Dietary nucleotides increase the mucosal IgA response and the secretion of transforming growth factor β from intestinal epithelial cells in mice

We have investigated the influence of dietary nucleotides on the intestinal immune system in ovalbumin (OVA)-specific T-cell receptor (TCR) transgenic mice (OVA-TCR Tg mice). When mice were supplied with water supplemented with 2% OVA ad libitum, the faecal OVA-specific immunoglobulin A (IgA) level significantly increased in those fed a nucleotide-supplemented diet (NT(+) diet) compared with those fed a nucleotide-free control diet (NT(–) diet). In the NT(+) diet-fed mice, secretion of transforming growth factor β (TGF-β), which is an isotype-specific switch factor for IgA, from intestinal epithelial cells (IECs) was significantly increased. Furthermore, an increased proportion of intestinal intraepithelial lymphocytes (IELs) bearing γδ TCR (TCRγδ⁺ IELs) and increased secretion from IECs of interleukin 7 (IL-7), which is essential for the development of TCRγδ⁺ IELs, were also observed in OVA-TCR-Tg mice fed the NT(+) diet, as we previously demonstrated using BALB/c mice (Nagafuchi et al., Biosci. Biotechnol. Biochem. 64: 1459-65 (2000)). Considering that TCRγδ⁺ T cells and TGF-β are important for an induction of the mucosal IgA response, our results suggest that dietary nucleotides augment the mucosal OVA-specific IgA response by increasing the secretion of TGF-β from IECs and the proportion of TCRγδ⁺ IELs. This revised version was published online in August 2006 with corrections to the Cover Date.     

The DNA damage sensors ataxia-telangiectasia mutated kinase and checkpoint kinase 2 are required for hepatitis C virus RNA replication

Cellular responses to DNA damage are crucial for maintaining genome integrity, virus infection, and preventing the development of cancer. Hepatitis C virus (HCV) infection and the expression of the HCV nonstructural protein NS3 and core protein have been proposed as factors involved in the induction of double-stranded DNA breaks and enhancement of the mutation frequency of cellular genes. Since DNA damage sensors, such as the ataxia-telangiectasia mutated kinase (ATM), ATM- and Rad3-related kinase (ATR), poly(ADP-ribose) polymerase 1 (PARP-1), and checkpoint kinase 2 (Chk2), play central roles in the response to genotoxic stress, we hypothesized that these sensors might affect HCV replication. To test this hypothesis, we examined the level of HCV RNA in HuH-7-derived cells stably expressing short hairpin RNA targeted to ATM, ATR, PARP-1, or Chk2. Consequently, we found that replication of both genome-length HCV RNA (HCV-O, genotype 1b) and the subgenomic replicon RNA were notably suppressed in ATM- or Chk2-knockdown cells. In addition, the RNA replication of HCV-JFH1 (genotype 2a) and the release of core protein into the culture supernatants were suppressed in these knockdown cells after inoculation of the cell culture-generated HCV. Consistent with these observations, ATM kinase inhibitor could suppress the HCV RNA replication. Furthermore, we observed that HCV NS3-NS4A interacted with ATM and that HCV NS5B interacted with both ATM and Chk2. Taken together, these results suggest that the ATM signaling pathway is critical for HCV RNA replication and may represent a novel target for the clinical treatment of patients with chronic hepatitis C.     

Cooperative effect of the attenuation determinants derived from poliovirus sabin 1 strain is essential for attenuation of enterovirus 71 in the NOD/SCID mouse infection model

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and is also associated with serious neurological disorders. An attenuated EV71 strain [EV71(S1-3′)] has been established in the cynomolgus monkey infection model; this strain contains the attenuation determinants derived from the type 1 poliovirus vaccine strain, Sabin 1 [PV1(Sabin)], in the 5′ nontranslated region (NTR), 3D polymerase, and 3′ NTR. In this study, we analyzed the effect of the attenuation determinants of PV1(Sabin) on EV71 infection in a NOD/SCID mouse infection model. We isolated a mouse-adapted EV71 strain [EV71(NOD/SCID)] that causes paralysis of the hind limbs in 3- to 4-week-old NOD/SCID mice by adaptation of the virulent EV71(Nagoya) strain in the brains of NOD/SCID mice. A single mutation at nucleotide 2876 that caused an amino acid change in capsid protein VP1 (change of the glycine at position 145 to glutamic acid) was essential for the mouse-adapted phenotype in NOD/SCID mice. Next, we introduced attenuation determinants derived from PV1(Sabin) along with the mouse adaptation mutation into the EV71(Nagoya) genome. In 4-week-old mice, the determinants in the 3D polymerase and 3′ NTR, which are the major temperature-sensitive determinants, had a strong effect on attenuation. In contrast, the effect of individual determinants was weak in 3-week-old NOD/SCID mice, and all the determinants were required for substantial attenuation. These results suggest that a cooperative effect of the attenuation determinants of PV1(Sabin) is essential for attenuated neurovirulence of EV71.     

Postnatal growth of the rat palatine gland

To elucidate how the palatine glands grow postnatally, the palatine glands of rats from 0 to 8 weeks of age were investigated histologically and immunohistochemically. Under light microscope, three dimensions of the right part of the palatine glands were measured and the total number of excretory ducts of the glands was counted from the parasagittal serial sections. Immunohistochemistry with anti-5-bromo-2'-deoxyuridine (BrdU) monoclonal antibody was also employed to detect the cellular proliferative activity. At birth (0 weeks), the palatine glands consisted of ducts and immature acini. The ducts in the glands were connected with excretory ducts. After 2 weeks, there was no duct in the glands. Most acinar cells became mature as mucous cells and took the form of tubulo-acini connected directly with excretory ducts. In the posterior region of the glands, serous acinar cells forming demilunes were occasionally seen. All three dimensions of the palatine glands became longer, and the number of excretory ducts tended to increase. Immunohistochemistry showed acinar and duct cells were highly proliferative in early stage of postnatal life and their proliferative activity decreased thereafter. This study demonstrated that immature rat palatine glands of newborn rats grow three-dimensionally during maturation, and that the parenchymal cell proliferation contributes to the growth of the rat palatine glands. In addition, it is suggested that the glandular tissue arises from the excretory ducts formed postnatally.     

The reaction of osteoclasts when releasing osteocytes from osteocytic lacunae in the bone during bone modeling

Osteocytes are released from the osteocytic lacunae when osteoclasts resorb the bone matrix during bone modeling and remodeling. It remains unknown how osteoclasts react when releasing osteocytes during bone modeling, and the fate of these released osteocytes is also unclear. Femoral mid-shafts of 2-day-old kittens were sectioned into serial 0.5 microm-thick semithin or 0.1 microm-thick ultrathin sections, and examined by light microscopy (LM) and transmission electron microscopy (TEM). The sections showed many osteoclasts at the endosteum but there were no osteoblasts. There were many half-released, fully released, half-exposed, and fully exposed osteocytes on the bone surfaces. Many cell-like structures were seen in the cell bodies of osteoclasts by LM, and some semithin sections were re-sectioned into ultrathin sections for re-observation by TEM. By TEM, these were determinated to be mononuclear cells. The serial ultrathin sections showed that the mononuclear cells appeared to be engulfed in osteoclasts on one section but that the cell was connected with the bone surface of the osteocytic lacuna on another section. These results show that the mononuclear cells in the osteoclasts were osteocytes. The present study suggests that osteoclasts engulf some osteocytes but do not engulf others when releasing osteocytes during bone modeling.     

Infection of human hepatocyte chimeric mouse with genetically engineered hepatitis C virus and its susceptibility to interferon

We developed a reverse genetics system of hepatitis C virus (HCV) genotypes 1a and 2a using infectious clones and human hepatocyte chimeric mice. We inoculated cell culture-produced genotype 2a (JFH-1) HCV intravenously. We also injected genotype 1a CV-H77C clone RNA intrahepatically. Mice inoculated with HCV by both procedures developed measurable and transmissible viremia. Interferon (IFN) alpha treatment resulted in greater reduction of genotype 2a HCV levels than genotype 1a, as seen in clinical practice. Genetically engineered HCV infection system should be useful for analysis of the mechanisms of resistance of HCV to IFN and other drugs.     

The Role of VP1 Amino Acid Residue 145 of Enterovirus 71 in Viral Fitness and Pathogenesis in a Cynomolgus Monkey Model

Enterovirus 71 (EV71), a major causative agent of hand, foot, and mouth disease, occasionally causes severe neurological symptoms. We identified P-selectin glycoprotein ligand-1 (PSGL-1) as an EV71 receptor and found that an amino acid residue 145 in the capsid protein VP1 (VP1-145) defined PSGL-1-binding (PB) and PSGL-1-nonbinding (non-PB) phenotypes of EV71. However, the role of PSGL-1-dependent EV71 replication in neuropathogenesis remains poorly understood. In this study, we investigated viral replication, genetic stability, and the pathogenicity of PB and non-PB strains of EV71 in a cynomolgus monkey model. Monkeys were intravenously inoculated with cDNA-derived PB and non-PB strains of EV71, EV71-02363-EG and EV71-02363-KE strains, respectively, with two amino acid differences at VP1-98 and VP1-145. Mild neurological symptoms, transient lymphocytopenia, and inflammatory cytokine responses, were found predominantly in the 02363-KE-inoculated monkeys. During the early stage of infection, viruses were frequently detected in clinical samples from 02363-KE-inoculated monkeys but rarely in samples from 02363-EG-inoculated monkeys. Histopathological analysis of central nervous system (CNS) tissues at 10 days postinfection revealed that 02363-KE induced neuropathogenesis more efficiently than that induced by 02363-EG. After inoculation with 02363-EG, almost all EV71 variants detected in clinical samples, CNS, and non-CNS tissues, possessed a G to E amino acid substitution at VP1-145, suggesting a strong in vivo selection of VP1-145E variants and CNS spread presumably in a PSGL-1-independent manner. EV71 variants with VP1-145G were identified only in peripheral blood mononuclear cells in two out of four 02363-EG-inoculated monkeys. Thus, VP1-145E variants are mainly responsible for the development of viremia and neuropathogenesis in a non-human primate model, further suggesting the in vivo involvement of amino acid polymorphism at VP1-145 in cell-specific viral replication, in vivo fitness, and pathogenesis in EV71-infected individuals.     

Reduced thymic size and numbers of splenic CD4+ and CD8+ cells in artificially reared mouse pups

The effect of early nutrition on the development of the immune tissue and T cells of mouse pups was examined. Newborn mice were divided into three experimental groups: mother-reared (MR) pups, pups that were fed on a milk substitute from the first day (AR-0), and the third day (AR-2), using a hand-feeding system. The average thymic size of the AR-2 pups was respectively significantly larger and smaller than that of the AR-0 and MR pups. In contrast, the splenic sizes of the AR-0 and AR-2 pups were greater than that of the MR pups. The numbers of CD4+CD8- and CD4-CD8+ cells in the spleen of the MR pups were significantly higher than those in the AR-0 pups. These results indicate that early nutrition affected the sizes of the thymus and spleen and the composition of CD4+CD8- or CD4-CD8+ T cells in the spleen.     

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.     

Formation of covalently closed circular DNA in Hep38.7-Tet cells,a tetracycline inducible hepatitis B virus expression cell line

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in chronic HBV infection. However, analysis of the molecular mechanism of cccDNA formation is difficult because of the low efficiency in tissue cultured cells. In this study, we developed a more efficient cccDNA expression cell, Hep38.7-Tet, by subcloning from a tetracycline inducible HBV expression cell, HepAD38. Higher levels of cccDNA were produced in Hep38.7-Tet cells compared to HepAD38 cells. In Hep38.7-Tet cells, the cccDNA was detectable at six days after HBV induction. HBV e antigen (HBeAg) secretion was dependent upon cccDNA production. We screened chemical compounds using Hep38.7-Tet cells and HBeAg secretion as a marker. Most of the hit compounds have already been reported as anti-HBV compounds. These data suggested that Hep38.7-Tet cells will be powerful tools for analysis of the molecular mechanism of cccDNA formation/maintenance and development of novel therapeutic agents to control HBV infection.