First identification of canine adenovirus 1 in mink and bioinformatics analysis of its 100 K protein.

Introduction: Animal trade favors the spreading of emerging canine adenovirus 1 (CAdV-1) in mink. Because the 100K protein is not exposed to the viral surface at any stage, it can be used to differentiate the vaccine from wild virus infection. However, no related research has been conducted. This study aimed to find evidence of CAdV-1 in mink and predict the character of the 100K protein in the current circulating CAdV-1 strain of mink. Method: In this experiment, the identification of CAdV-1, the phylogenetic tree, homology, and bioinformatics analysis of 100K were conducted. Results: The results showed that the CAdV-1 was identified in the mink and that its Fiber was located in a separate branch. It was closely related to strains isolated from Norwegian Arctic fox and Red fox. 100K was located in a separate branch, which had the closest genetic relationship with skunks, porcupines, raccoons, and hedgehogs and a far genetic relationship with the strains in dogs. 100K protein is an unstable and hydrophobic protein. It had evidence of selective pressure and recombination, 1 glycosylation site, 48 phosphorylation sites, 60 dominant B cell epitopes, and 9 peptides of MHC-I and MHC-II. Its subcellular localization was mainly in the endoplasmic reticulum and mitochondria. The binding sites of 100K proteins were DBP proteins and 33K proteins. Discussion: The stains in the mink were different from fox. The exploration of its genomic characteristics will provide us with a deeper understanding of the prevention of canine adenovirus.

Immunomodulatory Effect of Ginsenoside Rb2 Against Cyclophosphamide-Induced Immunosuppression in Mice.

Ginsenoside Rb2 (Rb2), a fundamental saponin produced and isolated from ginseng (Panax ginseng C.A. Meyer), has a wide range of biological actions. The objective of this investigation was to see if ginsenoside Rb2 has any immunomodulatory properties against cyclophosphamide (CTX)-induced immunosuppression. For the positive control group, levamisole hydrochloride (LD) was used. We discovered that intraperitoneal injection of Rb2 (5, 10, 20 mg/kg) could relieve CTX-induced immunosuppression by enhanced immune organ index, reduced the pathological characteristics of immunosuppression, promoted natural killer (NK) cells viability, improved cell-mediated immune response, boosted the IFN-γ (Interferon-gamma), TNF-α (Tumor necrosis factor-alpha), IL-2 (Interleukin-2), and IgG (Immunoglobulin G), as well as macrophage activity like carbon clearance and phagocytic index. Rb2 significantly elevated the mRNA expression of IL-4 (Interleukin-4), SYK (Tyrosine-protein kinase-SYK), IL-2, TNF-α, and IL-6 (Interleukin-6) in the spleen of CTX-injected animals. Molecular docking results showed that Rb2 had excellent binding properties with IL-4, SYK, IL-2, TNF, and IL-6, indicating the target protein might be strongly correlated with the immunomodulatory effect of Rb2. Taken together, ginsenoside Rb2 can improve the immune function that is declined in CTX-induced immunosuppressed mice, the efficacy maybe due to the regulation of related cytokine and mRNA expression.

Canine Adenovirus 1 Isolation Bioinformatics Analysis of the Fiber.

Canine adenovirus type 1 (CAdV-1) is a double-stranded DNA virus, which is the causative agent of fox encephalitis. The Fiber protein is one of the structural proteins in CAdV-1, which mediates virion binding to the coxsackievirus and adenovirus receptor on host cells. The suspected virus was cultured in the MDCK cells, and it was determined through the cytopathic effects, sequencing and electron microscopy. The informatics analysis of the Fiber was done using online bioinformatics servers. The CAdV-1-JL2021 strain was isolated successfully, and were most similar to the CAdV-1 strain circulating in Italy. The occurrence of negative selection and recombination were found in the CAdV-1-JL2021 and CAdV-2-AC_000020.1. Host cell membrane was its subcellular localization. The CAdV-1-JL2021 Fiber (ON164651) had 6 glycosylation sites and 107 phosphorylation sites, exerted adhesion receptor-mediated virion attachment to host cell, which was the same as CAdV-2-AC_000020.1 Fiber. The Fiber tertiary structure of the CAdV-1-JL2021 and CAdV-2-AC_000020.1 was different, but they had the same coxsackievirus and adenovirus receptor. "VATTSPTLTFAYPLIKNNNH" were predicted to be the potential CAdV-1 B cell linear epitope. The MHC-I binding peptide "KLGVKPTTY" were both presented in the CAdV-1-JL2021 and CAdV-2-AC_000020.1 Fiber and it is useful to design the canine adenovirus vaccine.

Difference Analysis Between Canine Adenovirus Types 1 And 2.

Canine adenoviruses (CAdVs) include type 1 (CAdV-1, virulent strain) and type 2 (CAdV-2, attenuated strain). In recent years, the incidences of CAdV infections are increasing. However, they are difficult to distinguish when the symptoms are untypical. It is pivotal to find the differences between the two virus types for scientific, epidemiological, and specific treatment. CAdV-1 (virulent strain) and CAdV-2 (attenuated strain) induced canine hepatitis (ICH) and tracheobronchitis (ITB), respectively, but the clinical symptom is not obvious. CAdV-1 and CAdV-2 have the same genome structure, diameter, morphological features, and cytopathic features, but the same character hinder the diagnose time of the serotypes. CAdV-1 and CAdV-2 have a difference in the genome sequence, coding proteins, viral activity, hemagglutination patterns. After infection, pathogenicity and transmission route are different between the two serotypes. Sequence alignment, PCR, Real time-PCR assay are useful methods to distinguish the two serotypes. The attenuated live CAdV-2 vaccine is currently used to protect against CAdV-1, but it also has a risk. The further research should focus on the pathogenicity mechanism and the useful vaccine for the two serotypes of canine adenovirus.

Immunogenicity of an Inactivated Canine Adenovirus Type 1 Vaccine for Foxes.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis. As with most viral agents, the best method of prevention is vaccination. In this study, the CAdV-1 strain F1301 strain was used to construct a new type 1 canine adenovirus inactivated vaccine candidate, and its safety and immunogenicity were evaluated in silver foxes. Next, animals were challenged and survival rates of animals vaccinated with either the commercially available or the current candidate vaccine were examined. The results confirmed that the inactivated CAdV-1 vaccine prepared in this study can effectively protect against challenge with virulent CAdV-1 in silver foxes, and the safety profile was improved relative to that of the commercial vaccine. This study confirmed that the fox CAdV-1 F1301 strain can be used as a platform for an inactivated CAdV-1 vaccine.

Identification and primary application of hybridomas cell secreting monoclonal antibodies against mink (Neovison vison) interferon-gamma.

Due to their susceptibility to several human viruses, the mink has been proposed as potential animal models for the study of human viral infections. However, there are no specific monoclonal antibody (mAbs) currently available for the detection of mink-specific interferon-gamma (miIFN-γ). The BALB/c mice were immunized intraperitoneally with purified recombinant miIFN-γ protein. The splenocytes were obtained and fused with murine myeloma cells. Five of 24 hybridoma clones were obtained to produce mAbs steadily with the strongest affinity to recombinant miIFN-γ protein. The isotype of the 31A, 31B and 31G were lgG 2b. The isotype of 44 and 46 were lgG 2a and 1. All five mAbs were κ light chains. Western blotting and indirect ELISA method showed that 5 mAbs were positive to miIFN-γ. Immunofluorescence showed that 2 mAbs (44 and 46) had a positive reaction to miIFN-γ. The hybridoma clone 46 had the highest sensitivity for the detection of miIFN-γ. Most importantly, our primary sandwich ELISA system (mAbs 46 and polyclonal antiserum) detected endogenous IFN-γ in mink lymphocytes infected with canine distemper virus (CDV). We have thus developed a novel mAbs could recognize miIFN-γ, and have demonstrated the first ELISA-based measurement of IFN-γ in lymphocyte of the mink.

The role of TGF-ß1/Smad3 signaling pathway and oxidative stress in the inhibition of osteoblast mineralization by copper chloride.

To explore the relationship of oxidative stress and TGF-ß 1/Smad3 pathway in the inhibition of osteoblast mineralization by copper chloride (CuCl2), the osteoblasts were treated with CuCl2 (0, 50 µM, 100 µM, 150 µM CuCl2 5H2O) for 24 h. We found that Cu impaired the osteoblast structure, inhibited the glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities, alkaline phosphatase (ALP) content, mRNA expression of collagen I (COL-I), osteocalcin (OCN), insulin-like growth factor I (IGF-I), bone morphogenetic protein-2 (BMP-2), transforming growth factor ß1 (TGF-ß1) and core-binding factor α1 (Cbfα1), promoted the reactive oxygen species (ROS) production, inactivated the TGF-ß1/Smad3 pathway. It indicates that the inactivated TGF-ß1/Smad3 pathway leads to osteoblast impairment by CuCl2. It will contribute to clarify the influence of CuCl2 on the osteoblast mineralization.

Identification of Two Novel Linear Neutralizing Epitopes within the Hexon Protein of Canine Adenovirus Using Monoclonal Antibodies.

Canine adenovirus (CAdV) has a high prevalence in canine populations. High affinity neutralizing antibodies against conserved epitopes can provide protective immunity against CAdV and protect against future outbreaks. In this study, we identified two CAdV-2-specific neutralizing monoclonal antibodies (mAbs), 2C1 and 7D7, which recognized two linear-dependent epitopes. MAb 2C1 potently neutralized CAdV-2 with a 50% neutralization titer (NT50) of 4096, and mAb 7D7 partially neutralized CAdV-2 with a 50% NT50 of 64. Immunoprecipitation, Western blot and protein spectral analysis indicated that both neutralizing mAbs recognized the hexon protein (Hex) of CAdV-2. Through a 12-mer random peptide phage display and synthetic peptides analysis, we finely mapped the neutralizing epitopes to two 10-amino acid (aa) peptides within the CAdV Hex: 634RIKQRETPAL643 located on the surface region; and 736PESYKDRMYS745 located in the inner region of the expected 3D structure of trimeric Hex. Importantly, the two epitopes are highly conserved among all CAdV isolates by sequence alignment analysis. Thus, these results provide insights into the interaction between virus and mAbs at the aa level and may have potential applications in the development of novel therapeutic or epitope-based vaccines, antibody therapeutics and a diagnostic method suitable for the rapid detection of all CAdVs.

The biological characteristics of the canine adenovirus type 1 from fox and the transcriptome analysis of the infected MDCK cell.

Canine adenovirus type 1 (CAdV-1) is the etiologic agent of fox encephalitis, and a virus strain from fox encephalitis is isolated and related research are conducted. In this experiment, the results showed that the F1301 strain was confirmed to be the CAdV-1. The whole genome of the CAdV-1 F1301 strain isolated from fox was 30,535 bp and had higher homology to the other reported CAdV-1 strains. After 0, 12, and 36 h of CAdV-1 infection, the difference gene of the 592 long noncoding RNA and 11,215 microRNA were involved in cell responses to CAdV-1 infection through the PI3K-AKT, Wnt, Herpes simplex, hepatitis C, and Epstein-Barr virus infection pathway in Madin-Darby canine kidney cell line (MDCK). The results indicate that the biological characterization of the CAdV-1 and the MDCK cell-CAdV-1 interaction are clarified.

Cytoprotective effect of deferiprone against aluminum chloride-induced oxidative stress and apoptosis in lymphocytes.

Aluminum (Al) is a toxic metal, and excessive Al accumulation causes immunosuppression. Deferiprone (DFP) is a well-known chelator and used in dialysis patients for removing Al from tissues. The present study aimed to investigate whether DFP treatment can attenuate immunotoxicity induced by aluminum chloride (AlCl3) in cultured lymphocytes. Lymphocytes were treated with 0 and 0.6 mmol/L AlCl3∙6H2O (pH 7.2) and/or 1.8 mmol/L DFP, respectively. Immune function of lymphocytes was assessed by T and B lymphocytes proliferation rates, T lymphocyte subpopulations and IL-2, IL-6 and TNF-α contents. In addition, lymphocyte damage was assessed by LDH activity, NO and MDA contents, NOS, SOD and GSH-Px activities, lymphocyte apoptosis index. These results showed that AlCl3 exposure reduced T and B lymphocyte proliferation rates, CD3+ and CD4+ T lymphocyte subpopulations, CD4+/CD8+ ratio, IL-2, IL-6 and TNF-α contents, SOD and GSH-Px activities, early and later lymphocyte apoptosis indexes while enhanced CD8+ T lymphocyte subpopulation, NO and MDA contents, LDH activity. DFP treatment attenuated the immunotoxicity of lymphocytes and reduced oxidative stress and lymphocyte apoptosis induced by AlCl3, indicating that DFP could protect lymphocytes against immunosuppression induced by AlCl3 through attenuating oxidative stress and apoptosis.

Aluminum Chloride Causes the Dysfunction of Testes Through Inhibiting the ATPase Enzyme Activities and Gonadotropin Receptor Expression in Rats.

The aim of this experiment is to explore the effects of aluminum chloride (AlCl3) on the ATPase enzymes and gonadotropin receptors in the testes. Eighty male Wistar rats were orally exposed to 0 mg/kg body weight (BW) (control group, CG), 64 mg/kg BW (low-dose group, LG), 128 mg/kg BW (mid-dose group, MG), or 256 mg/kg BW (high-dose group, HG) for 120 days. The microstructure and ultrastructure of testes; the activities of Na+-K+-ATPase, Mg2+-ATPase, and Ca2+-ATPase; and the mRNA and protein expressions of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptors (LHR) in the testes were examined. The results showed that the testes histological structure were damaged; the activities of Na+-K+-ATPase, Mg2+-ATPase, and Ca2+-ATPase, the mRNA and protein expressions of FSHR and LHR in the testes were all decreased in the rats with AlCl3 exposure. It indicates that AlCl3 causes the dysfunction of testes in rats.

Aluminum chloride caused liver dysfunction and mitochondrial energy metabolism disorder in rat.

Aluminum (Al) is known to exert hepatotoxicity. However, the mechanisms mostly are unclear. Liver is a metabolism organ that maintains the energy level and structural stability of body, mitochondria are the main sites of energy metabolism, thus, we hypothesized that mitochondrial energy metabolism disorder contributes to liver dysfunction in aluminum chloride (AlCl3) treatment rat. To verify the hypothesis, forty male Wistar rats were randomly allocated and orally exposed to 0, 64mg/kg, 128mg/kg and 256mg/kg body weight AlCl3 in drinking water for 120days, respectively. We found that AlCl3 exposure reduced the electron transport chain complexes I-V activities and adenosine triphosphate (ATP) level, as well as disturbed mitochondrial DNA transcript, presenting as the inhibited mRNA expressions of NADH dehydrogenase 1, NADH dehydrogenase 2, cytochrome b, cytochrome c oxidase subunit 1, cytochrome c oxidase subunit 3 and ATP synthase 6, indicating that AlCl3 exposure disturbs the mitochondrial energy metabolism, and it caused an increase in liver enzymes (Aspartate aminotransferase and Alanine aminotransferase) and histopathological lesions. Additionally, we found that reactive oxygen species accumulation and decreased superoxide dismutase activity in mitochondria, and increased 8-Hydroxydeoxyguanosine levels in mitochondrial DNA, demonstrating AlCl3 exposure promotes mitochondrial oxidative stress, which may be a contributing factor to mitochondrial energy metabolism disorder and liver dysfunction. The study displayed that mitochondria are the potential target of liver damage induced by AlCl3, providing considerable direction for the prevention and clinical intervention of liver diseases.

Protective Effect of Selenium on Aflatoxin B1-Induced Testicular Toxicity in Mice.

Aflatoxins have been considered as one of the major risk factors of male infertility, and aflatoxin B1 (AFB1) is the most highly toxic and prevalent member of the aflatoxins family. Selenium (Se), an essential nutritional trace mineral for normal testicular development and male fertility, has received extensive intensive on protective effects of male reproductive system due to its potential antioxidant and activating testosterone synthesis. To investigate the protective effect of Se on AFB1-induced testicular toxicity, the mice were orally administered with AFB1 (0.75 mg/kg) and Se (0.2 mg/kg or 0.4 mg/kg) for 45 days. We found that that Se elevated testes index, sperm functional parameters (concentration, malformation, and motility), and the level of serum testosterone in AFB1-exposed mice. Moreover, our results showed that Se attenuated the AFB1-induced oxidative stress and the reduction of testicular testosterone synthesis enzyme protein expression such as steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage (P450scc), and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) in AFB1-exposed mice. These results demonstrated that Se conferred protection against AFB1-induced testicular toxicity and can be attributed to its antioxidant and increased testosterone level by stimulating protein expression of StAR and testosterone synthetic enzymes.

Inhibition of bone formation in rats by aluminum exposure via Wnt/ß-catenin pathway.

The previous research found that aluminum trichloride (AlCl3) inhibited rat osteoblastic differentiation through inactivation of Wnt/ß-catenin signaling pathway in vitro. On that basis, the experiment in vivo was conducted in this study. Rats were orally exposed to 0 (control group) and 0.4 g/L AlCl3 (AlCl3-treated group) for 30, 60, 90 or 120 days, respectively. We found that mRNA expressions of type I collagen and insulin-like growth factor-1, mRNA and protein expressions of Runx2 and survivin, ratio of p-GSK3ß/GSK3ß and protein expression of ß-catenin were all decreased, whereas the mRNA and protein expressions Dkk1 and sFRP1 and the mRNA expressions and activity of Caspase-3 were increased in the AlCl3-treated group compared with the control group with time prolonged. These results suggest that AlCl3 inhibits bone formation and induces bone impairment by inhibiting the Wnt/ß-catenin signaling pathway in young growing rats.

Aluminum Trichloride Inhibited Osteoblastic Proliferation and Downregulated the Wnt/ß-Catenin Pathway.

Aluminum (Al) exposure inhibits bone formation. Osteoblastic proliferation promotes bone formation. Therefore, we inferred that Al may inhibit bone formation by the inhibition of osteoblastic proliferation. However, the effects and molecular mechanisms of Al on osteoblastic proliferation are still under investigation. Osteoblastic proliferation can be regulated by Wnt/ß-catenin signaling pathway. To investigate the effects of Al on osteoblastic proliferation and whether Wnt/ß-catenin signaling pathway is involved in it, osteoblasts from neonatal rats were cultured and exposed to 0, 0.4 mM (1/20 IC50), 0.8 mM (1/10 IC50), and 1.6 mM (1/5 IC50) of aluminum trichloride (AlCl3) for 24 h, respectively. The osteoblastic proliferation rates; Wnt3a, lipoprotein receptor-related protein 5 (LRP-5), T cell factor 1 (TCF-1), cyclin D1, and c-Myc messenger RNA (mRNA) expressions; and p-glycogen synthase kinase 3ß (GSK3ß), GSK3ß, and ß-catenin protein expressions indicated that AlCl3 inhibited osteoblastic proliferation and downregulated Wnt/ß-catenin signaling pathway. In addition, the AlCl3 concentration was negatively correlated with osteoblastic proliferation rates and the mRNA expressions of Wnt3a, c-Myc, and cyclin D1, while the osteoblastic proliferation rates were positively correlated with mRNA expressions of Wnt3a, c-Myc, and cyclin D1. Taken together, these findings indicated that AlCl3 inhibits osteoblastic proliferation may be associated with the inactivation of Wnt/ß-catenin signaling pathway.

Effects of aluminum trichloride on the cartilage stimulatory growth factors in rats.

Aluminum (Al) is considered to be a potentially toxic metal and inhibits cartilage formation. Transforming growth factor ß1 (TGF-ß1) and bone morphogenetic protein 2 (BMP-2) are cartilage stimulatory growth factors, which play important roles in regulating the cartilage formation. To investigate the effects of aluminum trichloride (AlCl3) on the regulation of cartilage formation. Eighty Wistar rats were orally exposed to 0 (control group), 0.4 g/L (low-dose group), 0.8 g/L (mid-dose group) and 1.6 g/L (high-dose group) AlCl3 for 120 days, respectively. The rats body weight were decreased, the cartilage histological structure were disrupted, the cartilage and serum contents of Al and the serum level of C-telopeptide of type II collagen were all increased, the serum level of type II collagen (Col II) and alkaline phosphatase (ALP), and the mRNA expressions of TGF-ß1, BMP-2, ALP and Col II were all decreased in the AlCl3-treated groups compared with those in control group. These results indicate that AlCl3 inhibits the cartilage formation through inhibition of the cartilage stimulatory growth factors expressions.

Aluminum trichloride induces bone impairment through TGF-ß1/Smad signaling pathway.

Aluminum (Al) is recognized worldwide as serious inorganic contaminants. Exposure to Al is associated with low BMD and an increased risk of osteoporosis. However, the precise molecular mechanisms remains unclear. Thus, in this study, rats were orally exposed to 0 (control group, CG) and 0.4g/L AlCl3 (AlCl3 treated group, AG) in drinking water for 120days; osteoblasts were treated with AlCl3 (0.12mg/mL) and/or TGF-ß1 (4.5ng/mL) for 24h. We found that AlCl3 decreased the BMD, damaged femoral ultrastructure, decreased the activities of GSH-Px and SOD, and increased the levels of ROS and MDA in bone, decreased the activity of B-ALP and content of PINP, and increased the activity of TRACP-5b and content of NTX-I in serum, decreased mRNA expressions of TGF-ß1, TßRI, TßRII and Smad4, protein expressions of TGF-ß1, p-Smad2/3 and Smad2/3/4 complex, and increased Smad7 mRNA expression in bone and in osteoblasts. Moreover, we found exogenous TGF-ß1 application reversed the inhibitory effect of AlCl3 on osteoblasts activity by activating the TGF-ß1/Smad signaling pathway and increasing the mRNA expressions of ALP and Col I in osteoblasts. These results demonstrate that AlCl3 induces bone impairment through inactivation of TGF-ß1/Smad signaling pathway.

The suppressive effects of aluminum chloride on the osteoblasts function.

Aluminum (Al) exposure impairs bone formation, and bone formation is mediated by the osteoblasts. But effects of Al on the osteoblasts function remain elusive. The osteoblasts were exposed to 0, 0.0252, 0.126, 0.252mg/mL AlCl3·6H2O for 24h. The osteoblasts viability, TGF-ß1, BMP-2, IGF-I and Cbfα1 mRNA expressions, and GSH-Px and SOD activities, ROS concentration were determined. The osteoblasts ultrastructural features were also observed. The results showed that AlCl3 suppressed the osteoblasts viability, TGF-ß1, BMP-2, IGF-I and Cbfα1 mRNA expressions, GSH-Px and SOD activities, and elevated ROS concentration compared with the CG. The ultrastructural features of osteoblasts in the HG showed mitochondrial swelling, foam-like structure, uneven distribution of chromatin, incomplete cell membrane and cytoplasm spillover compared with the CG. It indicates that AlCl3 inhibits osteoblasts viability, growth regulation factors mRNA expressions, anti-oxidative function, and damaged the osteoblasts histology structure, impairing the osteoblasts function.

Inhibition of osteoblast differentiation by aluminum trichloride exposure is associated with inhibition of BMP-2/Smad pathway component expression.

Bone morphogenetic protein-2 (BMP-2)/Smad signaling pathway plays an important role in regulating osteoblast (OB) differentiation. OB differentiation is a key process of bone formation. Aluminum (Al) exposure inhibits bone formation and causes Al-induced bone disease. However, the mechanism is not fully understood. To investigate whether BMP-2/Smad signaling pathway is associated with OB differentiation in aluminum trichloride (AlCl3)-treated OBs, the primary rat OBs were cultured and exposed to 0 (control group, CG), 1/40 IC50 (low-dose group, LG), 1/20 IC50 (mid-dose group, MG), and 1/10 IC50 (high-dose group, HG) of AlCl3 for 24 h, respectively. We found that the expressions of OB differentiation markers (Runx-2, Osterix and ALP) and BMP-2/Smad signaling pathway components (BMP-2, BMPR-IA, p-BMPR-IA, BMPR-II, p-Smad1/5/8 and p-Smad1/5/8/4) were all decreased in AlCl3-treated OBs compared with the CG. These results indicated that inhibition of OB differentiation by AlCl3 was associated with inhibition of BMP-2/Smad pathway component expression. Our findings provide a novel insight into the mechanism of AlCl3-induced bone disease.