TNF-α Concentrations Research Articles

Ozone Induces Oxidative Stress and Inflammation in Nasal Mucosa of Rats

Background: The development of the global economy has led to changes in air pollution patterns. The haze phenomenon characterized by high concentrations of particulate matter 2.5 (PM2.5) has changed to complex pollution, and photochemical pollution characterized by ozone (O3) has become increasingly prominent. Ozone pollution and its impact on human health has become an important topic that needs to be studied urgently. Objective: To investigate the effects of ozone on oxidative stress and inflammation in the nasal mucosa of a rat model. Methods: Thirty-two healthy female Sprague–Dawley rats, eight in each group, were divided into four groups using the randomized numeric table method: normal control group (NC group), normal rats with a low level of ozone inhalation exposure (NEL group, 0.5 ppm), medium ozone inhalation exposure (NEM group, 1 ppm), and high ozone inhalation exposure (NEH group, 2 ppm). The ozone inhalation exposure groups were placed in the ozone inhalation exposure system and exposed to different concentrations of ozone for 2 h each day for 6 weeks. Nasal secretion was measured, and nasal lavage and nasal mucosa were collected. Malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) activities were measured by colorimetric assay, and the nasal mucosa was analyzed by Western blot. Western blot (WB) was used to detect the expression of NF-κB p65 nuclear protein in nasal mucosa. The mRNA expression of NF-κB target genes IL-6 and IL-8 and tumor necrosis factor-α (TNF-α) was detected by real-time quantitative PCR (qRT-PCR), and the protein content of pro-inflammatory factors IL-6, IL-8, and TNF-α was detected by ELISA in serum and nasal lavage fluid. The nasal mucosa of rats was stained with hematoxylin-eosin (HE) to observe the pathological changes in the nasal mucosa. The data were analyzed by SPSS 20.0 software. Results: The amount of nasal secretion increased significantly in all groups after ozone exposure compared with that in the NC group. The MDA content of the nasal mucosa was significantly increased in the ozone-exposed group compared with the NC group, and the activity levels of SOD and GSH-Px in the nasal mucosa were lower in the ozone-exposed group than in the NC group. The mRNA expression of IL-6, IL-8, and TNF-α in the nasal mucosa of the ozone-exposed group was elevated, and the protein content of TNF-α, IL-6, and IL-8 in the nasal lavage fluid was elevated, and the content increased with the increase in ozone concentration. The expression of NF-κB p65 intracellular protein in the nasal mucosa of each ozone-exposed group was higher than that of the normal group, and the content increased with the increase in ozone concentration. Conclusions: Ozone inhalation exposure promotes oxidative stress and the release of inflammatory factors TNF-α, IL-6, and IL-8, leading to pathological damage of the nasal mucosa, the degree of which increases with increasing concentration. This pathological process may be related to the activation of the transcription factor NF-κB by ozone in the nasal mucosa of rats, which increases the expression of its target genes.

Identification of a novel immune infiltration-related gene signature, MCEMP1, for coronary artery disease.

This study aims to identify a novel gene signature for coronary artery disease (CAD), explore the role of immune cell infiltration in CAD pathogenesis, and assess the cell function of mast cell-expressed membrane protein 1 (MCEMP1) in human umbilical vein endothelial cells (HUVECs) treated with oxidized low-density lipoprotein (ox-LDL). To identify differentially expressed genes (DEGs) of CAD, datasets GSE24519 and GSE61145 were downloaded from the Gene Expression Omnibus (GEO) database using the R "limma" package with p < 0.05 and |log2 FC| > 1. Gene ontology (GO) and pathway analyses were conducted to determine the biological functions of DEGs. Hub genes were identified using support vector machine-recursive feature elimination (SVM-RFE) and least absolute shrinkage and selection operator (LASSO). The expression levels of these hub genes in CAD were validated using the GSE113079 dataset. CIBERSORT program was used to quantify the proportion of immune cell infiltration. Western blot assay and qRT-PCR were used to detect the expression of hub genes in ox-LDL-treated HUVECs to validate the bioinformatics results. Knockdown interference sequences for MCEMP1 were synthesized, and cell proliferation and apoptosis were examined using a CCK8 kit and Muse® Cell Analyzer, respectively. The concentrations of IL-1β, IL-6, and TNF-α were measured with respective enzyme-linked immunosorbent assay (ELISA) kits. A total of 73 DEGs (four down-regulated genes and 69 up-regulated genes) were identified in the metadata (GSE24519 and GSE61145) cohort. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis results indicated that these DEGs might be associated with the regulation of platelet aggregation, defense response or response to bacterium, NF-kappa B signaling pathway, and lipid and atherosclerosis. Using SVM-RFE and LASSO, seven hub genes were obtained from the metadata. The upregulated expression of DIRC2 and MCEMP1 in CAD was confirmed in the GSE113079 dataset and in ox-LDL-treated HUVECs. The associations between the two hub genes (DIRC2 and MCEMP1) and the 22 types of immune cell infiltrates in CAD were found. MCEMP1 knockdown accelerated cell proliferation and suppressed cell apoptosis for ox-LDL-treated HUVECs. Additionally, MCEMP1 knockdown appeared to decrease the expression of inflammatory factors IL-1β, IL-6, and TNF-α. The results of this study indicate that MCEMP1 may play an important role in CAD pathophysiology.

Knockdown of IGF2BP3 Down-Regulates PDCD4 Levels to Attenuate Hypoxic-Ischemic Brain Damage

Background: Hypoxic-ischemic brain damage (HIBD) is a prevalent brain injury with high mortality and morbidity. It results from hypoxia and ischemia of the brain due to various perinatal factors. A previous study showed that knockdown of programmed cell death factor 4 (PDCD4) could reduce infarction injury resulting from ischemia/reperfusion injury. However, exact mechanism by which PDCD4 acts in HIBD is not yet understood. Our aim in present investigation was to investigate the function and mechanism of PDCD4 in alleviating HIBD. Methods: An HIBD model was developed using neonatal rats. After 48 h of modeling, short-term neurological function was evaluated and the brain tissue removed for assessment of cerebral infarct volume and brain water content (BWC). A cell model of oxygen glucose deprivation/reoxygenation (OGD/R) was also constructed. Overexpression or knockdown of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) or PDCD4 was performed in pretreated cells. Results: The geotaxis reflex time, cerebral infarct volume, and BWC all increased after HIBD in this neonatal rat model. Additionally, the levels of PDCD4 and of the N6-Methyladenosine (m6A) reader protein IGF2BP3 were increased in HIBD rats and OGD/R-stimulated pheochromocytoma (PC12) cells relative to controls. Moreover, OGD/R-stimulated pheochromocytoma PC12 cells showed decreased cell viability, increased apoptosis, and elevated Interleukin 6 (IL-6), Interleukin 1 β (IL-1β), and tumor necrosis factor-α (TNF-α) contents. These features were reversed after knocking down IGF2BP3. The interaction between IGF2BP3 protein and PDCD4 mRNA was confirmed by RNA immunoprecipitation and RNA pull-down assays. Furthermore, knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells reduced cell damage via down-regulation of PDCD4. Finally, the IGF2BP3/PDCD4 axis alleviated OGD/R-induced cell injury in primary cortical neurons (PCNs). Conclusions: PDCD4 and m6A reader protein IGF2BP3 were up-regulated in an HIBD neonatal rat model. Knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells or PCNs alleviated cell damage through reducing PDCD4.

Effect of IL-7 and IL-7R blockade on cytokine production by peripheral blood t cells from patients with psoriatic arthritis

Psoriatic arthritis (PsA) is a chronic immune-mediated inflammatory joint disease, often associated with psoriasis. Interactions between immune cells, mainly T lymphocytes, and cells of the osteoarticular system are central in the pathogenesis of PsA. Th1, Th17 cytokines associated with the development of PsA contribute to an increase in the production of cytokines, chemokines, matrix metalloproteinases, adhesion molecules by immune cells, chondrocytes, fibroblasts, and induction of osteoclasts. That leads to the destruction of cartilage and bone tissue. Among the cytokines potentially involved in the pathogenesis of PsA, IL-7 is of particular interest. IL-7 is a T cell survival factor. However, it can promote the production of IFNγ and TNFα by T cells. IL-7 may indirectly promote osteoclast maturation and cartilage degradation. The aim was to investigate the effect of IL-7 and blockade of the α-chain of the IL-7 receptor (IL-7R) in vitro on the production of IL-5, IL-13, IL-2, IL-6, IL-9 IL-10, IFNγ, TNFα, IL-17A, IL-17F, IL-4, and IL-22 by T cells in norm and PsA.The study included 14 patients with PsA in the acute stage of the disease and 8 healthy individuals. To determine cytokines concentration, multiplex analysis was performed using flow cytometry.It was shown that IL-7 enhances the production of Th1, Th2, Th17, Th22, and Th9 cytokines in both patients with PsA and healthy individuals. The exception was IL-2 for both groups. Under the blockade of IL-7R with monoclonal antibodies, the level of IL-6 increases and production of IFNγ, TNFα, IL-17F, IL-10, IL-5, and IL-9 by donors’ cells and production of IFNγ, TNFα, IL-22, IL-2, IL-4, IL-5, IL-13, and IL-9 by cells from patients with PsA decreases relative to production of cells stimulated with IL-7. In donors, the blockade contributed to a change in the balance of Th1/Th2 cytokines: level of IL-4, IL-13 did not change on the background of a decrease in production of IFNγ, TNFα. In patients with PsA, under blockade of IL-7R the production of IL-10 remained at an increased level and concentration of IFNγ, TNFα and IL-2, which are actively involved in the tissue damage mechanisms, decreased. The obtained data indicate the prospect of using the IL-7R as a target for the treatment of psoriatic diseases.

Research on ameliorating pulmonary fibrosis in silicosis mice of Cordyceps cicadae polysaccharides

Objective: A mouse silicosis model was constructed by injecting silicon dioxide (SiO(2)) particles into the trachea to explore the effect and mechanism of Cordyceps cicadae polysaccharides (CCP) on ameliorating pulmonary fibrosis in silicosis mice. Methods: In May 2023, CCP were extracted and isolated, the monosaccharide composition and functional group composition were analyzed by high performance liquid chromatography and Fourier transform infrared spectroscopy. C57BL/6J mice were injected with 50 μl 50 mg/ml SiO(2) suspension to construct silicosis mouse model, which were then randomly divided into model group, CCP intervention groups [low dose group (LCCP group), medium dose group (MCCP group) and high dose group (HCCP group) ], the control group was administered by physiological saline, 8 mice in each group. Mice in the CCP intervention groups received oral gavage administration once daily with CCP solution (100, 200 and 400 mg/kg), while control group and model group received physiological saline, lasted for 30 days. The body weight of mice was recorded and the lung coefficient was calculated. The pathomorphological changes of mouse lung tissue were determined by HE and Masson staining. The contents of fibrosis indexes [hydroxyproline acid (HYP), connective tissue growth factor (CTGF) and matrix metallopeptidase 2 (MMP-2) ] of lung tissue and the pro-inflammatory factors[tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) ] of lung tissue and alveolar lavage fluid were determined by ELISA. The expression level of Collagen Ⅰ was determined by immunohistochemistry. The relative protein expression levels of transforming growth factor-β1 (TGF-β1), P-Smad2, α-smooth muscle actin (α-SMA), Toll-like receptor 4 (TLR4), nuclear factor kappa-B p65 (NF-κBp65) and myeloid differentiation primary response gene 88 (MyD88) in lung tissue were determined by Western blot. Results: The total sugar content of the CCP was 86.78%, composed of D-mannose, D-rhamnose, D-glucose and D-galactose, with a molar ratio of 12.71∶1.53∶1.00∶12.64. The infrared spectrum indicated the characteristic groups of its polysaccharides. Compared with the control group, the body weight of mice in the model group was decreased, lung coefficient was increased, the contents of HYP, CTGF and MMP-2 in lung tissue were increased, and the contents of TNF-α, IL-1β and IL-6 in lung tissue and alveolar lavage fluid were increased (P<0.05). The mice lung showed massive inflammatory cell infiltration and collagen fiber deposition, and the silicosis fibrosis was severe. The expression of CollagenⅠin lung tissue of model group was increased, and the proteins expression levels of TGF-β1, P-Smad2/Smad2, α-SMA, TLR4, NF-κBp65 and MyD88 were increased in mouse lung tissue (P<0.05). Compared with the model group, the body weights of mice in the MCCP and HCCP groups were increased, the lung coefficients were decreased, the contents of HYP, CTGF and MMP-2 in lung tissue were decreased, and the contents of TNF-α, IL-1β and IL-6 in lung tissue and alveolar lavage fluid were decreased (P<0.05). The inflammatory cell infiltration in the lung was reduced, and the degree of fibrosis was improved to varying degrees. The expression level of CollagenⅠwas down-regulated in the lung tissue of MCCP and HCCP groups, and the protein expression levels of TGF-β1, P-Smad2/Smad2, α-SMA, TLR4, NF-κBp65 and MyD88 were decreased in lung tissue (P<0.05) . Conclusion: The CCP could reduce the levels of fibrosis-related indicators and pro-inflammatory factors in lung tissue, ameliorating mouse lung inflammation and silicosis fibrosis caused by SiO(2) particles by inhibiting the activation of TGF-β1/Smad pathway and TLR4/nuclear factor kappa-B (NF-κB) pathway.

A Pilot Study on the Effects of Exercise Training on Cardiorespiratory Performance, Quality of Life, and Immunologic Variables in Long COVID.

Objectives: Fatigue is a prominent feature of long COVID (LC) and may be related to several pathophysiologic mechanisms, including immune hyperstimulation. Aerobic endurance exercise training may be a useful therapy, with appropriate attention to preventing post-exertional malaise. Methods: Fourteen participants completed a pilot study of aerobic exercise training (twenty 1.5 h sessions of over 10 weeks). Cardiorespiratory fitness, 6 min walk distance, quality of life, symptoms, 7-day physical activity, immunophenotype, and inflammatory biomarkers were measured before and after exercise training. Results: The participant characteristics at baseline were as follows: 53.5 ± 11.6 yrs, 53% f, BMI 32.5 ± 8.4, 42% ex-smokers, 15.1 ± 8.8 months since initial COVID-19 infection, low normal pulmonary function testing, V.O2peak 19.3 ± 5.1 mL/kg/min, 87 ± 17% predicted. After exercise training, participants significantly increased their peak work rate (+16 ± 20 W, p = 0.010) and V.O2peak (+1.55 ± 2.4 mL/kg/min, p = 0.030). Patients reported improvements in fatigue severity (-11%), depression (-42%), anxiety (-29%), and dyspnea level (-46%). There were no changes in 6MW distance or physical activity. The circulating number of CD3+, CD4+, CD19+, CD14++CD16, and CD16++CD14+ monocytes and CD56+ cells (assessed with flow cytometry) increased with acute exercise (rest to peak) and was not diminished or augmented by exercise training. Plasma concentrations of TNF-α, IL-6, IL-8, IL-10, INF-γ, and INF-λ were normal at study entry and not affected by training. Conclusions: Aerobic endurance exercise training in individuals with LC delivered beneficial effects on cardiorespiratory fitness, quality of life, anxiety, depression, and fatigue without detrimental effects on immunologic function.

Resistin alleviates lipopolysaccharide-induced inflammation in bovine alveolar macrophages by activating the AMPK/mTOR signaling pathway and autophagy

ObjectiveResistin (RETN) is an adipocyte-specific hormone that participates in metabolism and modulates cellular inflammation. Our study aimed to assess the effects of RETN treatment on autophagy and the underlying molecular and biological mechanisms in bovine alveolar macrophages (BAMs). MethodsThe optimal concentration of RETN + lipopolysaccharide (LPS) on macrophages was screened and then used to co-culture with alveolar macrophages. Autophagosomes in BAMs were examined using a transmission electron microscope (TEM). Quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression of microtubule-associated protein light chain 3 (LC3) and p62. Western blot (WB) was used to detect the protein expressions of LC3 and p62. The distribution of LC3 and p62 proteins in the cells was observed by immunofluorescence (IF). The concentrations of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) were detected using enzyme-linked immunosorbent assay (ELISA). The protein expression of adenosine-monophosphate-activated protein kinase (AMPK), p-AMPK, mammalian target of rapamycin (mTOR), and p-mTOR was detected using WB. ResultsThe treatment of BAMs with RETN or LPS increased the number of autophagosomes and the ratio of LC3II/LC3I and decreased the expression level of p62 protein. RETN treatment significantly triggered autophagy compared to LPS treatment. Moreover, the ratios of p-AMPK/AMPK and p-mTOR/mTOR were upregulated and downregulated, respectively, after RETN treatment, suggesting that AMPK/mTOR signaling pathway activation is required for RETN-mediated autophagy in BAMs. Additionally, the ratio of LC3-II/LC3-I was lower, and the concentrations of IL-1β, IL-6, and TNF-α significantly decreased in the LPS and RETN co-treatment groups compared to the single LPS treatment group. However, both autophagy- and LPS-induced inflammation were partially alleviated by RETN treatment. ConclusionRETN can promote autophagy in BAMs by activating the AMPK/mTOR signaling pathway, it may help prevent LPS-induced inflammation.

Prebiotic inulin controls Th17 cells mediated central nervous system autoimmunity through modulating the gut microbiota and short chain fatty acids

ABSTRACT Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination occurring in the central nervous system (CNS). Inulin is a common prebiotic that can improve metabolic disorders by modulating the gut microbiota. However, its capacity to affect CNS autoimmunity is poorly recognized. Experimental autoimmune encephalomyelitis (EAE) is a classical mouse model of MS. Herein, we found that oral administration of inulin ameliorated the severity EAE in mice, accompanied by reductions in inflammatory cell infiltration and demyelination in the CNS. These reductions were associated with decreased proportion and numbers of Th17 cells in brain and spleen. Consistent with the findings, the serum concentrations of IL-17, IL-6, and TNF-α were reduced in inulin treated EAE mice. Moreover, the proliferation of auto-reactive lymphocytes, against MOG35-55 antigen, was attenuated ex vivo. Mechanistically, inulin treatment altered the composition of gut microbiota. It increased Lactobacillus and Dubosiella whereas decreased g_Prevotellaceae_NK3B31_group at the genus level, alongside with elevated concentration of butyric acid in fecal content and serum. In vitro, butyrate, but not inulin, could inhibit the activation of MOG35-55 stimulated lymphocytes. Furthermore, fecal microbiota transplantation assay confirmed that fecal contents of inulin-treated normal mice had an ameliorative effect on EAE mice. In contrast, antibiotic cocktail (ABX) treatment diminished the therapeutic effect of inulin in EAE mice as well as the reduction of Th17 cells, while supplementation with Lactobacillus reuteri restored the amelioration effect. These results confirmed that the attenuation of inulin on Th17 cells and inflammatory demyelination in EAE mice was dependent on its modulation on gut microbiota and metabolites. Our findings provide a potential therapeutic regimen for prebiotic inulin supplementation in patients with multiple sclerosis.

Study on pharmacokinetic and tissue distribution of hyperin, astragalin, kaempferol-3-O-β-D-glucuronide from rats with multiple administrations of Semen Cuscutae processed with salt solution with effect of treating recurrent spontaneous abortion.

Semen Cuscutae is a traditional Chinese medicine (TCM) that tonifies the kidneys and prevents miscarriage. According to Chinese medicine theory, kidney deficiency is one of the main causes of recurrent spontaneous abortion (RSA). The previous studies showed that raw product of Semen Cuscutae (SP) and Semen Cuscutae processed with salt solution (YP) have ameliorative effects on RSA, and that YP is superior to SP. However, the active components of YP to ameliorate RSA remain unclear and require further studies. The objective of this study is to investigate the active components of YP in ameliorating RSA. First, a rat model of RSA was established using hydroxyurea in combination with mifepristone. Aqueous decoction of YP was given by gavage to rats. Second, pregnant rats were sampled on days 5, 7, 9, 10 and 12 during the modelling period. The content of Hyperin (HY), astragalin (AS) and kaempferol-3-O-β-D-glucuronide (KA) in blood and liver, heart, spleen, lung and kidney tissues were detected by liquid chromatography-mass spectrometry (LC-MS). The pharmacodynamic indicators including progesterone (P), chorionic gonadotropin β (β-HCG), estradiol (E2), tumor necrosis factor-α (TFN-α), interleukin 4 (IL-4), and tryptophan (TRP) were measured by enzyme-linked immunosorbent assay (ELISA) Pearson's correlation analysis and grey relational analysis were used to establish the relationship between the pharmacodynamic indexes and chemical constituents. The pharmacokinetic results showed that the area under curve (AUC) value of KA was the largest. The tissue distribution results showed that astragalin was widely distributed in liver, heart, spleen, lung and kidney in the RSA model rats, while HY was detected only in the uterus, and KA was detected only in the kidney. The pearson correlationl analysis showed that KA was significantly and positively correlated with the contents of E2, P, β-HCG and TRP. Both AS and HY were significantly negatively correlated with the content of TNF-α, respectively. This study reveals the pharmacokinetics and tissue distribution of KA, AS and HY in rats with RSA. It was elucidated that all three were involved in the regulation of progesterone levels and immune function. It initially revealed the mechanism of action of YP in enhancing the improvement of RSA, and it provided a theoretical basis for the quality assessment of YP.

Chlorogenic Acid Enhances the Intestinal Health of Weaned Piglets by Inhibiting the TLR4/NF-κB Pathway and Activating the Nrf2 Pathway.

Chlorogenic acid (CGA) is a natural polyphenol with potent antioxidant and anti-inflammatory activities. However, the exact role of it in regulating intestinal health under oxidative stress is not fully understood. This study aims to investigate the effects of dietary CGA supplementation on the intestinal health of weaned piglets under oxidative stress, and to explore its regulatory mechanism. Twenty-four piglets were randomly divided into two groups and fed either a basal diet (CON) or a basal diet supplemented with 200 mg/kg CGA (CGA). CGA reduced the diarrhea rate, increased the villus height in the jejunum, and decreased the crypt depth in the duodenum, jejunum, and ileum of the weaned piglets (p < 0.05). Moreover, CGA increased the protein abundance of Claudin-1, Occludin, and zonula occludens (ZO)-1 in the jejunum and ileum (p < 0.05). In addition, CGA increased the mRNA expression of pBD2 in the jejunum, and pBD1 and pBD2 in the ileum (p < 0.05). The results of 16S rRNA sequencing showed that CGA altered the ileal microbiota composition and increased the relative abundance of Lactobacillus reuteri and Lactobacillus pontis (p < 0.05). Consistently, the findings suggested that the enhancement of the intestinal barrier in piglets was associated with increased concentrations of T-AOC, IL-22, and sIgA in the serum and T-AOC, T-SOD, and sIgA in the jejunum, as well as T-AOC and CAT in the ileum caused by CGA (p < 0.05). Meanwhile, CGA decreased the concentrations of MDA, IL-1β, IL-6, and TNF-α in the serum and jejunum and IL-1β and IL-6 in the ileum (p < 0.05). Importantly, this study found that CGA alleviated intestinal inflammation and oxidative stress in the piglets by inhibiting the TLR4/NF-κB signaling pathway and activating the Nrf2 signaling pathway. These findings showed that CGA enhances the intestinal health of weaned piglets by inhibiting the TLR4/NF-κB pathway and activating the Nrf2 pathway.

PSIV-13 Changes in Interferon-γ and Tumor necrosis factor-α in the vaginal environment of cattle during early gestation

Abstract The embryo must create an immuno-tolerant uterine environment via cytokine signaling to prevent rejection and facilitate embryo attachment. Cytokines have a variety of roles in both reproductive and pregnancy-related processes. Cytokines and chemokines are important in inflammatory pathways for uterine remodeling and recruitment of immune cells during pregnancy. Interferon-γ (INFγ) and tumor necrosis factor-a (TNFα) influence T-helper cell immune responses, which can assist in early pregnancy establishment. Therefore, we hypothesized that INFγ and TNFα concentrations following breeding will be differentially expressed based on mating type, time, and pregnancy outcome. To test this, we completed two experiments in which INFγ and TNFα were quantified in vaginal fluid samples on d 0, d 7, d 14, d 21, d 28, and d 35 following mating. In experiment one, Angus heifers (n = 9) were used to determine cytokine concentrations in the vaginal environment between d 0 to d 35, and between artificial insemination (AI) and natural mating. In experiment 2, lactating dairy cows (n = 88) were used to determine if cytokines could be used as a potential biomarker for successful pregnancy. All vaginal flush samples were collected via Foley catheter in 20 mL of sterile saline. Statistical analysis was completed in SAS 9.4 to determine differences in cytokine concentrations based on day of gestation, mating type, and pregnancy outcome. In experiment 1, vaginal concentrations of TNFα were least (P = 0.05) in AI-bred heifers on d 35 (1.15 ± 2.97 pg/mL) and intermediate in non-pregnant heifers (4.39 ± 2.97 pg/mL) compared with natural service-bred heifers on d 7 (18.09 ± 2.97 pg/mL). INFγ was increased (P = 0.05) on d 7 following natural service (3.86 ± 0.76 pg/mL) compared with AI (0.86 ± 0.76 pg/mL). TNFα was increased (P = 0.02) on d 7 following natural service (18.09 ± 2.89 pg/mL) compared with AI (1.93 ± 2.89 pg/mL) and was increased (P = 0.02) by d 7 after a natural mating compared with a pregnancy resulting from AI mating. In experiment two, concentrations of IFNγ were greater (P = 0.04) on d 7 (1.36 ± 0.57 pg/mL) compared with d 8 (0.24 ± 0.57 pg/mL) of gestation. There was also a tendency for TNFα concentrations to decrease (P = 0.09) from d 7 (60.06 ± 26.94 pg/mL) to d 8 (19.67 ± 26.94 pg/mL) of gestation. In conclusion, concentrations of INFγ and TNFα may not be indicative of a viable embryo or pregnancy outcome but do differ based on day of gestation and mating type, which may aid in understanding the role these cytokines play during early gestation.

PSI-8 Inflammatory cytokine concentrations and gene expression in mature and aging sedentary horses

Abstract Chronic inflammation associated with aging, termed “inflamm-aging” may negatively impact overall animal health. However, the mechanisms of inflamm-aging and, therefore, effective interventions, remain unknown. Nuclear factor erythroid 2-related factor 2 (Nrf2; NFE2L2) is a transcription factor that controls antioxidant gene expression and could be a target for promoting anti-inflammatory pathways. To test the hypothesis that supplementation of nonenzymatic derived antioxidants (Protandim Nrf2 Synergizer, LifeVantage Corporation) would activate Nrf2 and mitigate inflammation, 40 mature, sedentary horses [32 mares, 8 geldings; 15.7 ± 4.9 yr of age, body weight (BW) = 519 ± 46 kg] were stratified by age, sex, and BW and randomly assigned to one of four dietary treatment groups for 56 d: 1) 0 mg (CON); 2) 675 mg (Pro1); 3) 2,025 mg (Pro3); or 4) 4,050 mg (Pro6) Protandim Nrf2 Synergizer per day (n = 10 per group). Blood samples were collected at d 0 (September, 27°C, 61.6% humidity), d 28 (October, 21.6°C, 55.8% humidity), and d 56 (November, 15.1°C, 73.8% humidity) of supplementation prior to the morning feeding. Plasma cytokine concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-6, IL-8, and IL-10 were quantified using custom Meso Scale Discovery U-Plex kits and whole blood gene expression of IL-1β, NFE2L2, and heme oxygenase 1 (HMOX1) were determined using qRT-PCR. Data were analyzed using PROC MIXED in SAS v9.4 with sex, time, treatment, age (under or over 15 yr), and time × treatment as fixed effects and time as a repeated effect with horse(treatment) as the subject. Day 0 was included as a covariate when treatment groups differed at d 0. Relationships between age and cytokine concentrations or mRNA expression were determined using PROC CORR. Plasma IL-4 (r = 0.4054) concentrations as well as whole blood HMOX1 (r = 0.3437) and NFE2L2 (r = 0.3382) expression on d 0 were positively correlated with age (P ≤ 0.04). Regardless of treatment or age, IL-1β and NFE2L2 expression and IL-4 and IL-8 concentrations decreased from d 0 to 28 (P ≤ 0.03) and remained depressed at d 56 (P ≤ 0.02) while IL-10 was not different from d 0 to 28 but decreased from d 0 to 56 (P = 0.02). TNFα concentrations were not different between treatments or over time. These data corroborate increased inflammatory markers with age and suggest a change in inflammatory status over time potentially due to seasonal environmental factors.

Impact of inflammatory skin conditions on the biological profile of plasma rich in growth factor

Plasma rich in growth factors (PRGF) can be used over patients suffering from dermatoses due to its anti-inflammatory effect. However, this population group might present soluble autoimmune components and there is limited information about the effect of chronic skin inflammation on PRGF bioactive properties. With the aim of characterizing PRGF composition, PRGF from healthy (H) donors and patients with atopic dermatitis (AD), psoriasis (PS), or lichen sclerosus (LS) was obtained. In order to reduce the inflammatory component, leukocyte exclusion and heat-inactivation (Immunosafe) were tested. Haematological-serological parameters, platelet functionality, clot microstructure, protein content and bioactivity were determined. Mean values and 95 % confidence intervals (mean[95 % CI]) were computed for key haematological parameters, such as platelet (410×103/mm3[371–449]) and leukocyte content (205×103/mm3[148−262]), platelet activation (resting: 4.3 %[3.1–5.5] and activated: 97.4 %[96.7–98.0]), the concentration of plasma proteins and morphogens, including immunoglobulins A (210.7 mg/dL[191.8–229.6]), G (933.1 mg/dL[887.2–978.9]), E (783.5 mg/dL[54.4–1512.6]), and M (115.0 mg/dL[97.1–133.0]), Complement Protein (31.6 mg/mL[26.6–36.6]), C-Reactive protein (3.1 mg/L[2.0–4.1]), TGF-β1 (35975.6 pg/mL[34221.3–37729.8]), fibronectin (146410.0 ng/mL[136518.3–156301.7]), PDGF-AB (13308.5 pg/mL[12401.0–14216.0]), CD40L (2389.3 pg/mL[1887.7–2890.8]), IL-4 (0.12 pg/mL[0.07–0.18]), IL-13 (35.4 pg/mL[21.0–49.7]), IL-1β (0.09 pg/mL[0.06–0.11]) and TNF-α (0.31 pg/mL[0.24–0.38]), and also for cell proliferation (332.9ngDNA/mL[317.4–348.3]), viability (135.6 %[132.0–139.2]) and migration (103.8cells/mm2[98.3–109.3]). Plasma from AD donors presented increased Immunoglobulin E (IgE) that was significantly reduced after Immunosafe along with the complement system and autoantibodies. Platelet functionality was altered for AD, but no microstructure differences were identified. Pathological groups presented reduced concentration of fibronectin (AD/LS) and Platelet-Derived Growth Factor (PDGF-AB) (P). Immunosafe treatment reduced Cluster of Differentiation 40 Protein (CD40L), interleukin 1β (IL-1β), and Tumor Necrosis Factor α (TNF-α) concentrations. Fibroblasts supplemented with PRGF obtained from pathological patients (PS/AD) showed reduced viability but Immunosafe increased cell proliferation and migration in SP (LS) and L-SP samples (PS/AD). In conclusion, PRGF derived from pathological patients present autoimmune components, but heat-inactivation or leukocyte exclusion could minimize local side effects.

PSVII-16 Recombinant bovine lactoferrin expressed in Schizochytrium sp. alleviates inflammation and oxidative damage in calf intestinal epithelial cells

Abstract This study aims to generate recombinant bovine lactoferrin (rbLF) by expressing it in Schizochytrium sp. (SZ) and evaluate its effects on calf intestinal epithelial cells (CIECs) exposed to lipopolysaccharide (LPS) or hydrogen peroxide-induced damage. The bovine lactoferrin expression cassette was transfected into Schizochytrium sp. via 18s rRNA gene recombination. Primary CIECs were utilized in this investigation, and the optimal dose of rbLF was evaluated by cell counting kit-8 assay after being treated with different concentrations of rbLF for 24 h. The optimal dose of rbLF was then used as a medium supplement before or after 24 h of 80 μg/mL LPS challenge or 4 h of 400 μM of hydrogen peroxide (H2O2) challenge. Cell viability, gene expression of tumor necrosis factor (TNF) -α, toll-like receptor (TLR) -4, interleukin (IL) -17A, zonula occludens-1 (ZO-1), and claudin-1, as well as protein levels of TNF-α, IL-17A, ZO-1, and claudin-1, were assessed to elucidate the preventive and therapeutic effects of rbLF. Data analysis was performed using SAS 9.4 by F-test, The statistical significance was declared at P &amp;lt; 0.05. Results indicate that rbLF demonstrated no cytotoxicity within the 50 to 1,000 μg/mL range on CIECs. Treatment with 175 μg/mL rbLF for 24 h increased cell viability, expression, and protein concentrations of ZO-1 and claudin following hydrogen peroxide challenge (P &amp;lt; 0.05). Moreover, rbLF pretreatment for 24 hours, despite decreasing cell viability after the H2O2 challenge, increased the expression and protein concentrations of ZO-1 and claudin-1 compared with non-rbLF-treated groups (P &amp;lt; 0.05). When used to prevent LPS-induced inflammation, rbLF enhanced the gene expression and protein concentrations of TNF-α, ZO-1, and claudin-1 compared with controls (P &amp;lt; 0.05). As a treatment for LPS-induced inflammation, rbLF decreased TLR4, TNF-α, and IL-17A expression levels (P &amp;lt; 0.05). These findings suggest that SZ-expressed rbLF is safe for calve intestinal cells and 175 μg/mL of rbLF may mitigate inflammation-induced intestinal barrier dysfunction.

335 Effects of feeding an immunomodulatory feed additive (NutraGen) on serum cytokine and antibody concentrations in calves following exposure to a combination bovine respiratory disease challenge

Abstract Two 89-d experiments (Exp 1 and Exp 2) evaluated the effects of feeding an immunomodulatory feed additive (NutraGen; NG) in calves following exposure to bovine viral diarrhea virus (BVDV) on d -3 and Mannheimia haemolytica (MH) infection on d 0. For each experiment, steers [body weight (BW = 251 ± 38.2 kg were randomly allocated to 1 of 3 treatments. Treatments included a placebo (CON; Exp 1 n = 5; Exp 2 n = 5), a placebo fed from d -18 to d -3 followed by NG fed from d -3 to d 28 (CHLG; Exp 1 n = 5; Exp 2 n = 6), and NG fed from d -18 to d 28 (PREC; Exp 1 n = 6; Exp 2 n = 5). Data were analyzed using the GLIMMIX procedure of SAS Version 9.4 (SAS Institute, Cary, N.C.). Animal served as the experimental unit and time served as a repeated measure. Treatment, time, and the treatment × time interaction served as fixed effects. Block was included as a random effect. Cytokine data were tested for normality using the Shapiro-Wilk test, determined to be non-normal, and logarithm (base 10) transformed as a result prior to statistical analysis. Multiple time effects and treatment × time interactions occurred for serum MH, BVDV 1, and BVDV 2 antibody concentrations in both experiments (P ≤ 0.05). In Exp 1, there was a tendency for an overall treatment effect for calves assigned to the CON treatment to exhibit the greatest MH serum antibody concentrations (P = 0.06). While no differences were detected between treatment groups, BVDV 1 and BVDV 2 antibody concentrations peaked on the same day in both experiments, d 28 post-challenge. There were no treatment × time interactions, treatment effects, or time effects for serum TNF-α, IFN-γ, or IL-17A concentrations (P ≥ 0.13) in either experiment. In Exp 1, there was a treatment effect for serum IL-10 (P ≤ 0.0001), where calves assigned to the PREC treatment had increased serum IL-10 concentrations when compared with calves in the other treatment groups. In Exp 2, there was a tendency for a treatment effect for IL-10 concentrations, where calves assigned to the PREC treatment displayed decreased IL-10 concentrations when compared with calves in the other treatment groups (P = 0.07). These data suggest that feeding NG to calves prior to a stress experience may alter cellular responses. Overall, these experiments suggest that NG impacted antibody concentrations and cytokine expression during a bovine respiratory disease challenge.

Exploring the synergistic effects of vitamin D and synbiotics on cytokines profile, and treatment response in breast cancer: a pilot randomized clinical trial.

This study was designed to investigate the effect of vitamin D and/or synbiotics on the response to treatment, cytokines profile and hormonal biomarkers in breast cancer patients undergoing neoadjuvant therapy. A total of 76 patients were recruited and completed the course of the intervention between 2019 and 2021 in Kerman, Iran. breast cancer patients were randomly enrolled in this study. Patients divided into four groups to receive one of the following regimens: placebo, vitamin D, synbiotics and a combination of vitamin D and synbiotics. clinicopathologic parameters, inflammatory and anti-inflammatory biomarkers and hormonal levels were measured at the baseline and four months after intervention. The study results found no clear link between the interventions and achieving pathological complete response (pCR), and a similar trend was observed in Ki-67 index examination. After neoadjuvant therapy, TNF-α concentrations decreased, with vitamin D supplementation moderating this decline. Vitamin D supplemented groups showed a significant increase in serum IL-6 levels. While IL-10 levels decreased in the placebo group, all intervention groups were protected from this decline. Moreover, there was a notable increase in the anti-inflammatory index, particularly in the group receiving both vitamin D and synbiotic supplementation, suggesting potential synergistic anti-inflammatory effects from their combined administration. The outcomes suggest a potential anti-inflammatory function of this combination. Consequently, more extensive studies with prolonged follow-up periods and substantial sample sizes are warranted to thoroughly evaluate their potential benefits for breast cancer patients.

The association between physical activity and delayed neurocognitive recovery in elderly patients: a mediation analysis of pro-inflammatory cytokines

BackgroundDelayed neurocognitive recovery (dNCR) can result in unfavorable outcomes in elderly surgical patients. Physical activity (PA) has been shown to improve cognitive function, potentially by reducing systemic inflammatory responses. However, there is a lack of supportive data indicating whether PA has a protective effect against dNCR.AimsTo examine the correlation between dNCR and PA, and to further analyze if pro-inflammatory cytokines mediate this relationship.MethodsThis study is a prospective nested case-control investigation of elderly patients who had knee replacement surgery. dNCR was defined as a decline in cognitive function compared with baseline by using a battery of neuropsychological tests. PA was assessed with the Physical Activity Scale for the Elderly (PASE). Enzyme-linked immunosorbent assay (ELISA) was used to measure the serum concentrations of IL-6, IL-1β, and TNF-α. Multivariable logistic regression analysis was conducted to assess the association between PA and dNCR. Mediation analysis was employed to evaluate whether pro-inflammatory cytokines mediate the relationship between them.ResultsA cohort of 152 patients was included, resulting in an incidence rate of dNCR of 23.68%. PA was associated with dNCR after full adjustment [OR = 0.199, (95% CI, 0.061; 0.649), P = 0.007]. Mediation analysis showed that the IL-6 mediated the statistical association between PA and dNCR, with mediation proportions (%) of 77.68 (postoperative concentration of IL-6) or 27.58 (the absolute change in IL-6 before and after surgery).ConclusionsPA serves as a protective factor against dNCR, possibly through the reduction of pro-inflammatory cytokine concentrations.The Chinese Clinical Trail Registry: www.http://chictr.org.cn, Registration No. ChiCTR2300070834, Registration date: April 24, 2023.

Involvement of interferon-gamma and tumor necrosis factor-alpha in the formation of unstable atherosclerotic plaque

Research on the role of various interleukins in atherosclerosis has shown that pro-inflammatory cytokines contribute to disease progression and destabilization of atherosclerotic plaques. The review presents the latest data from the scientific literature and the study’s own findings on the effects of pro-inflammatory cytokines INF-γ and TNF-α on the formation of unstable atherosclerotic lesions. It has been shown that the deterioration of the unstable plaque cap strength and its destruction can be associated with an increase in concentration, activation and action of powerful pro-inflammatory cytokines INF-γ and TNF-α in the vascular wall.

Spontaneous and Stimulated Production of Cytokines by Blood Cells Ex Vivo as a Biomarker of Initially High or Low Hypoxia Resistance in Rats.

Spontaneous and stimulated production of cytokines by peripheral blood cells obtained from the caudal vein of male Wistar rats was assessed before testing their resistance to oxygen deficiency in a decompression chamber. To study the spontaneous production of cytokines, heparinized blood cells were incubated in a culture medium (24 h, 5% CO2, 37°C) and the content of proinflammatory cytokines IL-6 and TNFα and anti-inflammatory IL-10 in the culture medium was assessed by ELISA. To stimulate cytokine production, blood cells were incubated for 24 h with LPS, phytohemagglutinin, and concanavalin A at final concentrations of 2, 4, and 4 μg, respectively. Two weeks after blood sampling, individual resistance of the animals to hypoxia in a decompression chamber was determined. In animals with low resistance to hypoxia, the levels of spontaneous production of all three cytokines were significantly higher, while after stimulation, the level of IL-1β increased by more than 2 times. The animals with spontaneous production of IL-10>50 pg/ml, IL-6>10 pg/ml, and TNFα>10 pg/ml, as well as with the increase in IL-1β production by more than 2 times upon stimulation were classified as low-resistant. At IL-10<15 pg/ml, IL-6<9 pg/ml, and TNFα<7 pg/ml, as well as the absence of the increase in IL-1β production upon stimulation, they were classified as high-resistant. The identified features of spontaneous and stimulated production of cytokines can be used as non-invasive biomarkers to determine the resistance to hypoxia without exposure to sublethal hypoxia in a decompression chamber.

TNFα and IL-8 vitreous concentrations variations with two antidiabetic therapies in patients with proliferative diabetic retinopathy: an observational study

BackgroundAntidiabetic therapies are effective, but could indirectly modify the inflammatory response in the ocular microenvironment; therefore, a study was developed to evaluate the inflammatory cytokine profile in the vitreous humor of diabetic patients with retinopathy under treatment with antidiabetic drugs.MethodsObservational, comparative, retrospective, cross-sectional study. Interleukins 1β, 6, 8, 10, and tumor necrosis factor-alpha (TNFα) were evaluated in the vitreous humor obtained from patients with type 2 diabetes mellitus, proliferative diabetic retinopathy, and concomitant retinal detachment or vitreous hemorrhage, and who were already on antidiabetic treatment with insulin or metformin + glibenclamide. The quantification analysis of each cytokine was performed by the cytometric bead array (CBA) technique; medians and interquartile ranges were obtained, and the results were compared between groups using the Mann-Whitney U test, where a p-value < 0.05 was considered significant.ResultsThirty-eight samples; quantification of TNFα concentrations was higher in the group of patients administered insulin, while interleukin-8 was lower; in the metformin + glibenclamide combination therapy group, it occurred inversely. In the stratified analysis, the highest concentrations of interleukin-8 and TNFα occurred in patients with vitreous hemorrhage; however, the only statistical difference existed in patients with retinal detachment, whose TNFα concentration in the combined therapy group was the lowest value found (53.50 (33.03–86.66), p = 0.03). Interleukins 1β, 6, and 10 were not detected.ConclusionInterleukin-8 and TNFα concentrations are opposite between treatment groups; this change is more accentuated in patients with proliferative diabetic retinopathy and vitreous hemorrhage, where the highest concentrations of both cytokines are found, although only TNFα have statistical difference.