Necrosis Factor Ligand Superfamily Member Research Articles

A novel dual-cytokine–antibody fusion protein for the treatment of CD38-positive malignancies

A novel dual-cytokine-antibody fusion protein, consisting of an antibody directed against CD38 [a tumor-associated antigen mainly expressed on the surface of multiple myeloma (MM) cells], simultaneously fused to both tumor necrosis factor ligand superfamily member 10 (TRAIL) and interleukin-2 (IL2), was designed, expressed and purified to homogeneity. The novel fusion protein, termed IL2-αCD38-αCD38-scTRAIL, was able to selectively recognize its cognate antigen expressed on the surface of MM and lymphoma cell lines, as evidenced by flow cytometry analysis. Moreover, the targeted version of TRAIL was able to induce cancer cell death in vitro, both with MM cell lines and with fresh isolates from the bone marrow of MM patients. The experiments provide a rationale for possible future applications of IL2-αCD38-αCD38-scTRAIL for the treatment of patients with MM or other CD38-positive malignancies.

The power of precise bioinformatics prediction of miRNA:mRNA interactions:miR-4699 as a potential inducer of Wnt signaling pathway.

microRNAs have attracted interest because of their regulatory effects on gene expression. Experimental detection of potential targets of miRNAs is a laborious task. Considering the expensive techniques of detection, computational approaches for miRNA target prediction can be used as the first step in miRNA research. A large number of tools and algorithms have been developed during the last two decades, led to problems such as confusion in selecting an appropriate tool and false positive or negative results. Therefore, one of the most frequent problems and critical issues of miRNA research is finding a reliable miRNA target prediction tool. In this study, we have proposed a research direction and introduced user-friendly and current databases and tools with the highest accuracy. To verify whether our proposed research direction is practical, we have provided a case example of predicting a miRNA which can target negative regulators of osteogenesis and experimentally evaluated the accuracy of the prediction results by Real-Time PCR and Luciferase assay. The results of RT-qPCR and Luciferase assay indicated a significant decline in expression of Dickkopf-related protein 1 (DKK1) and tumor necrosis factor ligand superfamily member 11 (TNFSF11) as the key negative regulators of osteogenesis upon overexpression of miR-4699-3p. The results emphasize the validity and importance of accurate in silico investigation as the first step in experimental studies. This is the first report detailing the prediction and validation of miR-4699-3p target genes. We suggest hsa-miR-4699 for further investigation as an osteogenic miRNA for therapeutics purposes.

Association of TNFSF4 Polymorphisms with Neuromyelitis Optica Spectrum Disorders in a Chinese Population.

The tumor necrosis factor ligand superfamily member 4 (TNFSF4) gene encodes a vital co-stimulatory molecule of the immune system and has been identified as a susceptibility locus for systemic lupus erythematosus, systemic sclerosis, and primary Sjögren's syndrome. However, the association of TNFSF4 polymorphisms with neuromyelitis optica spectrum disorders (NMOSD), an inflammatory, demyelinating autoimmune disease of the central nervous system, has not yet been investigated. To evaluate whether TNFSF4 polymorphisms contribute to risk of NMOSD, four single-nucleotide polymorphisms (SNPs) (rs1234315, rs2205960, rs704840, and rs844648) were selected and genotyped in a cohort of 312 patients with NMOSD and 487 healthy controls. Our study showed that rs844648 was associated with an increased risk of NMOSD, according to the allelic model (OR=1.30, 95% CI 1.06-1.59, P=0.011, Pcorr=0.044). Significant associations of rs844648 (OR=1.67, 95% CI 1.17-2.38, P=0.005, Pcorr=0.02) and rs704840 (OR=1.75, 95% CI 1.17-2.63, P=0.007, Pcorr=0.027) with NMOSD occurrence were also observed under the recessive model. Moreover, linkage disequilibrium analysis revealed two blocks within TNFSF4; in one block, the haplotype Ars844648Grs704840 significantly increased the risk of NMOSD, whereas Grs844648Trs704840 reduced the risk. This study demonstrates an association between TNFSF4 polymorphisms and susceptibility for the development of NMOSD in the Chinese population.

TRAIL mutant membrane penetrating peptide alike (TMPPA) TRAIL‑Mu3 enhances the antitumor effects of TRAIL invitro and invivo.

The aim of the present study was to prepare a tumor necrosis factor ligand superfamily member 10 (TRAIL) mutant membrane penetrating peptide alike (TMPPA), TRAIL‑Mu3, and to investigate its antitumor effects in colorectal cancer invitro and invivo. The pMD19/TRAIL plasmid with designed primers was amplified to construct the target gene; it was ligated with an expression plasmid and the expression was confirmed. Subsequently, TRAIL‑Mu3 was purified and further confirmed by western blot analysis. Immunofluorescence analysis was used to detect the distribution of TRAIL‑Mu3 in colorectal cancer cells. In addition, the present study investigated the antitumor effects of TRAIL‑Mu3 on colorectal cancer invitro and invivo. A novel TMPPA, TRAIL‑Mu3, was synthesized in the present study. Following a series of detection experiments, it was confirmed that the TRAIL‑Mu3 gene was obtained and was able to express TRAIL‑Mu3 successfully. The immunofluorescence analysis demonstrated that TRAIL‑Mu3 exhibited a markedly enhanced affinity to the colorectal cancer cell surface. In addition, TRAIL‑Mu3 exerted stronger antitumor effects, compared with TRAIL, on colorectal cancer invitro and invivo.

Combination of oncolytic adenovirus and luteolin exerts synergistic antitumor effects in colorectal cancer cells and a mouse model.

In recent years, oncolytic viruses have attracted increasing interest due to their potent antitumor effects. Luteolin, a natural product, has additionally been observed to exhibit various pharmacological antitumor activities. Previously, a novel dual-targeting oncolytic adenovirus, complement decay-accelerating factor (CD55)-tumor necrosis factor ligand superfamily member 10 (TRAIL), was constructed, which exhibited significant growth inhibitory effects in various types of tumor cell. The present study investigated whether the combination of luteolin and CD55-TRAIL was able to exert a synergistic antitumor effect in colorectal carcinoma (CRC) cells. The cytotoxicity and tumor cell apoptosis mediated by combination treatment in CRC cells were detected via an MTT assay, Hoechst staining and western blotting, respectively. Tumor growth in vivo was examined in a CRC mouse xenograft model following various treatments. The results demonstrated that the addition of luteolin enhanced oncolytic adenovirus-mediated enhanced green fluorescent protein, early region 1A and TRAIL expression. The combination of CD55-TRAIL with luteolin synergistically inhibited tumor growth and promoted CRC cellular apoptosis in vitro and in vivo. Additionally, the combination of CD55-TRAIL with luteolin significantly decreased cytotoxicity in lung/bronchial normal epithelial cells, compared with single treatment. Therefore, the combination of CD55-TRAIL with luteolin may be a novel efficient therapeutic strategy for CRC in the future.

Knockdown of Indian hedgehog protein induces an inhibition of cell growth and differentiation in osteoblast MC3T3‑E1 cells.

Indian hedgehog protein (Ihh) is evolutionarily conserved and serves important roles in controlling the differentiation of progenitor cells into osteoblasts. Ihh null mutant mice exhibit a failure of osteoblast development in endochondral bone. Although studies have demonstrated that Ihh signaling is a potent local factor that regulates osteoblast differentiation, the specific transcription factors that determine osteoblast differentiation remain unclear. Further studies are required to determine the precise mechanism through which Ihh regulates osteoblast differentiation. In the present study, Ihh was knocked down in osteoblast MC3T3-E1 cells using short hairpin RNA, to investigate the function of Ihh in osteoblast proliferation and differentiation and to examine the potential mechanism through which Ihh induces osteoblast apoptosis and cell cycle arrest. It was observed that the knockdown of Ihh induced a marked inhibition of cell growth and increased the apoptosis rate compared with the negative control osteoblasts. Downregulation of Ihh resulted in a cell cycle arrest at the G1 to S phase boundary in osteoblasts. In addition, the knockdown of Ihh decreased the alkaline phosphatase activity and mineral deposition of osteoblasts. The inhibitory roles of Ihh downregulation in osteoblast growth and differentiation may be associated with the transforming growth factor-β/mothers against decapentaplegic homolog and tumor necrosis factor receptor superfamily member 11B/tumor necrosis factor ligand superfamily member 11 signaling pathways. Manipulating either Ihh expression or its signaling components may be of benefit for the treatment of skeletal diseases.

Down-regulation of MBD4 contributes to hypomethylation and overexpression of CD70 in CD4+ T cells in systemic lupus erythematosus

BackgroundSystemic lupus erythematosus (SLE) is an autoimmune disease that is characterized by lymphocytic infiltration and overproduction of autoantibodies, leading to significant morbidity and mortality. However, the pathogenesis of this disorder has not yet been completely elucidated. It has been reported that CD70, a B cell costimulatory molecule encoded by the gene TNFSF7 (tumor necrosis factor ligand superfamily member 7), is overexpressed in CD4+ T cells from patients with SLE due to the demethylation of its promoter. We aimed to investigate the expression patterns of MBD4 (methyl-CpG binding domain protein 4) in CD4+ T cells and its contribution to the pathogenesis of SLE by increasing CD70 expression through epigenetic regulation.ResultsOur results showed that the expression of MBD4 was significantly decreased in CD4+ T cells from SLE patients. We verified that transfection of MBD4 siRNA into healthy CD4+ T cells upregulated expression of CD70 and decreased the methylation level of the CD70 promoter. Overexpression of MBD4 inhibited CD70 expression and enhanced the DNA methylation level of CD70 in CD4+ T cells of SLE patients.ConclusionOur results indicated that downregulation of MBD4 contributed to overexpression and hypomethylation of the CD70 gene in SLE CD4+ T cells. This modulation of MBD4 may provide a novel therapeutic approach for SLE.

Presymptomatic Diagnosis of Celiac Disease in Predisposed Children: The Role of Gene Expression Profile.

The prevalence of celiac disease (CD) has increased significantly in recent years, and risk prediction and early diagnosis have become imperative especially in at-risk families. In a previous study, we identified individuals with CD based on the expression profile of a set of candidate genes in peripheral blood monocytes. Here we evaluated the expression of a panel of CD candidate genes in peripheral blood mononuclear cells from at-risk infants long time before any symptom or production of antibodies. We analyzed the gene expression of a set of 9 candidate genes, associated with CD, in 22 human leukocyte antigen predisposed children from at-risk families for CD, studied from birth to 6 years of age. Nine of them developed CD (patients) and 13 did not (controls). We analyzed gene expression at 3 different time points (age matched in the 2 groups): 4-19 months before diagnosis, at the time of CD diagnosis, and after at least 1 year of a gluten-free diet. At similar age points, controls were also evaluated. Three genes (KIAA, TAGAP [T-cell Activation GTPase Activating Protein], and SH2B3 [SH2B Adaptor Protein 3]) were overexpressed in patients, compared with controls, at least 9 months before CD diagnosis. At a stepwise discriminant analysis, 4 genes (RGS1 [Regulator of G-protein signaling 1], TAGAP, TNFSF14 [Tumor Necrosis Factor (Ligand) Superfamily member 14], and SH2B3) differentiate patients from controls before serum antibodies production and clinical symptoms. Multivariate equation correctly classified CD from non-CD children in 95.5% of patients. The expression of a small set of candidate genes in peripheral blood mononuclear cells can predict CD at least 9 months before the appearance of any clinical and serological signs of the disease.

Influence of High-Fat Diet on Bone Tissue: An Experimental Study in Growing Rats.

The relationship between obesity and bone tissue remains contradictory, especially when the effect of high-fat diet is assessed in experimental models. The aim of this study was to evaluate the effects of high-fat diet on bone metabolism of growing rats. Twenty weaned female Wistar rats were equally divided into two groups: SD (standard diet) and HFD (high-fat diet with 60 % of energy as fat). After five weeks of the two diets, the rats were euthanized, and the liver, blood and bones extracted. The liver was analysed for malondialdehyde (MDA) and reduced glutathione (GSH) concentrations. Blood was analysed by the ELISA method for osteoprotegerin (OPG) and tumour necrosis factor ligand superfamily member 11 (TNFSF11/RANKL). The bone tissue was analysed by dual-energy X-ray absorptiometry (DXA), mechanical tests, computed microtomography, histological quantitative analysis and scanning electron microscopy. The gene expressions of PPAR-γ Runx-2, RANKL and Cathepsin-K were also evaluated. HFD caused an increase in the MDA concentration, indicating oxidative stress. It also increased the expression of PPAR-γ, which is the gene that is related to adipocyte differentiation. There was an increase in BMD of the tibia of animals fed with the HFD, but other microstructural and mechanical properties were maintained unaltered. In addition, there were no changes in the gene expressions related to the differentiation of osteoblasts and osteoclasts, as well as no changes to the biochemical markers of bone formation and bone resorption. Liver and gene parameters are changed in response to the HFD. However, although there was an increase in BMD, the microstructure and function of the bone did not change after a 5-week HFD.

Sanguinarine protects against ovariectomy‑induced osteoporosis in mice.

Natural compounds are alternative agents that have therapeutic potential for preventing and treating osteoporosis. Traditionally, sanguinarine has been used clinically due to its diverse biological properties, including antimicrobial, anti-inflammatory and anticancer effects. Recently, for the first time, it was reported that sanguinarine inhibits osteoclast differentiation and bone resorption by suppressing the tumor necrosis factor ligand superfamily member 11-induced nuclear factor-κB and extracellular signal-regulated kinase signaling pathways in vitro. Therefore, the present study further investigated the pharmacological effect of sanguinarine on osteoporosis in vivo. Micro-computed tomography and histomorphometry analysis demonstrated that sanguinarine, at low and high concentrations, prevents ovariectomy (OVX)-induced bone loss. In addition, further investigation of the cellular response in vivo revealed that sanguinarine inhibited osteoclastic bone resorption and promoted osteoblastic bone formation in a dose-dependent manner. Therefore, the present study demonstrated that sanguinarine protected mice from OVX-induced osteoporosis by modulating bone remodeling, indicating that sanguinarine may have potential in the treatment of osteoporosis.

<em>In silico</em> investigations revealed four potential colon cancer drugs from phytochemicals in <em>Zingiber officinale</em>

<p align="left">Cancer is a difficult disease to treat, and few effective drugs are available. Hence, it is of great importance to develop effective anti-cancer therapeutic agents with well-defined pharmacokinetic properties.<strong> </strong>Although, ginger (<em>Zingiber officinale</em>) has a number of proven pharmacological activities, its effect on colon cancer has not received much attention. This study therefore investigated the potential colon cancer drug of compounds found in ginger. Dihydropyrimidine dehydrogenase was modeled using comparative homology modeling and virtual screening was performed locally on a Linux platform using AutoDock Vina<sup>®</sup>. The results showed that human dihydropyrimidine dehydrogenase is a homolog of pig dihydropyrimidine dehydrogenase. The leads of potential colon cancer drugs were beta-sitosterol, 6-Shogoal, Alloaromadedrene, and Zingiberol. They had similar binding site with levamisole for tumor necrosis factor ligand superfamily member 6 with His 148 and Tyr 192 common at their binding site whereas they had different binding sites from 5-fluorouracil for dihydropyrimidine dehydrogenase. The leads had better bioactivities compared with reference drugs (5-flourouracil and Levamisole) approved clinically for the treatment of colon cancer. <em>In vitro</em>, <em>ex vivo</em> and/or <em>in vivo</em> validations of the leads against colon cancer are recommended. <strong></strong></p>

Identification of human serum protein targets of Qianggu Decoction () in primary type I osteoporosis based on tandem mass tag labeling and liquid chromatography-tandem mass spectrometry technology.

To investigate the serum protein targets of Qianggu Decoction (, QGD) on treating osteoporosis by the proteomics analysis using tandem mass tag (TMT) and liquid chromatographytandem mass spectrometry (LC-MS/MS). Twenty serum protein samples were recruited (10 patients with primary type I osteoporosis before and after QGD treatment) and the high abundance ratios protein was removed, two serum samples were extracted and labeled with TMT reagent. Then, mass spectrometric detection, identification of differentially expressed proteins and bioinformatics analysis of differentially expressed proteins were carried out. A total of 60 proteins were identified, within a 99% confidence interval, to be differentially regulated of which, 34 proteins were up-regulated and 26 proteins were down-regulated. Differentially expressed proteins analyzed by Gene Ontology (GO) annotation mainly get involved in 12 different biological processes, 7 types of cellular components, and 6 kinds of molecular functions. Angiotensinogen (AGT), stromelysin-1 (MMP3), heparanase (HPSE) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were screened as candidate protein targets of QGD treatment, which were related to metabolic mechanism of bone remodeling and/or bone collagen of osteoporosis. By the utilization of the protein-protein interaction network analysis tool named STRING10.0, it showed that AGT, MMP3, HPSE and GAPDH were located in the key node of the protein-protein interactions network. Furthermore, AGT, MMP3, HPSE and GAPDH were found to be directly related to BMP, MAPK, Wnt, SMAD and tumor necrosis factor ligand superfamily member 11 (TNFSF11) families. The proteomics analysis by using TMT combined with LC-MS/MS was a feasible method for screening the potential therapeutic targets associated with QGD treatment. It suggests that AGT, MMP3, HPSE and GAPDH may be candidate protein targets of QGD treatment which can be used as therapeutic effect monitor and early diagnosis of primary type I osteoporosis.

The combination of blueberry juice and probiotics reduces apoptosis of alcoholic fatty liver of mice by affecting SIRT1 pathway.

PurposeTo explore the effects of the combination of blueberry juice and probiotics on the apoptosis of alcoholic fatty liver disease (AFLD).MethodsHealthy C57BL/6J mice were used in the control group (CG). AFLD mice models were established with Lieber–DeCarli ethanol diet and evenly assigned to six groups with different treatments: MG (model), SI (SIRT1 [sirtuin type 1] small interfering RNA [siRNA]), BJ (blueberry juice), BJSI (blueberry juice and SIRT1 siRNA), BJP (blueberry juice and probiotics), and BJPSI (blueberry juice, probiotics, and SIRT1 siRNA). Hepatic tissue was observed using hematoxylin and eosin (HE) and Oil Red O (ORO) staining. Biochemical indexes of the blood serum were analyzed. The levels of SIRT1, caspase-3, forkhead box protein O1 (FOXO1), FasL (tumor necrosis factor ligand superfamily member 6), BAX, and Bcl-2 were measured by reverse transcription-polymerase chain reaction and Western blotting.ResultsHE and ORO staining showed that the hepatocytes were heavily destroyed with large lipid droplets in MG and SI groups, while the severity was reduced in the CG, BJ, and BJP groups (P<0.05). The levels of superoxide dismutase (SOD), reduced glutathione (GSH), and high-density lipoprotein-cholesterol (HDL-C) were increased in BJ and BJP groups when compared with the model group (P<0.05). In contrast, the levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT), total triglycerides (TGs), total cholesterol, low-density lipoprotein-cholesterol (LDL-C), and malondialdehyde (MDA) were lower in BJ and BJP groups than in the model group (P<0.05). The level of SIRT1 was increased, while the levels of FOXO1, phosphorylated FOXO1, acetylated FOXO1, FasL, caspase-3, BAX, and Bcl-2 were decreased in CG, BJ, and BJP groups (P<0.05). Meanwhile, SIRT1 silence resulted in increase of the levels of FOXO1, phosphorylated FOXO1, acetylated FOXO1, FasL, caspase-3, BAX, and Bcl-2.ConclusionThe combination of blueberry juice and probiotics reduces apoptosis in AFLD by suppressing FOXO1, phosphorylated FOXO1, acetylated FOXO1, FasL, caspase-3, BAX, and Bcl-2 via the upregulation of SIRT1.

Hepatic lipase inactivation decreases atherosclerosis in insulin resistance by reducing LIGHT/Lymphotoxin β-Receptor pathway

Coexistence of insulin resistance (IR) and metabolic syndrome (MetS) increases the risk of cardiovascular disease (CVD). Genetic studies in diabetes have linked Hepatic Lipase (HL) to an enhanced risk of CVD while others indicate a role of HL in inflammatory cells. Thus, we explored the role of HL on atherosclerosis and inflammation in a mouse model of MetS/IR, (apoE-/-Irs2+/- mice) and in patients with MetS and IR. HL-deficiency in apoE-/-Irs2+/- mice reduced atheroma size, plaque vulnerability, leukocyte infiltration and macrophage proliferation. Compared with apoE-/-Irs2+/-HL+/+ mice, MCP1, TNFα and IL6 plasma levels, pro-inflammatory Ly6Chi monocytes and activated(CD69+)-T lymphocytes were also decreased in apoE-/-Irs2+/-HL-/- mice. The LIGHT (Tumour necrosis factor ligand superfamily member 14, TNFSF14)/Lymphotoxin β-Receptor(LTβ-R) pathway, which is involved in T-cell and macrophage activation, was diminished in plasma and in apoE-/-Irs2+/-HL-/- mouse atheromas. Treatment of apoE-/-Irs2+/-HL-/- mice with LIGHT increased the number of Ly6Chi-monocytes and lesion size. Acutely LIGHT-treated apoE-/- mice displayed enhanced proliferating Ly6Chi-monocytes and increased activation of the mitogen-activated protein kinase p38, suggesting that LIGHT/LTβ-R axis might promote atherogenesis by increasing proinflammatory monocytes and proliferation. Notably, MetS-IR subjects with increased atherosclerosis displayed up-regulation of the LIGHT/LTβ-R axis, enhanced inflammatory monocytes and augmented HL mRNA expression in circulating leukocytes. Thus, HL-deficiency decreases atherosclerosis in MetS/IR states by reducing inflammation and macrophage proliferation which are partly attributed to reduced LIGHT/LTβ-R pathway. These studies identify the LIGHT/LTβ-R axis as a main pathway in atherosclerosis and suggest that its inactivation might ameliorate inflammation and macrophage proliferation associated with atherosclerosis burden in MetS/IR.

Use of a proximity extension assay proteomics chip to discover new biomarkers for human atherosclerosis.

We used a proteomics array to simultaneously measure multiple proteins that have been suggested to be associated with atherosclerosis and related them to plaque prevalence in carotid arteries in a human population-based study. In the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS; n = 931, 50% women, all aged 70 years), the number of carotid arteries with plaques was recorded by ultrasound. Levels of 82 proteins were assessed in plasma by a proximity extension assay (Proseek Multiplex CVD, Olink Bioscience, Uppsala, Sweden) and related to carotid measures in a regression framework. Following adjustment for multiple testing with Bonferroni correction, seven of the proteins were significantly related to the number of carotid arteries affected by plaques in sex-adjusted models (osteoprotegrin, T-cell immunoglobulin and mucin domain (TIM)-1, growth/differentiation factor 15 (GDF-15), matrix metalloprotease-12 (MMP-12), renin, tumor necrosis factor ligand superfamily member 14 (TNFSF14) and growth hormone). Of these, renin (odds ratio [OR], 1.30; 95% confidence interval [CI], 1.13-1.49 per standard deviation increase), growth hormone (OR, 1.24; 95% CI, 1.08-1.43), osteoprotegerin (OR, 1.22; 95% CI, 1.05-1.43) and TNFSF14 (OR, 1.17; 95% CI, 1.01-1.35) were related to plaque prevalence independently of each other and traditional cardiovascular risk factors. A novel targeted proteomics approach using the proximity extension technique discovered several new associations of candidate proteins with carotid artery plaque prevalence in a large human sample.

The Naïve Murine Cornea as a Model System to Identify Novel Endogenous Regulators of Lymphangiogenesis: TRAIL and rtPA

In the murine cornea, which is an established model for analyzing pathologic lymphatic vessel growth, phenotypic heterogeneity of the endogenous lymphatic vessels in the limbus of the cornea was previously described. In this study, the cornea of BALB/c, C57BL/6, and FVB mice with different limbal lymphangiogenic phenotypes was analyzed to identify novel candidates potentially influencing lymphatic vessel growth. Pathway specific expression analysis of the cornea was performed to identify novel candidate genes. Corneal protein expression of the respective candidates was analyzed by fluorescent immunohistochemistry. The effect of the candidates on proliferation of human dermal lymphatic endothelial cells (HDLECs) was analyzed by BrdU proliferation ELISA. Thirteen genes were differentially regulated in corneas of mouse strains with more endogenous limbal lymphatic vessels (high-lymphangiogenic) (C57BL/6) compared to mouse strains with less endogenous limbal lymphatic vessels (low-lymphangiogenic) (BALB/c, FVB). Two candidates, Tumor necrosis factor (ligand) superfamily member 10 (Tnfsf10/Trail) and Plasminogen activator, tissue (Plat/tPA) were expressed in the cornea of BALB/c and C57BL/6 mice on the protein level. In vitro, Trail and recombinant tPA inhibited the proliferation of human dermal lymphatic endothelial cells. Molecular analysis of the naive cornea in mouse strains with different limbal lymphatic phenotypes is a valuable model to identify novel endogenous regulators of lymphangiogenesis.

Effects of fermented soybean meal on innate immunity-related gene expressions in nursery pigs acutely challenged with lipopolysaccharides.

This experiment was to determine if replacing soybean meal with fermented soybean meal (FSBM) would reduce the innate immune response after lipopolysaccharide challenge and the changes of gene expression profiles associated with this response. Forty-eight 21 day-old pigs were housed individually and fed three diets for 15 days: CON (a diet without FSBM or spray-dried plasma protein; SDPP), PP7 (a diet with 7% SDPP), and FS10 (a diet with 10% FSBM). Pigs were fitted with a jugular vein catheters receiving lipopolysaccharide challenge (25 μg/kg body weight (BW)) on day 15. Blood was collected for 5 h at 30-min intervals to measure cortisol. Expressions of gene transcripts in total RNA from leukocytes were compared using an oligonucleotide microarray at 210 min after lipopolysaccharides injection. Cortisol of FS10 was lower (P < 0.05) than CON after lipopolysaccharides challenge. The expression levels of 17 transcripts, including cytosolic glutathione peroxidase and glutathione S-transferase A4-4 were increased (P < 0.05), whereas 23 genes including adiponectin, neonatal Fc receptor and tumor necrosis factor ligand superfamily member 5 were decreased (P < 0.05) in FS10. This study suggests that FSBM-fed pigs can modulate expression of genes related to inflammatory response and anti-oxidant activity which can be a potential reason for reduced serum cortisol.

A TNFSF15 disease-risk polymorphism increases pattern-recognition receptor-induced signaling through caspase-8-induced IL-1.

Inflammatory diseases are characterized by dysregulated cytokine production. Altered functions for most risk loci, including the inflammatory bowel disease and leprosy-associated tumor necrosis factor ligand superfamily member 15 (TNFSF15) region, are unclear. Regulation of pattern-recognition-receptor (PRR)-induced signaling and cytokines is crucial for immune homeostasis; TNFSF15:death receptor 3 (DR3) contributions to PRR responses have not been described. We found that human macrophages expressed DR3 and that TNFSF15:DR3 interactions were critical for amplifying PRR-initiated MAPK/NF-κB/PI3K signaling and cytokine secretion in macrophages. Mechanisms mediating TNFSF15:DR3 contributions to PRR outcomes included TACE-induced TNFSF15 cleavage to soluble TNFSF15; soluble TNFSF15 then led to TRADD/FADD/MALT-1- and caspase-8-mediated autocrine IL-1 secretion. Notably, TNFSF15 treatment also induced cytokine secretion through a caspase-8-dependent pathway in intestinal myeloid cells. Importantly, rs6478108 A disease risk-carrier macrophages demonstrated increased TNFSF15 expression and PRR-induced signaling and cytokines. Taken together, TNFSF15:DR3 interactions amplify PRR-induced signaling and cytokines, and the rs6478108 TNFSF15 disease-risk polymorphism results in a gain of function.

Novel biomarkers of mercury-induced autoimmune dysfunction: A cross-sectional study in Amazonian Brazil

Mercury is a ubiquitous environmental contaminant, causing both neurotoxicity and immunotoxicity. Given its ability to amalgamate gold, mercury is frequently used in small-scale artisanal gold mining. We have previously reported that elevated serum titers of antinuclear autoantibodies (ANA) are associated with mercury exposures of miners in gold mining. The goal of this project was to identify novel serum biomarkers of mercury-induced immunotoxicity and autoimmune dysregulation.We conducted an analysis of serum samples from a cross-sectional epidemiological study on miners working in Amazonian Brazil. In proteomic screening analyses, samples were stratified based on mercury concentrations and ANA titer and a subset of serum samples (N=12) were profiled using Immune Response Biomarker Profiling ProtoArray protein microarray for elevated autoantibodies. Of the up-regulated autoantibodies in the mercury-exposed cohort, potential target autoantibodies were selected based on relevance to pro-inflammatory and macrophage activation pathways. ELISAs were developed to test the entire sample cohort (N=371) for serum titers to the highest of these autoantibodies (anti-glutathione S-transferase alpha, GSTA1) identified in the high mercury/high ANA group.We found positive associations between elevated mercury exposure and up-regulated serum titers of 3760 autoantibodies as identified by ProtoArray. Autoantibodies identified as potential novel biomarkers of mercury-induced immunotoxicity include antibodies to the following proteins: GSTA1, tumor necrosis factor ligand superfamily member 13, linker for activation of T cells, signal peptide peptidase like 2B, stimulated by retinoic acid 13, and interferon induced transmembrane protein. ELISA analyses confirmed that mercury-exposed gold miners had significantly higher serum titers of anti-GSTA1 autoantibody [unadjusted odds ratio=89.6; 95% confidence interval: 27.2, 294.6] compared to emerald miners (referent population).Mercury exposure was associated with increased titers of several autoantibodies in serum including anti-GSTA1. These proteins play a wide variety of roles, including as antioxidants, in the regulation of pro- and anti-inflammatory cytokines, as well as danger and oxidative stress signaling. Dysregulation of these proteins and pathways is believed to play a role in autoimmune diseases such as rheumatoid arthritis, Sjögren׳s syndrome, and multiple sclerosis. Taken together, these results suggest that mercury exposure can induce complex autoimmune dysfunction and the immunotoxic effects of this dysfunction may be measured by serum titers to autoantibodies such as anti-GSTA1.

High Expression of TNFSF13 in Tumor Cells and Fibroblasts Is Associated With Poor Prognosis in Non–Small Cell Lung Cancer

To examine high expression of tumor necrosis factor ligand superfamily member 13 (TNFSF13), which is correlated with several malignancies. TNFSF13 messenger RNA expression in tumor cells and fibroblasts in a cohort of patients with non-small cell lung cancer (NSCLC) was analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry using a tissue microarray. TNFSF13 expression was significantly higher in lung adenocarcinomas compared with squamous cell carcinomas (P = .022). High TNFSF13 expression in NSCLC stroma was related with low differentiation (P = .045) and sex (male > female, P = .005). Cox proportional hazards regression univariate and multivariable analysis revealed TNFSF13 expression in NSCLC tumor cells (P = .007) or fibroblasts (P = .027) as an independent prognostic factor in the 5-year overall survival rate. Our findings indicate TNFSF13 is a prognostic factor in NSCLC and suggest TNFSF13 may be a novel therapeutic target for NSCLC.