Primary goose kidney tubular epithelial cells for goose astrovirus genotype 2 infection: establishment and RNA sequencing analysis.

Goose astrovirus genotype 2 (GAstV-2) mainly causes gout in goslings; therefore, it is a major pathogen threatening to goose flocks. However, the mechanisms underlying host-GAstV-2 interactions remain unclear because host cells suitable for GAstV-2 replication have been unavailable. We previously noted that GAstV-2 is primarily located in goose renal epithelial cells, where it causes kidney damage. Therefore, here, we derived goose primary renal tubular epithelial (RTE) cells (GRTE cells) from the kidneys of goose embryos after collagenase I digestion. After culture in Dulbecco's modified Eagle medium/Nutrient mixture F-12 with 10% fetal bovine serum (FBS), the isolated cells had polygonal with roadstone-like morphology; they were identified to be epithelial cells based on the presence of cytokeratin 18 expression detected through immunofluorescence assay (IFA). GAstV-2 infection in GRTE cells led to no obvious cytopathic effects; the maximum amounts of infectious virions were observed 48 h post infection through IFA and quantitative PCR. Next, RNA-seq was performed to identify and map post-GAstV-2 infection differentially expressed genes. The downregulated pathways were mainly related to metabolism, including tryptophan metabolism, drug metabolism by cytochrome P450, xenobiotic metabolism by cytochrome P450, retinol metabolism, butanoate metabolism, starch and sucrose metabolism, ascorbate and aldarate metabolism, and drug metabolism by other enzymes and peroxisome. In contrast, the upregulated pathways were mostly related to the host cell defense and proliferation, including extracellular matrix-receptor interaction, complement and coagulation cascades, phagosome, PI3K-Akt signaling pathway, human T-lymphotropic virus 1 infection, lysosome, and tumor necrosis factor signaling pathway. In conclusion, we developed a GRTE cell line for GAstV-2 replication and analyzed the potential host-GAstV-2 interactions through RNA-seq; our results may aid in further investigating the pathogenic mechanisms underlying GAstV-2 infection and provide strategies for its prevention and control.

Effects of Goose Astrovirus Type 2 Infection on Peripheral Blood Lymphocyte and Macrophage Activity In Vitro.

Goose astrovirus type 2 (GAstV-2) is a novel pathogen causing visceral gout in goslings; it not only causes necrosis of renal epithelial cells but also causes spleen damage, indicating that GAstV-2 induces immunosuppression in goslings. However, to date, the interaction between GAstV-2 and immune cells remains unclear. In this study, peripheral blood lymphocytes and macrophages were isolated from goslings without GAstV-2 infection and then inoculated in vitro with GAstV-2, and the virus localization in the lymphocytes and macrophages, proliferation and apoptosis of lymphocytes, and phagocytic activity, reactive oxygen species (ROS) and nitric oxide (NO) production, and cell polarity in macrophages were determined. The results showed that GAstV-2 was observed in the cytoplasm of CD4 and CD8 T cells and macrophages, indicating that GAstV-2 can infect both lymphocytes and macrophages. GAstV-2 infection reduced the lymphocyte proliferation induced by Concanavalin A and lipopolysaccharide stimulation and increased the lymphocyte apoptosis rate and mRNA expression of Fas, demonstrating that GAstV-2 causes damage to lymphocytes. Moreover, GAstV-2 infection enhanced phagocytic activity and production of ROS and NO and induced a proinflammatory phenotype in macrophages (M1 macrophages), indicating that macrophages play an antiviral role during GAstV-2 infection. In conclusion, these results demonstrate that GAstV-2 infection causes damages to lymphocytes, and host macrophages inhibit GAstV-2 invasion during infection.

Repurposing maduramicin as a novel anticancer and anti-metastasis agent for triple-negative breast cancer as enhanced by nanoemulsion.

Triple-negative breast cancer (TNBC) is featured by aggression and metastasis and remains an unmet medical challenge due to high death rate. We aimed to repurpose maduramicin (MAD) as an effective drug against TNBC, and develop a nanoemulsion system to enhance anticancer efficacy of MAD. MDA-MB-231 and 4 T1 cells were used as in vitro model, and cell viability was determined by performing cell counting kit-8 and a colony-formation assay. Furthermore, MAD loaded nanoemulsion (MAD-NEs) was manufactured and characterized by a series of tests. The anticancer and anti-metastasis mechanism of MAD-NEs were assessed by performing cell cycle, apoptosis, wound-healing, transwell assay and Western blotting assays. Herein, MAD was firstly demonstrated to be an effective agent to suppress growth of TNBC cells. Subsequently, the optimized MAD-NEs were shown to have stability and high encapsulation efficiency, and could arrested cells in G0/G1 phase and induced apoptosis in TNBC cells. More importantly, MAD-NEs significantly impeded the metastasis of tumor cells, which was further demonstrated by the significant altered expression of epithelial-mesenchymal transition and extracellular matrix markers in vitro and in vivo. Moreover, compared to MAD, MAD-NEs exhibited higher efficacy in shrinking breast tumor size and repressing liver and lung metastasis in vivo, and showed excellent biocompatibility in tumor-bearing mice. The successfully prepared MAD-NEs are expected to be harnessed to suppress tumor growth, invasion and metastasis in the battle against malignant TNBC.

Orally Administered Halofuginone-Loaded TPGS Polymeric Micelles Against Triple-Negative Breast Cancer: Enhanced Absorption and Efficacy with Reduced Toxicity and Metastasis.

Background: Halofuginone (HF)-loaded TPGS polymeric micelles (HTPM) were successfully fabricated using the thin-film hydration technique. HTPM via intravenous injection have been demonstrated to exert an excellent anticancer effect against triple-negative breast cancer (TNBC) cells and subcutaneous xenografts. In the present study, we further explored the potential treatment effect and mechanism of orally administered HTPM alone and in combination with surgical therapy on TNBC in subcutaneous and orthotopic mouse models. Methods: Herein, the stability and in vitro release behavior of HTPM were first evaluated in the simulated gastrointestinal fluids. Caco-2 cell monolayers were then used to investigate the absorption and transport patterns of HF with/without encapsulation in TPGS polymeric micelles. Subsequently, the therapeutic effect of orally administered HTPM was checked on subcutaneous xenografts of TNBC in nude mice. Ultimately, orally administered HTPM, combined with surgical therapy, were utilized to treat orthotopic TNBC in nude mice. Results: Our data confirmed that HTPM exhibited good stability and sustained release in the simulated gastrointestinal fluids. HF was authenticated to be a substrate of P-glycoprotein (P-gp), and its permeability across Caco-2 cell monolayers was markedly enhanced via heightening intracellular absorption and inhibiting P-gp efflux due to encapsulation in TPGS polymeric micelles. Compared with HF alone, HTPM showed stronger tumor-suppressing effects in subcutaneous xenografts of MDA-MB-231 cells when orally administered. Moreover, compared with HTPM or surgical therapy alone, peroral HTPM combined with partial surgical excision synergistically retarded the growth of orthotopic TNBC. Fundamentally, HTPM orally administered at the therapeutic dose did not cause any pathological injury, while HF alone led to weight loss and jejunal bleeding in the investigated mice. Conclusion: Taken together, HTPM could be applied as a potential anticancer agent for TNBC by oral administration.

Radiography, CT, and MRI Diagnosis of Enzootic Nasal Tumor in Goats Infected With Enzootic Nasal Tumor Virus.

The aim of this study was to describe radiography, computed tomography (CT), and magnetic resonance imaging (MRI) findings of enzootic nasal tumors in goats infected with enzootic nasal tumor viruses. Five of six goats with a mean age of 2 years, showed clinical signs of respiratory disease. Head radiographs showed increased density of the unilateral or bilateral nasal cavity in four goats, and a CT scan showed that the space-occupying lesion of the nasal cavity originated from the ethmoid bone and was enhanced homogeneously postcontrast in all goats. The nasal concha was destroyed and the paranasal sinus mucosa was thickened and filled with fluid in some goats. On MRI, the mass exhibited equal or slightly higher signal intensity on T2 weighted images, equal signal intensity on T1 weighted images, a high signal on fluid-attenuated inversion recovery images and heterogeneous enhancement postcontrast. After dissection, histopathological examination of the mass and virus genome detection of the nasal secretions confirmed that the intranasal mass was a low-grade adenocarcinoma and that the goats were infected with enzootic nasal tumor virus type 2. In conclusion, CT and MRI have high diagnostic values for enzootic nasal tumors because they match the postmortem findings and are more accurate than radiography.

Goose Nephritic Astrovirus Infection of Goslings Induces Lymphocyte Apoptosis, Reticular Fiber Destruction, and CD8 T-Cell Depletion in Spleen Tissue.

The emergence of a novel goose nephritic astrovirus (GNAstV) has caused economic losses to the Chinese goose industry. High viral load is found in the spleen of goslings infected with GNAstV, but pathological injuries to the spleen due to GNAstV are largely unknown. In this study, 50 two-day-old goslings were infected orally with GNAstV, and 50 goslings were treated with PBS as control. Spleens were collected at different times following infection to assess damage. GNAstV infection caused visceral gout and urate deposition in joints, and resulted in 16% mortality. GNAstV was found in the lymphocytes and macrophages within the spleen. Lymphocyte loss, especially around the white pulp, and destruction and decline in the number of reticular fibers was observed in GNAstV-infected goslings. Moreover, in GNAstV-infected goslings, ultrahistopathological examination found that splenic lymphocytes exhibited condensed chromatin and apoptotic bodies, and reticular cells displayed damage to plasma membrane integrity and swollen mitochondria. Furthermore, TUNEL staining confirmed apoptosis of lymphocytes, and the mRNA levels of Fas and FasL were significantly increased in the GNAstV-infected goslings. In addition, GNAstV infection reduced the number and protein expression of CD8. In conclusion, GNAstV infection causes lymphocyte depletion, reticular cell necrosis, reticular fiber destruction, lymphocyte apoptosis, and reduction in CD8 levels, which contribute to spleen injury.

Immune-related gene expression in the kidneys and spleens of goslings infected with goose nephritic astrovirus.

Goose nephritic astrovirus (GNAstV) was first isolated in 2018, causing great economic losses to the goose industry. However, little is known about host immune response to GNAstV infection. In this study, forty 2-day-old goslings were randomly divided into 2 groups: infection and negative control groups. Each gosling in the infection group was challenged with 0.5 mL GNAstV-JSHA intramuscularly, whereas the gosling in the negative control group was inoculated with the same amount of PBS. Histopathological changes and virus location in the spleen and kidney were examined, and the expression of immune-related genes was determined by qPCR at 7 and 14 d after infection. Our results showed that GNAstV infection induced degeneration and necrosis of splenic lymphocytes and renal epithelial cells, and these cells were positive for the virus. In addition, GNAstV infection induced the activation of pattern recognition receptors (RIG-I, MDA-5, and TLR3) and key adaptor molecules (MyD88, MAVS, and IRF7) in the spleen and kidney, and upregulated the gene expression of interferon-α in the spleen and antiviral proteins (MX1, OASL, and IFITM3) in the spleen and kidney. Moreover, high expression levels of interleukin (IL)-1ß and IL-8 in the spleen and iNOS in the spleen and kidney were found. These results indicated that GNAstV infection activated host innate immune response. Furthermore, GNAstV infection increased the expression levels of CD8+, MHCI, and MHCII, indicating that adaptive immune response was activated. Besides, TGF-ß was highly expressed in the spleen and kidney, which may be an immune evasion strategy of GNAstV to cause infection. Interestingly, both IL-1ß and IL-6 mRNA levels were decreased in the kidney, which may help reduce kidney lesions. This is the first study to report changes in immune-related gene expression in response to GNAstV infection, and our results provide insights into viral pathogenesis.

Pseudorabies virus glycoprotein gE suppresses interferon-ß production via CREB-binding protein degradation.

Cyclic GMP-AMP synthase (cGAS) is a main sensor used to detect microbial DNA in the cytoplasm, which subsequently induces the production of interferon (IFN) via the cGAS/STING/IRF3 signaling pathway, leading to an antiviral response. However, some viruses have evolved multiple strategies to escape this process. Pseudorabies virus (PRV) is a double-stranded DNA virus belonging to the Alphaherpesvirinae subfamily, which can cause serious damage to the porcine industry. Many herpesvirus components have been reported to counteract IFN production, whereas little is known of PRV. In the present study, we found that PRV glycoprotein E (gE) was involved in counteracting cGAS/STING-mediated IFN production. Ectopic expression of gE decreased cGAS/STING-mediated IFN-ß promoter activity and the level of mRNA expression. Moreover, gE targeted at or downstream of IRF3 was found to inhibit IFN-ß production. However, gE did not affect the phosphorylation, dimerization and nuclear translocation of IRF3. Furthermore, gE is located on the nuclear membrane and could subsequently degrade CREB-binding protein (CBP). MG132, a proteasome inhibitor, decreased CBP degradation and restored the IFN-ß production induced by gE. Finally, gE-deleted PRV induced a higher level of IFN-ß production and reduced CBP degradation compared to wild-type PRV. Together, these results demonstrate that PRV gE can inhibit cGAS/STING-mediated IFN-ß production by degrading CBP to interrupt the enhanced assembly of IRF3 and CBP.

Goose astrovirus infection affects uric acid production and excretion in goslings.

In 2018, a new goose astrovirus (GAstrV) was reported in China, which causes 2 to 20% deaths in 4- to 16-day-old goslings causing great damages to the livestock industry. Gout is the typical feature of GAstrV infection in goslings. However, the mechanism of gout formation remains unclear. In the present study, 2-day-old goslings were infected intramuscularly with GAstrV for 14 D. One quarter of the infected goslings died, and typical gout pathological changes were found in the dead infected goslings. Pathological changes were observed in the morphology of the kidney and liver, such as degeneration, necrosis, and inflammatory cell infiltration. Accordingly, a high virus load was found in both organs. The serum level of uric acid in the inoculated goslings was higher, whereas no differences were found in levels of creatinine, calcium, and phosphorus. Moreover, the xanthine dehydrogenase (XOD) and adenosine deaminase (ADA) activities and the mRNA levels of xanthine dehydrogenase, adenosine deaminase, phosphoribosyl pyrophosphate amidotransferase, and phosphoribosyl pyrophosphate synthetase 1 in livers increased, wheres the multidrug resistance-associated protein 4 mRNA level and Na-K-ATPase activity in the kidneys decreased. These results showed that GAstrV infection could cause lesions on the liver and kidney and then increase the expression or activity of enzymes related to uric acid production in the liver and decrease renal excretion function, which contribute to hyperuricemia and gout formation.

PCV2 infection activates the cGAS/STING signaling pathway to promote IFN-ß production and viral replication in PK-15 cells.

PCV2 is a single-stranded DNA virus that we previously found to induce IFN-ß production via RIG-I and MDA-5. cGAS is known to be the most important DNA sensor for the recognition of cytosolic DNA; however, it remains unclear whether the interferon production induced by PCV2 is associated with cGAS. In the present study, PCV2 infection was found to increase the level of cGAS and STING expression, promote the release of cyclic dinucleotide cGAMP, and induce STING dimerization and translocation into the nucleus of PK-15 cells. These findings indicate that PCV2 infection activates both cGAS and STING. Furthermore, the knockdown of cGAS and STING decreased both the mRNA expression and promoter activity of IFN-ß, demonstrating that the cGAS/STING signaling pathway contributes to the production of IFN-ß. In addition, a knockdown of cGAS and STING also decreased the PCV2 viral load and infectivity. Therefore, PCV2 infection activates the cGAS/STING signaling pathway to induce IFN-ß production and the knockdown of cGAS and STING decreases viral replication in PK-15 cells. These results provide further insight into the relationship between PCV2 and host innate immunity.

Porcine circovirus type 2 inhibits inter-ß expression by targeting Karyopherin alpha-3 in PK-15 cells.

Interferon (IFN)-mediated antiviral response is an important part of host defense. Previous studies reported that porcine circovirus type 2 (PCV2) inhibits interferon production, but the mechanism is still poorly understood. In this study, PCV2 suppresses IFN-ß and IRF3 promoters and mRNA level of IFN-ß induced by ISD or Poly(I:C), but has no effect on the activation of AP-1 and NF-κB. Furthermore, PCV2 decreases the mRNA level of IFN-ß and IFN-ß promoter activity driven by STING, TBK1, IRF3, and IRF3/5D, and causes a reduction in the protein level of nuclear p-IRF3. In addition, PCV2 interrupts the interaction of KPNA3, rather than KPNA4, with p-IRF3. Overexpression of KPNA3 restores IFN-ß promoter activity. These results indicate that PCV2 disrupts the interaction of KPNA3 with p-IRF3 and blocks p-IRF3 translocation to the nucleus, thereby inhibiting IFN-ß induction in PK-15 cells.

RIG-1 and MDA-5 signaling pathways contribute to IFN-ß production and viral replication in porcine circovirus virus type 2-infected PK-15 cells in vitro.

Type I Interferons (IFNs) is known for its antiviral activity; however, it is surprising that in vitro treatment of IFN-α and IFN-γ enhanced the replication of porcine circovirus type 2 (PCV2), indicating a complex relationship between interferon and PCV2. To date, it remains poorly understood how the interferon is produced during PCV2 infection and whether the interferon induced by PCV2 itself can promote viral replication. In this study, PCV2 induced the up-regulation of IFN-ß in PK-15 cells, while treatment of PCV2-infected cells with the interferon regulatory factor-3 (IRF3) inhibitor, BX795, decreased the expression of IFN-ß, whereas treatment with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibitor, BAY11-7082, did not. These findings indicate that PCV2 can induce IFN-ß production via the IRF3-mediated rather than the NF-κB-mediated signal pathway. Moreover, PCV2 increased the protein expression levels of phosphorylation-IRF3 (p-IRF3), mitochondria antiviral-signaling protein (MAVS), retinoic acid-inducible gene I (RIG-1) and melanoma differentiation-associated protein 5 (MDA-5), and the knockdown of RIG-1 and MDA-5 decreased the expression level of IFN-ß in PK-15 cells. Therefore, PCV2 induces IFN-ß production via the RIG-1/MDA-5/MAVS/IRF signaling pathway. Furthermore, the PCV2 load and PCV2 infectivity decreased after knockdown of RIG-1 and MDA-5, indicating that RIG-1 and MDA-5 signaling pathways contribute to PCV2 replication. In conclusion, PCV2 induces the production of IFN-ß via the RIG-1 and MDA-5 signaling pathways, and the IFN-ß produced during PCV2 infection facilitates viral replication. These results will help us further understand the pathogenic mechanisms of PCV2.

Effector functions of memory CTLs can be affected by signals received during reactivation.

Memory cytotoxic T lymphocytes (CTLs) are able to provide protections to the host against repeated insults from intracellular pathogens. However, it has not been completely understood how the effector functions of memory CTLs are induced upon antigen challenge, which is directly related to the efficacy of their protection. Third signal cytokines, such as IL-12 and type I interferon, have been suggested to be involved in the protective function of memory CTLs, but direct evidence is warranted. In this report, we found that memory CTLs need to be reactivated to exert effector functions. Infusion of a large population of quiescent memory CTLs did not lead to cancer control in tumor-bearing mice, whereas infusion of a reactivated memory CTL population did. This reactivation of memory CTLs requires cytokines such as IL-12 in addition to antigen but was less dependent upon costimulation and IL-2 compared to naive CTLs. Memory CTLs responded more quickly and with greater strength than their naive counterparts upon stimulation, which is associated with higher upregulation of important transcription factors such as T-bet and phosphorylated STAT4. In addition, memory CTLs underwent less expansion than naive CTLs upon pathogen challenge. In conclusion, effector functions of established memory CTLs may be affected by certain cytokines such as IL-12 and type I IFN. Thus, a pathogen's ability to induce cytokines could contribute to the efficacy of protection of an established memory CTL population.

Pathogenicity and horizontal transmission studies of caprine parainfluenza virus type 3 JS2013 strain in goats.

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals, but goat infection has been rarely reported. In 2014, one novel PIV3 strain was first isolated from goats suffered respiratory diseases in Jiangsu and Anhui provinces of eastern China and named as caprine PIV3 (CPIV3) JS2013. In order to systematically evaluate the pathogenicity and horizontal transmission ability of this new virus, experimental infection of goats with the CPIV3 strain was done. The virus-inoculated goats (challenge control (CC) group) displayed coughing and nasal discharges from 3days post infection (dpi) and lasted for about 2 weeks. Two goats in group CC showed fever between 7 and 12dpi. As detected by a TaqMan real time quantitative RT-PCR (qRT-PCR), viremia was detected during 3-11dpi, peaked at 6dpi; and virus shedding from nasal discharge and faeces were confirmed during 3-21dpi and 4-21dpi, respectively. Virus-specific HI antibodies and neutralizing antibodies (NAs) became positive since 7dpi and 14dpi; peaked at 14dpi and 28dpi, respectively; and lasted at least 70days. Pathological lesions were mainly found on the lungs and tracheas. In addition, viruses were also detected in part of the tracheal secretion and lung samples, and the viral load in tracheal secretion was higher than that in lungs. Goats in horizontal infected group (hCC, kept in different cages in the same house with CC group) showed to be horizontally infected, with slightly milder clinical signs and pathological changes; and slightly shorter period of viremia and virus shedding. This was the first report of the detailed pathogenicity characterization of the novel CPIV3 and demonstrated its horizontal transmission ability. The results would be helpful for further studies on the preventive and control strategies for CPIV3 infections.

Ovarian Toxicity in Female Rats after Oral Administration of Melamine or Melamine and Cyanuric Acid.

Although the toxicity of melamine to the kidneys and testes is well known, few studies have investigated the effects of melamine on female reproductive organs. Therefore, this study explores the effects of oral administration melamine or melamine and cyanuric acid for 28 days on the ovaries of female rats. Rats that were exposed to the mixture exhibited reduced ovarian and uterine weights, a shorter estrous cycle, and reduced serum estrogen and progesterone levels compared to rats that were exposed to melamine and control rats. Furthermore, morphological analysis revealed pathological changes in the ovaries of rats exposed to melamine or the mixture, such as more atretic follicles and necrosis of oocytes and granulosa cells. TUNEL staining revealed that the exposed groups had a higher proportion of TUNEL-positive granulosa cells than the control group, and the mRNA expressions of SOD1, GPX1, GPX2, P450scc, 17ß-HSD I, and 17ß-HSD II were reduced in the exposure groups compared with the control group. These results indicated that exposure to melamine alone or to the melamine-cyanuric acid mixture could damage the ovaries in rats.

Oxidative stress and hypoxia observed in the kidneys of mice after a 13-week oral administration of melamine and cyanuric acid combination.

Both melamine and cyanuric acid have low toxicity, but together they may cause serious lesions to the kidney, via an unknown mechanism. This study was aimed to estimate whether lesions to the kidney were relative to oxidative damage and hypoxia in the kidney after mice exposed to 1mg/kg/day, 5mg/kg/day or 25mg/kg/day of a mixture of melamine and cyanuric acid for 13 weeks. Pathological changes to the kidneys, oxidative stress and energy parameters and hypoxia-inducible factor-1α (HIF-1α) change in the kidneys were evaluated. Pathological changes were found in the distal tubules of kidneys, such as crystals, proteinaceous casts and compensatory expansion, indicating that the mixture induced toxicity to the kidney. The activities of total antioxidant capacity (TAC) and superoxide dismutase (SOD) and the concentration of glutathione (GSH) decreased, while the concentrations of lipid peroxidation (MDA) and protein carbonyl groups (PC) increased after exposure to the mixture, demonstrating that the mixture resulted in imbalance of antioxidant and reactive oxygen species (ROS) and excessive ROS induced oxidant damage to lipid and proteins in kidneys. The activities of malate dehydrogenase (MDH) and succinate dehyrogenase (SDH) decreased, however, the activity of lactic dehydrogenase (LDH) and the concentration of HIF-1α increased after exposure to the mixture. Accordingly, it was concluded that the mixture resulted in a hypoxic state in kidneys and that both oxidative stress and hypoxia contributed to the lesion of kidneys. The exact cause of oxidative damage and hypoxia is not clear, it might be caused by either a direct effect or by an indirect effect, which is secondary to substantial renal damage caused by tubular obstruction due to crystal formation.

Effect on morphology, oxidative stress and energy metabolism enzymes in the testes of mice after a 13-week oral administration of melamine and cyanuric acid combination.

Cases of pet poisoning and infant renal calculus have attracted much attention to the toxicity of melamine and its derivatives, such as cyanuric acid. Although individually melamine and cyanuric acid have low toxicity, their simultaneous presence can cause severe damage. Little is known about their adverse effects on the reproductive system. In this study, mice were orally administrated 1, 5 or 25 mg/kg/d of both melamine and cyanuric acid for 13 weeks. Lethargy, rough hair, and reduction of food and water intake and of body and testis weight were found after exposure to the combination, and pathological changes were found in the morphology of the testes, such as disruption of the seminiferous tubule structure, decrease of the spermatogenic cell series and coagulation necrosis. Total antioxidant capacity and superoxide dismutase activities and glutathione concentration was lower and malondialdehyde concentration was higher than in control mice. The activities of malate dehydrogenase, lactate dehydrogenase and Na(+)/K(+)-ATPase were also lower in combination treated mice than in control mice. These results indicate that the combined exposure to both melamine and cyanuric acid damaged testes in mice by either a direct or indirect effect, which may be related to renal failure and secondary anorexia. Oxidative stress and lower energy production levels both contributed to the testicular damage.

Apoptosis induced in chicken embryo fibroblasts in vitro by a polyinosinic:polycytidylic acid copolymer.

This study investigated apoptosis induced in chicken embryo fibroblast (CEF) cells by the toxic effects of polyinosinic:polycytidylic acid copolymer (Poly I:C). Along with cell viability and morphological changes, other indicators were measured in vitro after incubation of the cells with Poly I:C, including phosphatidylserine translocation, the formation of DNA fragments, activity of caspase-3, caspase-8 and caspase-9, and the expression levels of RIPK1 mRNA and TNRSF8 mRNA. An inhibition of proliferation was found with increasing levels of Poly I:C, showing that Poly I:C was toxic to the cells. The findings of phosphatidylserine translocation and formation of DNA fragmentation suggested that Poly I:C induced apoptosis. With increased incubation time, the activities of caspase-3 and caspase-8 increased, while there was no significant change in caspase-9 activity. Accordingly, it is concluded that the apoptosis induced by Poly I:C involves a cell death receptor-mediated pathway. The transcription level of RIPK1 mRNA decreased, while that of TNFRSF8 mRNA increased, indicating that Poly I:C-induced apoptosis was related to upregulation of TNFRSF8. These observations provide insight into the potential mechanism of Poly I:C-induced toxicity.