Liquid Chromatography/Tandem Mass Spectrometry Analysis of Sophora flavescens Aiton and Protective Effects against Alcohol-Induced Liver Injury and Oxidative Stress in Mice.

In this study, we investigated the hepatoprotective effects of an ethanol extract of Sophora flavescens Aiton (ESF) on an alcohol-induced liver disease mouse model. Alcoholic liver disease (ALD) was caused by the administration of ethanol to male C57/BL6 mice who were given a Lieber-DeCarli liquid diet, including ethanol. The alcoholic fatty liver disease mice were orally administered ESF (100 and 200 mg/kg bw/day) or silymarin (50 mg/kg bw/day), which served as a positive control every day for 16 days. The findings suggest that ESF enhances hepatoprotective benefits by significantly decreasing serum levels of aspartate transaminase (AST) and alanine transaminase (ALT), markers for liver injury. Furthermore, ESF alleviated the accumulation of triglyceride (TG) and total cholesterol (TC), increased serum levels of superoxide dismutase (SOD) and glutathione (GSH), and improved serum alcohol dehydrogenase (ADH) activity in the alcoholic fatty liver disease mice model. Cells and organisms rely on the Kelch-like ECH-associated protein 1- Nuclear factor erythroid 2-related factor 2 (Keap1-Nrf2) system as a critical defensive mechanism in response to oxidative stress. Therefore, Nrf2 plays an important role in ALD antioxidant responses, and its level is decreased by increased reactive oxidation stress (ROS) in the liver. ESF increased Nrf2, which was decreased in ethanol-damaged livers. Additionally, four polyphenol compounds were identified through a qualitative analysis of the ESF using LC-MS/MS. This study confirmed ESF's antioxidative and hangover-elimination effects and suggested the possibility of using Sophora flavescens Aiton (SF) to treat ALD.

Ziziphus jujuba Miller Ethanol Extract Restores Disrupted Intestinal Barrier Function via Tight Junction Recovery and Reduces Inflammation.

Inflammatory bowel disease (IBD) is a chronic inflammatory condition caused by the disruption of the intestinal barrier. The intestinal barrier is maintained by tight junctions (TJs), which sustain intestinal homeostasis and prevent pathogens from entering the microbiome and mucosal tissues. Ziziphus jujuba Miller (Z. jujuba) is a natural substance that has been used in traditional medicine as a therapy for a variety of diseases. However, in IBD, the efficacy of Z. jujuba is unknown. Therefore, we evaluated ZJB in Caco2 cells and a dextran sodium sulfate (DSS)-induced mouse model to demonstrate its efficacy in IBD. Z. jujuba extracts were prepared using 70% ethanol and were named ZJB. ZJB was found to be non-cytotoxic and to have excellent antioxidant effects. We confirmed its anti-inflammatory properties via the down-regulation of inflammatory factors, including inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). To evaluate the effects of ZJB on intestinal barrier function and TJ improvement, the trans-epithelial electrical resistance (TEER) and fluorescein isothiocyanate-dextran 4 kDa (FITC-Dextran 4) permeability were assessed. The TEER value increased by 61.389% and permeability decreased by 27.348% in the 200 µg/mL ZJB group compared with the 50 ng/mL IL-6 group after 24 h. Additionally, ZJB alleviated body weight loss, reduced the disease activity index (DAI) score, and induced colon shortening in 5% DSS-induced mice; inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 were down-regulated in the serum. TJ proteins, such as Zonula occludens (ZO)-1 and occludin, were up-regulated by ZJB in an impaired Caco2 mouse model. Additionally, according to the liquid chromatography results, in tandem with mass spectrometry (LC-MS/MS) analysis, seven active ingredients were detected in ZJB. In conclusion, ZJB down-regulated inflammatory factors, protected intestinal barrier function, and increased TJ proteins. It is thus a safe, natural substance with the potential to be used as a therapeutic agent in IBD treatment.

Research Note: Characterization of monoclonal antibodies and development of sandwich ELISA for detecting chicken IL7.

IL7 is a hematopoietic growth factor required for development and maintenance of lymphocytes including T cells, B cells, and natural killer cells. Recently, chicken IL7 (chIL7) has been cloned and studied in viral and parasite infection models. However, no monoclonal antibodies (mAb) that specifically detect chIL7 have been developed so far. In this study, recombinant chIL7 that expressed for immunization and mAb against chIL7 were developed and characterized to assess their immunologic properties. Five mAb exhibiting specific binding to chIL7 were generated and investigated for their applicability by Western blot, ELISA, and neutralization assays. A sandwich ELISA mAb pair that enables the measurement of chIL7 protein levels in biological samples from Eimeria-infected chickens was identified and several mAb neutralized chicken primary thymocyte proliferation mediated by chIL7. The mAb developed in this study will be valuable reagents for fundamental and applied immunological studies in poultry.

Development of antigen sandwich ELISA to detect interferon-alpha (IFN-α) using monoclonal antibodies in chicken.

Interferon alpha (IFN-α) belongs to the type I interferon family which mediates an early innate immune response to viral infections. In the present study, we developed sandwich ELISA using specific mouse monoclonal antibodies (mAbs) to measure IFN-α production in chickens. Recombinant chicken IFN-α (chIFN-α) expressed in yeast were purchased from Kingfisher Biotech, and used to immunize the mice. Five mAbs which specifically recognize chicken IFN-α antigen were selected and characterized. For sandwich ELISA development, mAbs were labeled with biotin, followed by a pairing test to identify the best capture and detection antibodies. Two sets of mouse anti-chIFN-α mAb pairs were determined and a standard curve was established using recombinant chIFN-α. The sandwich ELISA effectively detected an increased IFN-α production in chicken macrophage cells stimulated by polyinosinic:polycytidylic acid (poly I:C), and its minimum detectable level was about 25 pg/mL. The anti-viral activity of chIFN-α against vesicular stomatitis virus was characterized in avian embryonic fibroblast and the mouse anti-chIFN-α mAbs which neutralize its activity were identified. The newly developed antigen sandwich ELISA developed in this study will be a useful tool to monitor IFN-α production in chickens.

Development and characterization of novel mouse monoclonal antibodies against chicken chemokine CC motif ligand 4.

Chemokine (C-C motif) ligand (CCL) 4 is a CC chemokine subfamily member defined by the sequential positioning of conserved cysteine residues. Upon the binding of G-protein-coupled receptors on the cell surface, CCL4 mediates a diverse set of biological processes including chemotaxis, tumorigenesis, homeostasis and thymopoiesis. Although the physiological roles of mammalian CCL4s were elucidated >20 years ago, there is limited information on the biological activities of chicken CCL4 (chCCL4). In the present study, we developed and characterized mouse monoclonal antibodies (mAbs) against chCCL4 to characterize better the immunological properties of chCCL4. Out of initial screening of >400 clones, two mAbs detecting chCCL4, 1A12 and 15D9, were identified and characterized using western blotting and chCCL4-specific antigen-capture enzyme-linked immunosorbent assay, and their neutralizing activity was validated by chCCL4-induced peripheral blood mononuclear cell chemotaxis assay. Furthermore, the intracellular expression of chCCL4 in various chicken cells by immunocytochemistry and flow cytometry was confirmed using 1A12 and 15D9 mAbs. These results collectively indicate that 1A12 and 15D9 mAbs specifically detect chicken CCL4 and they will be valuable immune reagents for basic and applied studies in avian immunology.

Trop2 is upregulated in the transition to dysplasia in the metaplastic gastric mucosa.

Intestinal-type gastric adenocarcinoma arises in a field of pre-existing metaplasia. While biomarkers of cancer and metaplasia have been identified, the definition of dysplastic transition as a critical point in the evolution of cancer has remained obscure. We have evaluated Trop2 as a putative marker of the transition from metaplasia to dysplasia in the stomach in multiple mouse models of metaplasia induction and progression. In addition, TROP2 expression was evaluated in human samples by immunostaining tissue microarrays for metaplasia, dysplasia, and gastric cancer. Dysplastic mouse organoids were evaluated in vitro following shRNA knockdown of Trop2 expression. In mouse models, no Trop2 was observed in the normal corpus and Trop2 was not induced in acute models of metaplasia induction with either L635 or DMP-777. In Mist1-Kras mice, Trop2 expression was not observed in metaplasia at 1 month after Kras induction, but was observed in dysplastic glands at 3-4 months after Kras induction. In human tissues, no Trop2 was observed in normal corpus mucosa or SPEM, but Trop2 expression was observed in incomplete intestinal metaplasia, with significantly less expression in complete intestinal metaplasia. Trop2 expression was observed in all dysplastic and 84% of gastric cancer lesions, although expression levels were variable. Dysplastic mouse organoids from Mist1-Kras mice expressed Trop2 strongly. Knockdown of Trop2 with shRNA markedly reduced organoid growth and budding behavior, and induced the upregulation of apical villin expression. We conclude that Trop2 is upregulated in the transition to dysplasia in the stomach and promotes dysplastic cell behaviors. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Development and characterization of monoclonal antibodies specific for chicken interleukin-13 and their neutralizing effects in chicken primary monocytes.

Compared with mammals, the functionality of chicken cytokines is not well understood because of the unavailability of immune reagents. Mammalian interleukin (IL)-13 is an important Th2 type cytokine with well-known biological functions through its 2 receptors, IL-13 receptor (IL-13R)-α1 and IL-13Rα2. In the present study, we developed mouse monoclonal antibodies (mAb) against chIL-13 and further investigated their specificity in detecting endogenously produced chIL-13. Upon characterization of mAb using indirect ELISA and Western blot, the capture ELISA was developed for detecting chIL-13. Neutralizing effects were tested by measuring nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in primary chicken monocytes stimulated with chIL-13, lipopolysaccharide (LPS), chIL-13+LPS, or chIL-13+LPS+mAb. In addition, gene expression of chIL-13Rα1, chIL-13Rα2, and TGF-ß1 was tested in chicken monocytes treated with chIL-13 or chIL-13+mAb. Based on indirect ELISA, 5 mAb that detected recombinant chIL-13 were identified, and all of them specifically detected recombinant chIL-13 protein by Western blotting. An optimal signal was obtained with 2 mAb (#9B11 and #10A2) in a pairing assay, and these 2 mAb were used in a capture assay. A neutralization assay further revealed that chIL-13 reduced LPS-stimulated NO production and iNOS expression in monocytes and macrophage cells, and the 2 mAb (#9B11 and #10A2) abrogated these effects. In addition, chIL-13-induced expressions of chIL-13Rα2 and TGF-ß1 were neutralized by the 2 mAb. In summary, the present study showed that chIL-13 may be involved in the alternative activation of primary monocytes in chickens and that chIL-13 signaling may be regulated through chIL-13Rα2 binding and TGF-ß1 secretion. Importantly, the newly developed anti-chIL-13 mAb will serve as valuable immune reagents for future studies on the biological activity of chIL-13 and its receptors.

Downregulation of common cytokine receptor γ chain inhibits inflammatory responses in macrophages stimulated with Riemerella anatipestifer.

Th17-cell-mediated inflammation is affected by the soluble form of common cytokine receptor γ chain (γc). We previously suggested that inflammatory cytokines including interleukin (IL)-17A are associated with Riemerella anatipestifer infection, which a harmful bacterial pathogen in ducks. Here, the expression profiles of membrane-associated γc (duγc-a) and soluble γc (duγc-b) in R. anatipestifer-stimulated splenic lymphocytes and macrophages, and in the spleens and livers of R. anatipestifer-infected ducks, were investigated. In vitro and in vivo results indicated that the expression levels of both forms of γc were increased, showing that marked increases were detected in the expression of the duγc-b form rather than the duγc-a form. Treatment with γc-specific siRNA downregulated mRNA expression of Th17-related cytokines, including IL-17A and IL-17F, in duck splenic macrophages stimulated with R. anatipestifer, whereas the expressions of interferon (IFN)-γ and IL-2 were enhanced. The results showed that the upregulation of γc, especially the duγc-b form, was associated with expression of Th17-related cytokines during R. anatipestifer infection.

Evaluation of the Immunomodulatory Activity of the Chicken NK-Lysin-Derived Peptide cNK-2.

Chicken NK-lysin (cNK-lysin), the chicken homologue of human granulysin, is a cationic amphiphilic antimicrobial peptide (AMP) that is produced by cytotoxic T cells and natural killer cells. We previously demonstrated that cNK-lysin and cNK-2, a synthetic peptide incorporating the core α-helical region of cNK-lysin, have antimicrobial activity against apicomplexan parasites such as Eimeria spp., via membrane disruption. In addition to the antimicrobial activity of AMPs, the immunomodulatory activity of AMPs mediated by their interactions with host cells is increasingly recognized. Thus, in this study, we investigated whether cNK-lysin derived peptides modulate the immune response in the chicken macrophage cell line HD11 and in chicken primary monocytes by evaluating the induction of chemokines, anti-inflammatory properties, and activation of signalling pathways. cNK-2 induced the expression of CCL4, CCL5 and interleukin(IL)-1ß in HD11 cells and CCL4 and CCL5 in primary monocytes. We also determined that cNK-2 suppresses the lipopolysaccharide-induced inflammatory response by abrogating IL-1ß expression. The immunomodulatory activity of cNK-2 involves the mitogen-activated protein kinases-mediated signalling pathway, including p38, extracellular signal-regulated kinase 1/2 and c-Jun N-terminal kinases, as well as the internalization of cNK-2 into the cells. These results indicate that cNK-2 is a potential novel immunomodulating agent rather than an antimicrobial agent.

Upregulation of duck interleukin-17A during Riemerella anatipestifer infection.

Although IL-17 cytokines play critical roles in host defense immunity, dysregulated expression of these cytokines is associated with inflammation and autoimmune diseases. Riemerella anatipestifer is the most important infectious bacterium in the duck industry. Interestingly, not all avian species are equally susceptible to R. anatipestifer infection. This paper reports the first description of mortality rate, bacterial burden, and expression profiles of immune-related genes between ducks and chickens infected with R. anatipestifer. Ducks exhibited increased susceptibility to R. anatipestifer infection compared to chickens, as determined by mortality rate and bacterial burden. Comparative expression analyses of immune-related genes in R. anatipestifer-infected tissues obtained from both species revealed that TLR3, TLR7, IL-2, IL-4, and IFN-γ transcript levels were higher in chickens, whereas TLR4 and IL-17A transcript levels were higher in ducks. Marked increases in expression of IL-17A and IL-6, but not TGF-ß, were associated with Th17 cell differentiation in duck splenic lymphocytes, but not in chicken splenic lymphocytes, stimulated with R. anatipestifer. Moreover, upregulation of IL-1ß, IL-6, and IL-17A mRNA expressions, but not TGF-ß, was confirmed in the liver and spleen of ducks infected with R. anatipestifer, indicating that IL-17A is strongly associated with Riemerella infection in ducks.

Molecular cloning, characterization and mRNA expression of duck interleukin-17F.

Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 510-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17. duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37-46% with corresponding mammalian homologues, and 53.5% with the previously described duck IL-17A (duIL-17A). The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes but not in poly I:C stimulated cells. duIL-17F forms heterodimers with duIL-17A. Recombinant duIL-17F, like duIL-17A, induced IL-1ß, IL-6, and IL-8 expression in duck embryonic fibroblasts (DEFs). duIL-17A, but not duIL-17F expression, was significantly up-regulated in the liver and spleen of Salmonella Typhimurium-infected ducks. Further analysis of the contributions of IL-17F to different Salmonella spp. or other disease models will be required to expand our understanding of its biological functions.

Different strategies for producing naturally soluble form of common cytokine receptor γ chain.

The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens.

Downregulation of chicken interleukin-17 receptor A during Eimeria infection.

Both interleukin-17A (IL-17A) and IL-17F are proinflammatory cytokines that have an important role in intestinal homeostasis via receptor signaling. These cytokines have been characterized in chickens, but very little is known about their receptors and their functional activity. We provide here the first description of the sequence analysis, bioactivity, and comparative expression analysis of chicken IL-17RA (chIL-17RA) in chickens infected with Salmonella and Eimeria, two major infectious agents of gastrointestinal diseases of poultry of economic importance. A full-length chIL-17RA cDNA with a 2,568-bp coding region was identified from chicken thymus cDNA. chIL-17RA shares ca. 46% identity with mammalian homologues and 29.2 to 31.5% identity with its piscine counterparts. chIL-17RA transcript expression was relatively high in the thymus and in the chicken macrophage cell line HD11. The chIL-17RA-specific small interfering RNA inhibits interleukin-6 (IL-6), IL-8, and IL-1ß mRNA expression in chicken embryo fibroblast cells (but not in DF-1 cells) stimulated with chIL-17A or chIL-17F. Interaction between chIL-17RA and chIL-17A was confirmed by coimmunoprecipitation. Downregulation of chIL-17RA occurred in concanavalin A- or lipopolysaccharide-activated splenic lymphocytes but not in poly(I·C)-activated splenic lymphocytes. In Salmonella- and Eimeria-infected chickens, the expression levels of the chIL-17RA transcript were downregulated in intestinal tissues from chickens infected with two Eimeria species, E. tenella or E. maxima, that preferentially infect the cecum and jejunum, respectively. However, chIL-17RA expression was generally unchanged in Salmonella infection. These results suggest that chIL-17RA has an important role in mucosal immunity to intestinal intracellular parasite infections such as Eimeria infection.

Chicken IL-17F: identification and comparative expression analysis in Eimeria-infected chickens.

Interleukin-17F (IL-17F) is a proinflammatory cytokine, which plays an important role in gut homeostasis. A full-length chicken IL-17F (chIL-17F) cDNA with a 510-bp coding region was identified from ConA-activated chicken splenic lymphocytes. ChIL-17F shares 53% amino acid sequence identity with the previously described chicken IL-17 (chIL-17A) and 38-43% with mammalian homologues. The locus harboring chIL-17 and chIL-17F displayed inverted order compared to those of mammals. ChIL-17F transcript expression was high in lymphoblast cell line CU205 and at moderate levels in small and large intestines and liver. ChIL-17F and chIL-17 expression profiles were examined by quantitative real-time RT-PCR in mitogen-stimulated splenic lymphocytes and intestinal areas affected by Eimeria maxima and Eimeria tenella infections. Expression levels of chIL-17F, like chIL-17, were elevated in mitogen-activated splenic lymphocytes. ChIL-17F, but not chIL-17, expression was upregulated in intestinal tissues affected by E. maxima and E. tenella infections. Recombinant chIL-17F biological activities were similar to that of chIL-17 in primary chicken embryonic fibroblasts. These results suggest that chIL-17F is a unique member of the IL-17 family of cytokines.

Predictive factors for lymph node metastasis and endoscopic treatment strategies for undifferentiated early gastric cancer.

BACKGROUND AND AIM: Although more than 80% of undifferentiated early gastric cancers (EGC) are not associated with lymph node metastasis, endoscopic mucosal resection is not generally accepted as a means of curative treatment because of an abundance of conflicting data concerning clinicopathological characteristics and prognoses. The aim of this study was to define a subgroup of undifferentiated EGC that could be cured by endoscopic treatment without the risk of lymph node metastasis. METHOD: A total of 591 patients surgically resected for undifferentiated EGC between January 1999 and March 2005 were reviewed. Associations between various clinicopathological factors and the presence of lymph node metastasis were analyzed to identify the risk factors of lymph node metastasis. RESULTS: Lymph node metastasis was found in 79 patients (13.4%). By multivariate logistic regression analysis, a tumor diameter 2.5 cm or larger, invasion into the middle third of the submucosal layer or deeper, and lymphatic involvement were identified as independent risk factors of lymph node metastasis (P < 0.001, respectively). Lymph node metastasis was not found in any patient with undifferentiated EGC smaller than 2.5 cm confined to the mucosa or upper third of the submucosal layer without lymphatic involvement. CONCLUSIONS: Undifferentiated intramucosal EGC smaller than 2.5 cm without lymphatic involvement was not associated with lymph node metastasis. Thus, we propose in this circumstance that endoscopic mucosal resection could be considered a definitive treatment without compromising the possibility of cure.