Prevalence and genetic features of rabbit hepatitis E virus in Korea.

Hepatitis caused by hepatitis E virus (HEV) is a public health concern worldwide. HEV strains have been isolated from several animal species, some of which induce zoonosis. Recently, the isolation of HEV from rabbits was reported. Here, the partial capsid gene (320 bp) of HEV was detected in rabbit feces via reverse transcriptase-polymerase chain reaction (RT-PCR). Rabbit HEV was found in two of six rabbit farms and 17 of 264 rabbit fecal samples (6.4%). A phylogenetic analysis of the partial capsid gene classified the 17 HEV isolates into the putative rabbit HEV clade. A full genomic sequence, KOR-Rb-1, was obtained from one rabbit HEV isolate by 5' and 3' rapid amplification of cDNA ends-PCR and RT-PCR, and comprised 7275 bp excluding the 3' poly(A) tail. It shared 77.5-86.8%, 86.6%, and 80.2-84.3% nucleotide identities with rabbit HEV isolates from China, the US, and France, respectively. It also shared 72.3-73.0%, 71.4%, 76.7-78.3%, 72.8-73.3%, and 47.1-47.2% nucleotide identities with representative strains of HEV-1, HEV-2, HEV-3, HEV-4, and avian HEV, respectively. A full-genome phylogenetic analysis classified KOR-Rb-1 into the provisional rabbit HEV clade. This isolate could be used to study the pathogenesis and zoonotic potential of rabbit HEV.

A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Taurine Distribution in Various Tissues With and Without Taurine Supplementation.

Taurine, a sulfur containing amino acid, has various physiological functions including development of the eye and brain, immune function, reproduction, osmo-regulatory function as well as anti-oxidant and anti-inflammatory activities. In order to understand the physiological role, we developed taurine deficient mice deleting a rate-liming enzyme, cysteine sulfinic acid decarboxylase (CSAD) for biosynthesis of taurine. Taurine was measured in various tissues including the liver, brain, lung, spleen, thymus, pancreas, heart, muscle and kidney as well as plasma from CSAD knock-out mice (CSAD KO) with and without treatment of taurine in the drinking water at the age of 2 months (2 M). Taurine was determined using HPLC as a phenylisothiocyanate derivative of taurine at 254 nm. Taurine concentrations in the liver and kidney from homozygotes of CSAD KO (HO), in which CSAD level is high, were 90% and 70% lower than WT, respectively. Taurine concentrations in the brain, spleen and lung, where CSAD level is low, were 21%, 20% and 28% lower than WT, respectively. At 2 M, 1% taurine treatment of HO restored taurine concentrations in all tissues compared to that of WT. To select an appropriate taurine treatment, HO were treated with various concentrations (0.05, 0.2, 1%) of taurine for 4 months (4 M). Restoration of taurine in all tissues except the liver, kidney and lung requires 0.05% taurine to be restored to that of WT. The liver and kidney restore taurine back to WT with 0.2% taurine. To examine which enzymes influence taurine concentrations in various tissues from WT and HO at 2 M, expression of five taurine-related enzymes, two antioxidant enzymes as well as lactoferrin (Lft) and prolactin receptor (Prlr) was determined using RT2 qPCR. The expression of taurine transporter in the liver, brain, muscle and kidney from HO was increased except in the lung. Our data showed expression of glutamate decarboxylase-like 1(Gadl-1) was increased in the brain and muscle in HO, compared to WT, indicating taurine in the brain and muscle from HO was replenished through taurine transporter and increased biosynthesis of taurine by up-regulated Gadl-1. The expression of glutathione peroxidase 3 was increased in the brain and peroxireductase 2 was increased in the liver and lung, suggesting taurine has anti-oxidant activity. In contrast to newborn and 1 month CSAD KO, Ltf and Prlr in the liver from CSAD KO at 2 M were increased more than two times and 52%, respectively, indicating these two proteins may be required for pregnancy of CSAD KO. Ltf in HOT1.0 was restored to WT, while Prlr in HOT1.0 was increased more than HO, explaining improvement of neonatal survival with taurine supplementation.These data are essential for investigating the role of taurine in development of the brain and eye, immune function, reproduction and glucose tolerance.

A Novel Cysteine Sulfinic Acid Decarboxylase Knock-Out Mouse: Immune Function (II).

Taurine deficient mice lacking cysteine sulfinic acid decarboxylase (CSAD KO) were developed for investigating the various physiological roles of taurine including the development of the brain and eye as well as immune function. Due to severe abnormalities of immune function in a taurine deficient cat, the immune function including adoptive and innate immunity in taurine-deficient mice have been studied. Previously we demonstrated that B cell function in CSAD KO was reduced in both females and males. However, T cell function was significantly reduced only in females. In this study, we have examined innate immunity using macrophage activation with LPS or/and IFN-γ and polymorphonuclear leukocytes (PMN) activation with phorbol myristate acetate (PMA). Pro- and anti-inflammatory cytokines including IL-6, TNF-α and IL-10 as well as nitric oxide (NO) were determined using ELISA and Griess reagent, respectively. Peritoneal macrophages were activated with 1 µg/mL of lipopolysaccharide (LPS) and/or 50 U/mL of IFN-γ. In addition, superoxide anion was measured using peritoneal PMN activated with PMA in the presence and absence of superoxide dismutase. Superoxide anion production in activated PMN from CSAD KO homozygotes (HO) was not significantly different from wild-type (WT) with and without 25 mM taurine. IL-10 and TNF-α production in both female and male CSAD KO were not significantly different. IL-6 and NO were significantly lower only in females as previously observed in Con A-activated cellular proliferation of splenocytes. Cytokine production with 10 mM of taurine was not different, indicating the reduction of NO and IL-6 in females may be due to the absence of the CSAD gene, not due to low taurine concentrations.These data indicate that some measures of innate immunity were altered in female CSAD mice.

Inflammatory mediators are inhibited by a taurine metabolite in CpG oligodeoxynucleotide and IFN-r activated macrophage cell line.

Taurine plays an important role in brain and retinal development, and has an antiinflammatory and antioxidant function. Taurine chloramine (Tau-Cl) is produced in polymorphonuclear leukocytes via the myeloperoxidase/halide system. We previously demonstrated that Tau-Cl inhibits the production of nitric oxide (NO) and TNF-α in human and murine macrophages activated with IFN-γ in combination with individual Toll-like receptor (TLR) ligands including those for TLR2 and/or TLR4. In the current study, we further explored the effects of Tau-Cl in RAW 264.7 cells stimulated with the TLR9 ligand CpG oligodeoxynucleotide (ODN). Specifically, we examined the effect of CpG ODN plus IFN-γ on the production of NO and TNF-α, and the effect of Tau-Cl on this process. Our findings show that CpG ODN plus IFN-γ-activated RAW 264.7 cells secrete high levels of NO and TNF-α, and that Tau-Cl (0.8 mM) inhibits this effect in a dose-dependent manner, more potently inhibiting the production of NO (99% inhibition) than that of TNF-α (48% inhibition). Nitric oxide synthase (iNOS) protein was also induced by CpG ODN plus IFN-γ, and was also inhibited by Tau-Cl. Furthermore, while CpG ODN plus IFN-γ induced TNF-α and iNOS mRNAs, Tau-Cl transiently suppressed this effect. Taurine itself had no effects on any of these processes. Our findings in a macrophage cell line demonstrate that Tau-Cl inhibits proinflammatory mediators resulting from TLR9 activation, and have implications for the utility of Tau-Cl in scenarios where such activation is deleterious such as in autoimmune conditions or infections in which overwhelming inflammation may occur. CpG ODNs and Tau-Cl both have potential for topical treatment of autoimmune conditions, including psoriasis, vitiligo, and alopecia areata. As CpG ODNs may, under some conditions, up-regulate Tregs, addition of Tau-Cl to CpG ODN topical formulations has potential for improving cancer immunotherapy.

Taurine chloramine inhibits NO and TNF-α production in zymosan plus interferon-γ activated RAW 264.7 cells.

Taurine is present abundantly in various tissues, especially in leukocytes embattled to foreign invaders such as microorganisms or oxidants. Taurine-chloramine (Tau-Cl) is produced from taurine at the site of inflammation via the myeloperoxidase-halide pathway in leukocytes induced by oxidants and/or infectious materials. Previously, our data demonstrated that Tau-Cl inhibited nitric oxide (NO) production and TNF-α secretion induced by lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR-4) ligand or lipoarabinomannan (LAM), a TLR-2 ligand plus interferon-γ (IFN-γ) in peritoneal macrophages or RAW 264.7 cells. Zymosan, a ß-glucan of yeast cell wall, is a ligand for TLR-2 and dectin-1 and stimulates macrophages to produce proinflammatory mediators such as NO and TNF-α. Based on our previous data, we examined the effect of zymosan and IFN-γ induced production of NO and TNF-α in the absence or presence of Tau-Cl or taurine using RAW 264.7 cells. Production of NO and secretion of TNF-α is increased when zymosan is combined with IFN-γ. Tau-Cl inhibited production of NO and secretion of TNF-α in zymosan plus IFN-γ activated RAW 264.7 cells in a dose-dependent manner (99% vs. 48% using 0.8mM Tau-Cl). Taurine was without effect. Nitric oxide synthase protein (iNOS), induced by zymosan plus IFN-γ, was inhibited by Tau-Cl (0.8mM) as measured using western blot analysis. NOS mRNA was inhibited by Tau-Cl at four, eight and 16 hours post activation, but not at 24 hours. TNF-α mRNA was inhibited at four hours and eight hours, but not at 16 and 24 hours. These data suggest that expression of both iNOS and TNF-α mRNAs are inhibited by treatment with Tau-Cl within four and eight hours, but not at later time points. Transient suppression of activation of RAW 264.7 cells induced by zymosan may play a critical physiological role for taurine in protecting against tissue injury from initial overt inflammation. This study indicates that tropical treatment of taurine may ameliorate inflammatory dermatoses caused by an environmental yeast or abnormal immune function.

Accelerated prion disease pathogenesis in Toll-like receptor 4 signaling-mutant mice.

Prion diseases such as scrapie involve the accumulation of disease-specific prion protein, PrP(Sc), in the brain. Toll-like receptors (TLRs) are a family of proteins that recognize microbial constituents and are central players in host innate immune responses. The TLR9 agonist unmethylated CpG DNA was shown to prolong the scrapie incubation period in mice, suggesting that innate immune activation interferes with prion disease progression. Thus, it was predicted that ablation of TLR signaling would result in accelerated pathogenesis. C3H/HeJ (Tlr4(Lps-d)) mice, which possess a mutation in the TLR4 intracellular domain preventing TLR4 signaling, and strain-matched wild-type control (C3H/HeOuJ) mice were infected intracerebrally or intraperitoneally with various doses of scrapie inoculum. Incubation periods were significantly shortened in C3H/HeJ compared with C3H/HeOuJ mice, regardless of the route of infection or dose administered. At the clinical phase of disease, brain PrP(Sc) levels in the two strains of mice showed no significant differences by Western blotting. In addition, compared with macrophages from C3H/HeOuJ mice, those from C3H/HeJ mice were unresponsive to fibrillogenic PrP peptides (PrP residues 106 to 126 [PrP(106-126)] and PrP(118-135)) and the TLR4 agonist lipopolysaccharide but not to the TLR2 agonist zymosan, as measured by cytokine production. These data confirm that innate immune activation via TLR signaling interferes with scrapie infection. Furthermore, the results also suggest that the scrapie pathogen, or a component(s) thereof, is capable of stimulating an innate immune response that is active in the central nervous system, since C3H/HeJ mice, which lack the response, exhibit shortened incubation periods following both intraperitoneal and intracerebral infections.

Identification of single-nucleotide polymorphisms of the prion protein gene in sika deer (Cervus nippon laiouanus).

Polymorphisms of the prion protein gene (PRNP) have been detected in several cervid species. In order to confirm the genetic variations, this study examined the DNA sequences of the PRNP obtained from 33 captive sika deer (Cervus nippon laiouanus) in Korea. A total of three single-nucleotide polymorphisms (SNPs) at codons 100, 136 and 226 in the PRNP of the sika deer were identified. The polymorphic site located at codon 100 has not been reported. The SNPs detected at codons 100 and 226 induced amino acid substitutions. The SNP at codon 136 was a silent mutation that does not induce any amino acid change. The genotype and allele frequencies were determined for each of the SNPs.

Anti-inflammatory activity of herbal medicines: inhibition of nitric oxide production and tumor necrosis factor-alpha secretion in an activated macrophage-like cell line.

Houttuynia cordata Thunb. (HC), Glycyrrhiza uralensis Fischer (GU), Forsythia suspense (Thunb.) Vahl (FS), and Lonicera japonica Thunb. (LJ) are Chinese herbs known to possess anti-inflammatory properties. The effects of aqueous extracts of these herbs on the production of the pro-inflammatory mediators, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) were examined in an activated macrophage-like cell line, RAW 264.7 cells. Aqueous extracts from FS at 0.0625-2.0 mg/ml inhibited in vitro production of NO and secretion of TNF-alpha in a dose-dependent manner. FS at 1.0-2.0 mg/ml and 0.125-2.0 mg/ml significantly inhibited NO production and TNF-alpha, respectively. An extract of LJ demonstrated potent inhibition of both NO production and TNF-alpha secretion in a dose-dependent manner. An aqueous extract from HC inhibited NO production in a dose-dependent manner, but minimally (approximately 30%) inhibited TNF-a secretion at 0.0625 and 0.125 mg/ml. In contrast, an aqueous extract of GU had a minimal effect on both the production of NO and the secretion of TNF-alpha. Viability of cells at all concentrations studied was unaffected as determined by MTT cytotoxicity assay and trypan blue dye exclusion. These results suggest that aqueous extracts from FS, LJ and HC have anti-inflammatory actions as measured by inhibition of NO production and/or TNF-alpha secretion.

Platycodin D and D3 isolated from the root of Platycodon grandiflorum modulate the production of nitric oxide and secretion of TNF-alpha in activated RAW 264.7 cells.

Platycodon D (PD) and D3 (PD3) isolated from Platycodon grandiflorum has been previously reported to show anti-inflammatory activities in rats. In this study, the production of proinflammatory cytokines, nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) was examined in a macrophage like cell line, RAW 264.7 cells, in the presence of PD and PD3, oligosaccharide derivatives of oleanolic acid. RAW 264.7 cells activated with lipopolysaccharide (LPS; 1 microg/ml) and recombinant interferon-gamma (rIFN-gamma; 50 U/ml) were treated with various doses of PD and PD3 for 24 h. Supernatants were analyzed for the production of NO and TNF-alpha using Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. NO was inhibited in a dose-dependent manner by PD and PD3 (IC50 of platycodin D approximately 15 uM, IC50 PD3 approximately 55 uM). The expression of inducible NOS (iNOS) was inhibited by these compounds, as measured by Western blot analysis, as well as the expression of iNOS mRNA, as measured by Northern blot analysis. RAW 264.7 cells were treated at various times after LPS and activation with PD. Treatment with PD up to 8 h after activation showed significant inhibition of NO, indicating that early signal transduction of NOS synthesis may be inhibited by PD. In contrast to NO, secretion of TNF-alpha as well as expression of TNF-alpha mRNA was increased by PD and PD3. TNF-alpha secretion from RAW 264.7 cells was measured at various times after LPS and rIFN-gamma activation. Secretion of TNF-alpha was also increased up to 8 h postactivation, suggesting that PD may stimulate TNF-alpha synthesis or inhibit degradation of TNF-alpha mRNA. Oleanolic acid was without effect on both the production of NO and secretion of TNF-alpha. These data suggest a dichotomous regulation of these important proinflammatory mediators by PD and PD3.

Altered lymphocyte proliferation and innate immune function in scrapie 139A- and ME7-infected mice.

Lymphoid organs play an important role in prion disease development and progression. While the role of lymphoid organs and changes in immune-related genes have been extensively investigated in scrapie-infected animals, innate immunity has not. Previous studies examined lymphocyte function in scrapie-infected C3H/HeJ mice, which exhibit defects in lipopolysaccharide (LPS) response now known to result from a mutation in Toll-like receptor (TLR) 4. We examined immune function in scrapie-infected CD1 mice, which are LPS responders. Lymphocyte proliferation from CD1 mice infected with either 139A or ME7 scrapie was measured in response to concanavalin (Con) A or LPS at 1 and 3 months after infection. Following LPS exposure, mice infected 3 months with ME7, but not 139A, demonstrated significantly decreased lymphocyte proliferation compared to controls. After Con A exposure, lymphocyte proliferation in scrapie-infected mice did not differ from controls. Gender-specific comparison of lymphocyte proliferation showed significant decreases in mitogenic responses in females infected 3 months with either 139A or ME7, compared to controls. Males infected for 3 months with ME7, but not 139A, showed significantly decreased proliferation after lymphocyte exposure to LPS, but not Con A. Neither gender showed changes in lymphocyte proliferation after 1 month of scrapie infection. Innate immune activation of peritoneal macrophages was determined via production of nitric oxide (NO), IL-6, and TNF-α after exposure to TLR ligands. TNF-α and IL-6 production were reduced in macrophages from females infected with either scrapie strain for 3 months, while NO production after TLR agonist plus IFN-γ exposure was decreased in both females and males infected for 3 months with 139A, compared to ME7. These data demonstrated altered innate immunity, suggesting hormonal and/or other gender-specific regulation may contribute to gender differences in some immune functions. Our data demonstrate lymphocyte proliferation and innate immune functioning in scrapie-infected mice deteriorate with disease progression.

Discrimination of animal species using polymorphisms of the nuclear gene zinc finger protein 238.

We screened 3750 single exonic genes listed in the intronless genes in the eukaryotes (SEGE) database and performed bioinformatic analyses to identify candidate genes for new species-specific markers. A set of PCR primers for the conserved regions of ZNF238 was developed and used to amplify the 823 bp DNA fragment. We compared nucleotide variations of the PCR products among 20 species plus two subspecies of animals, which led to the identification of interspecies nucleotide variations. To establish a simple method for the analysis of species-specific DNA polymorphisms using ZNF238, we developed a PCR-RFLP method using HhaI and HpyCH4IV restriction enzymes for 13 species. For the remaining species, the direct sequencing of PCR products provided additional SNPs, enabling precise species classification. As a result, we report here that a new nuclear DNA marker, ZNF238, can be used to increase the accuracy of species identification among euteleostomi (bony vertebrates).

Synergistic interaction of xyloglucan and xanthan investigated by rheology, differential scanning calorimetry, and NMR.

A new synergistic interaction between tamarind seed xyloglucan and xanthan was found and investigated by rheology, differential scanning calorimetry (DSC), and NMR. The effect of the acetyl and pyruvate groups in the side chain in xanthan on the synergistic interaction was also examined. The shear moduli G' and G' ' of the mixture solution of xyloglucan and native (or acetate-free) xanthan increased steeply at around 22 degrees C upon cooling. An exothermic DSC peak appeared at the same temperature. A drastic decrease in the of the acetyl and pyruvate groups of the xanthan side chain was observed from 1H NMR spectra only in the mixture at low temperatures (<25 degrees C). It was found that the pyruvate group is more restricted in the mixture solution compared with the acetyl group. The mixture of xyloglucan and pyruvate-free xanthan showed no synergistic interaction. We concluded that this synergistic interaction is caused by the intermolecular binding between xyloglucan and xanthan, and, in the heterotypic junction zones, the xanthan side chain becomes a new state that is different from both the coil and helix states.