not only acts as a neurotransmitter but also elicits a broad spectrum of biological action via the PACAP-specific receptor, PAC1. However, no studies have investigated PACAP and PAC1 in the periodontal ligament (PDL), so we aimed to perform this investigation in rats after tooth luxation. In the PDL of an intact first molar, there are few osteoclasts and osteoblasts. However, at days 3 and 5 after luxation, large PAC1-positive cells, thought to be osteoclasts because of their expression of the osteoclast marker, tartrate-resistant acid phosphatase, were detected in appreciable numbers. Osteoblast numbers increased dramatically on day 7 after luxation, and PAC1-positive mononuclear small cells were increased at day 14, many of which expressed the osteoblast marker, alkaline phosphatase. PACAP-positive nerve fibers were rarely detected in the PDL of intact first molars, but were increasingly evident at this site on days 5 and 7 after luxation. Double-immunofluorescence analysis demonstrated the relationship between PACAP-positive nerve fibers and PAC1-positive osteoclasts/-blasts in the PDL. At 5 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoclasts. At 7 days after luxation, PACAP-positive nerve fibers appeared in close proximity to PAC1-positive osteoblasts. These results suggest that PACAP may have effects on osteoclasts and osteoblasts in the PDL after tooth luxation and thus regulate bone remodeling after these types of injury.