IFN-γ mRNA Research Articles

Effects of rutin supplementation on intestinal morphology, antioxidant capacity, immunity, and gut microbiota of laying hens fed a diet containing stored soybean meal

Protein oxidation of soybean meal (SBM) during storage may have adverse effects on the intestinal health of laying hens. Moreover, rutin has anti-inflammatory and antioxidant properties which might be used as a feed additive to mitigate the intestinal damage caused by oxidised protein of SBM. This study aimed to investigate the effects of rutin supplementation on intestinal morphology, antioxidant capacity, immunity and caecal microbiota in laying hens fed a diet containing stored SBM. A total of 384 Hy-Line Brown laying hens (220 days) were randomly allocated into four groups with eight replicates of 12 laying hens each according to a 2 × 2 factorial arrangement with 2 types of SBM (FSM: SBM was stored in the cold storage warehouses at −20 °C for 45 days, and was considered as fresh and control SBM; RTSM: SBM was stored in room temperature warehouse (15 °C to 25 °C), average temperature was 20 °C for 45 days) and 2 levels of rutin (0 and 500 mg/kg). The results showed that the RTSM diet decreased the ileal glutathione peroxidase (GSH-Px) activity, the jejunal superoxide dismutase 2 (SOD2) and ileal NAD(P)H: quinone oxidoreductase 1 (NQO1) mRNA expression levels (p < 0.05), and tended to decrease the jejunal superoxide dismutase 1 (SOD1), ileal glutathione peroxidase 1 (GPX1) and increase the jejunal interleukin-1β (IL-1β) and interleukin-4 (IL-4) mRNA expression levels. Dietary rutin decreased the jejunal crypt depth (CD) (p < 0.05), increased the jejunal total antioxidant capacity (T-AOC) and GSH-Px activities (p < 0.05), decreased the content of interferon-γ (IFN-γ) in the jejunum (p < 0.05), and significantly reduced levels of immunoglobulin G (IgG), IL-1β, tumour necrosis factor-α (TNF-α), IFN-γ, and IL-4 in the ileal mucosa (p < 0.05). Dietary rutin increased SOD2, nuclear factor E2-related factor 2 (Nrf2) and NQO1 mRNA expression levels in the jejunum and GPX1, Nrf2 and NQO1 mRNA expression levels in the ileum, and decreased nuclear factor-κB (NF-κB), IL-1β, IFN-γ and IL-4 mRNA expression in the jejunum and NF-κB, IL-1β, TNF-α, IFN-γ and IL-4 mRNA expressions in the ileum. What’s more, dietary rutin changed the caecal microbiota. PCoA analysis indicated significant structural differences among four groups (p < 0.05), and SBM × Rutin interactions were found in Actinobacteriota and unclassifiedk_norank_d_ Bacteria at the phylum level (p < 0.05). These results suggested that RTSM had slight adverse effects on the intestinal health, and dietary rutin improved intestinal morphology, exerted antioxidant and anti-inflammatory effects via Nrf2 and NF-κB signal pathways, and changed the composition of caecal microbiota. Highlights The RTSM diet has adverse effects on the intestinal health of laying hens compared with the FSM diet. Dietary rutin improved the intestinal health by increasing the intestinal morphology, antioxidant capacity and anti-inflammatory effects. Dietary soybean meal and rutin changed the composition of caecal microbiota.

The Exposure to Lead (Pb) Exacerbates Immunological Abnormalities in BTBR T+ Itpr3tf/J Mice through the Regulation of Signaling Pathways Relevant to T Cells.

Autism spectrum disorder (ASD) is a common neurodevelopmental illness characterized by abnormal social interactions, communication difficulties, and repetitive and limited behaviors or interests. The BTBR T+ Itpr3tf/J (BTBR) mice have been used extensively to research the ASD-like phenotype. Lead (Pb) is a hazardous chemical linked to organ damage in the human body. It is regarded as one of the most common metal exposure sources and has been connected to the development of neurological abnormalities. We used flow cytometry to investigate the molecular mechanism behind the effect of Pb exposure on subsets of CD4+ T cells in the spleen expressing IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Furthermore, using RT-PCR, we studied the effect of Pb on the expression of numerous genes in brain tissue, including IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, AhR, IL-10, and Foxp3. Pb exposure increased the population of CD4+IFN-γ+, CD4+T-bet+, CD4+STAT1+, CD4+STAT4+, CD4+IL-9+, CD4+IRF4+, CD4+IL-22+, and CD4+AhR+ cells in BTBR mice. In contrast, CD4+IL-10+ and CD4+Foxp3+ cells were downregulated in the spleen cells of Pb-exposed BTBR mice compared to those treated with vehicle. Furthermore, Pb exposure led to a significant increase in IFN-γ, T-bet, STAT1, STAT4, IL-9, IRF4, IL-22, and AhR mRNA expression in BTBR mice. In contrast, IL-10 and Foxp3 mRNA expression was significantly lower in those treated with the vehicle. Our data suggest that Pb exposure exacerbates immunological dysfunctions associated with ASD. These data imply that Pb exposure may increase the risk of ASD.

Impact of a direct-fed microbial supplementation on intestinal permeability and immune response in broiler chickens during a coccidia challenge.

Maintaining intestinal health supports optimal gut function and influences overall performance of broilers. Microlife® Prime (MLP) contains a unique combination of four strains of Bacillus spp. selected to support a healthy gut which may improve performance. The aim of this study was to determine the effects of MLP supplementation on intestinal health and immunity of broilers challenged with a mixed coccidia infection during peak [0 to 6-day post-infection (dpi)] and recovery phases (6 to 13 dpi). A total of 120 male, 4 days-old Ross 708, broiler chicks were allotted to 3 treatment groups (8 replicate cages; 5 birds/cage) in a randomized complete block design. Treatments included a non-challenge (NEG), a coccidia challenge (POS), and coccidia challenge fed MLP (5 × 105 CFU/g of diet). Diets were corn-soybean meal-based. At 11 days of age, all birds, except for NEG, were orally gavaged with 15 doses (3 × the recommended commercial dose). On 6, 9, and 13 dpi, birds were orally gavaged with fluorescein isothiocyanate conjugate dextran (FITC-d). Plasma and mid-jejunum tissues were collected 2 h later. On 6 dpi, duodenal lesions from 2 birds/cage were scored and droppings were collected for oocyst enumeration. Body weight gain (BWG) and feed conversion ratio (FCR) were calculated over the experimental period. Data were analyzed with GLIMMIX procedure of SAS. During the peak phase, POS birds had reduced BWG (23%) and FCR (15%) compared to NEG birds (P < 0.05), while birds fed MLP had similar BWG (209 and 208 g) and FCR (1.17 and 1.21) compared to NEG (P > 0.05). On 6 dpi, POS birds had higher lesion scores and oocyst shedding, 2 × increase in serum FITC-d, and higher jejunum IL-10, and IFN-γ mRNA compared to NEG (P < 0.05). Birds fed MLP had reduced plasma FITC-d compared to POS birds (P < 0.05) and similar IL-10 and IFN-γ mRNA. On 13 dpi, birds fed MLP had lower plasma FITC-d, jejunum IL-10 and IFN-γ mRNA compared to POS birds (P < 0.05), but similar IL-10 to NEG birds (P > 0.05). This study confirms MLP improves intestinal health and positively modulates mucosal immune response post-coccidia challenge.

Preventive effect of Lactobacillus plantarum YS1 isolated from naturally fermented yoghurt on carrageenan-induced thrombosis in mice

AbstractThis work used a carrageenan-based thrombosis model to determine the preventative effects of Lactobacillus plantarum YS1 (LPYS1) on thrombus. In thrombotic mice, LPYS1 improved the activated partial thromboplastin time (APTT), while decreasing the thrombin time (TT), prothrombin time (PT), and fibrinogen (FIB) content. In thrombotic mouse serum, LPYS1 decreased the levels of malondialdehyde (MDA), tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), nuclear factor kappa-B (NF-κB), and interleukin-1 beta (IL-1β), while also increasing the activities of superoxide dismutase (SOD) and catalase (CAT). Moreover, LPYS1 upregulated the mRNA expression levels of copper/zinc-SOD (Cu/Zn-SOD), manganese-SOD (Mn-SOD), and CAT in the colon tissues of thrombotic mice, while downregulating those of NF-κB p65, IL-6, TNF-α, and interferon-gamma (IFN-γ) mRNA. In tail vein vascular tissues, LPYS1 suppressed the mRNA expression levels of NF-κB p65, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin. The abundances of both beneficial and pathogenic bacteria were altered by LPYS1. These findings show that LPYS1 has the capacity to protect mice from thrombosis, while also revealing some of the underlying mechanisms of this effect.

KERS-Inspired Nanostructured Mineral Coatings Boost IFN-γ mRNA Therapeutic Index for Antitumor Immunotherapy.

Tumor-associated macrophage (TAM) reprogramming is a promising therapeutic approach for cancer immunotherapy; however, its efficacy remains modest due to the low bioactivity of the recombinant cytokines used for TAM reprogramming. mRNA therapeutics are capable of generating fully functional proteins for various therapeutic purposes but accused for its poor sustainability. Inspired by kinetic energy recovery systems (KERS) in hybrid vehicles, a cytokine efficacy recovery system (CERS) is designed to substantially augment the therapeutic index of mRNA-based tumor immunotherapy via a "capture and stabilize" mechanism exerted by a nanostructured mineral coating carrying therapeutic cytokine mRNA. CERS remarkably recycles nearly 40% expressed cytokines by capturing them onto the mineral coating to extend its therapeutic timeframe, further polarizing the macrophages to strengthen their tumoricidal activity and activate adaptive immunity against tumors. Notably, interferon-γ (IFN-γ) produced by CERS exhibits ≈42-fold higher biological activity than recombinant IFN-γ, remarkably decreasing the required IFN-γ dosage for TAM reprogramming. In tumor-bearing mice, IFN-γ cmRNA@CERS effectively polarizes TAMs to inhibit osteosarcoma progression. When combined with the PD-L1 monoclonal antibody, IFN-γ cmRNA@CERS significantly boosts antitumor immune responses, and substantially prevents malignant lung metastases. Thus, CERS-mediated mRNA delivery represents a promising strategy to boost antitumor immunity for tumor treatment.

ET-1 receptor type B (ETBR) overexpression associated with ICAM-1 downregulation leads to inflammatory attenuation in experimental autoimmune myocarditis.

An experimental autoimmune myocarditis rat model was established by subcutaneous injection of porcine myocardial myosin (PCM). The effect of ET-1 receptor type B (ETBR) overexpression on autoimmune myocarditis was observed via tail vein injection of ETBR overexpression lentivirus in rats. We further investigated the mechanisms involved in the regulation of autoimmune myocarditis by ETBR overexpression. Six rats were randomly selected from 24 male Lewis rats as the NC group, and the remaining 18 rats were injected with PCM on Day 0 and Day 7, to establish the experimental autoimmune myocarditis (EAM) rat model. The 18 rats initially immunized were randomly divided into three groups: the EAM group, ETBR-oe group, and GFP group. On Day 21 after the initial immunization of rats, cardiac echocardiography and serum brain natriuretic peptide (BNP) analysis were performed to evaluate cardiac function, myocardial tissue HE staining was performed to assess myocardial tissue inflammatory infiltration and the myocarditis score, and mRNA expression of IFN-γ, IL-12, and IL-17 was detected by qRT-PCR. Subsequently, immunohistochemical analysis was performed to detect the localization and expression of the ETBR and ICAM-1 proteins, and the expression of ETBR and ICAM-1 was verified by qRT-PCR and western blotting methods. On Day 21 after initial immunization, left ventricular end-diastolic diameter (LVEDd), left ventricular end-systolic diameter (LVEDs), and serum BNP concentrations increased in the hearts of rats in the EAM group compared with the NC group (P < 0.01), and ejection fraction (EF) and fractional shortening (FS) decreased compared with those of the normal control (NC) group (P < 0.01). LVEDd, LVEDs, and serum BNP concentrations decreased in the ETBR-oe group compared with the EAM group, while EF and FS increased significantly (P < 0.01). HE staining showed that a large number of inflammatory cell infiltrates, mainly lymphocytes, were observed in the EAM group, and the myocarditis score was significantly higher than that of the NC group (P < 0.01). Compared with that of the EAM group, myocardial tissue inflammatory cell infiltration was significantly reduced in the ETBR-oe group, and the myocarditis scores were significantly lower (P < 0.01). The mRNAs of the inflammatory factors IFN-γ, IL-12 and IL-17 in myocardial tissue of rats in the EAM group exhibited elevated levels compared with those of the NC group (P < 0.01) while the mRNAs of IFN-γ, IL-12 and IL-17 were significantly decreased in the ETBR-oe group compared with the EAM group (P < 0.01). Immunohistochemistry showed that the staining depth of ETBR protein in myocardial tissue was greater in the EAM group than in the NC group, and significantly greater in the ETBR-oe group than in the EAM group, while the staining depth of ICAM-1 was significantly greater in the EAM group than in the NC group, and significantly lower in the ETBR-oe group than in the EAM group. The ICAM-1 expression level was significantly higher in the EAM group than in the NC group (P < 0.01), and was significantly lower in the ETBR-oe groupthan in the EAM group (P < 0.01).

Relation between interferon-gamma gene expression and its serum level with thyroid hormone status and Ferritin level in Iraqi sickle cell anemia patients

The hereditary hemoglobinopathy known as sickle cell disease is characterized by abnormal hemoglobin synthesis, hemolytic anemia, and intermittent obstruction of tiny blood arteries. The current investigation aims to study the relationship between interferon-gamma gene expression and serum levels with thyroid hormone status and Ferritin levels in sickle cell anemia patients. Blood samples were collected from 50 patients suffering from SCA and 50 healthy volunteers as a control group. The results of the relation of IFN-γ serum level with T3, T4 and TSH levels showed a positive relationship between IFN-γ serum and TSH levels. At the same time, an inverse association to T3 and T4 levels with high significance also showed high IFN-γ gene expression (10.78 ±5.06 Fold) in the patient group as compared with control (2.079 ±0.52 Fold) at a significant difference, as well as the results found acquire strong positive association between IFN-γ serum level and IFN-γ mRNA expression in the patient group compared with the control group. The current study concluded that there was an inverse association among T3, T4 and IFN-γ serum with high significance, as high gene expression of IFN-γ, and a strong positive association between IFN-γ serum level and IFN-γ mRNA expression. Keywords: IFN-γ, SCA, Ferritin, TSH, T3, T4.

MAP kinase inhibitor PD98059 regulates Th1, Th9, Th17, and natural T regulatory cells in an experimental autoimmune encephalomyelitis mouse model of multiple sclerosis

Experimental autoimmune encephalitis (EAE), an animal model of multiple sclerosis (MS), provides significant insights into the mechanisms that initiate and drive autoimmunity. MS is a chronic autoimmune disease of the central nervous system, characterized by inflammatory infiltration associated with demyelination. T lymphocyte cells play a crucial role in MS, whereas natural T regulatory (nTreg) cells prevent autoimmune inflammation by suppressing lymphocyte activity. This study sought to investigate the role of PD98059, a selective MAP kinase inhibitor, in Th1, Th9, Th17, and nTreg cells using the SJL/J mouse model of EAE. Following EAE development, the mice were intraperitoneally administered PD98059 (5 mg/kg for two weeks) daily. We evaluated the effects of PD98059 on Th1 (IFN-γ and T-bet), Th9 (IL-9 and IRF4), Th17 (IL-17A and RORγT), and nTreg (FoxP3 and Helios) cells in the spleen using flow cytometry. Moreover, we explored the effects of PD98059 on the IFN-γ, T-bet, IL-9, IRF4, IL-17A, RORγT, FoxP3, and Helios mRNA and protein levels in brain tissues using qRT-PCR and Western blot analyses. PD98059 treatment significantly decreased the proportion of CD4+IFN-γ+, CD4+T-bet+, CD4+IL-9+, CD4+IRF4+, CD4+IL-17A+, CD4+RORγT+, CD4+IL-17A+, and CD4+RORγT+ cells while increasing that of CD4+FoxP3+ and CD4+Helios+ cells. In addition, PD98059 administration decreased the mRNA and protein levels of IFN-γ, T-bet, IL-9, IRF4, IL-17A, and RORγT but increased those of FoxP3 and Helios in the brain tissue of EAE mice. Our findings suggest that PD98059 corrects immune dysfunction in EAE mice, which is concurrent with the modulation of multiple signaling pathways.

Cyclohexylethanoid derivative and rearranged abietane diterpenoids with anti-inflammatory activities from Clerodendrum bungei and C. inerme

One new dimeric cyclohexylethanoid derivative (1) and one new rearranged abietane diterpenoid (2) were isolated from Clerodendrum bungei and C. inerme, respectively, together with 11 known compounds. The structures of the isolated compounds were elucidatedby detailed spectroscopic and ECD analyses. The absolute configuration of 2 was unequivocally determined using single-crystal X-ray diffraction analysis. Compound 1 showed the greatest inhibitory effect on NO production in RAW 264.7 murine macrophages induced by lipopolysaccharide (LPS) with the IC50 value of 6.328 µM. Compound 1 also suppressed the proinflammatory cytokines IFN-γ, TNF-α, IL-1β, iNOS and IL-6 mRNA expression, while increased the mRNA expression of the anti-inflammatory cytokines IL-4, IL-10 and Arg-1 through NF-κB signaling pathway. The further experiments confirmed that compound 1 demonstrated the strong in vivo anti-inflammatory effect by reducing mortality, the serum TNF-α, IL-6 and IFN-γ levels, and tissue toxicity in CLP-induced septic mice. Collectively, these findings suggested that compound 1might serve as a potential anti-inflammatory agent candidate for the sepsis treatment.

Hepatobiliary and pancreatic: Multi-donor fecal microbiota transplantation attenuated high-fat diet-induced hepatic steatosis in mice by remodeling the gut microbiota.

Fecal microbiota transplantation (FMT) can improve the symptoms of nonalcoholic fatty liver disease (NAFLD) by restoring the gut microbiota. This study was aimed to evaluate the therapeutic effects of single-donor (SD) or multi-donor (MD) FMT in a mouse model of hepatic steatosis and explore the underlying mechanisms. Fecal samples were collected from NAFLD patients and healthy controls with similar baseline characteristics, with gut microbiota analyzed. Mice were fed either a normal-chow diet (NCD) or a high-fat diet (HFD) for 3weeks and then administered fecal microbiota collected from healthy SDs or MDs for 12weeks. Fecal samples from NAFLD patients showed significantly lower microbial diversity than those from healthy controls. MD-FMT reduced liver fat accumulation and body weight and significantly improved serum and liver biochemical indices in HFD-fed mice. Compared to untreated HFD-fed mice, MD-FMT significantly decreased the relative expression of IL-1β, IL-6, TNF-α, IFN-γ, and IL-1β mRNAs in the liver. The relative protein level of intestinal barrier components, including claudin-1, occludin, and E-cadherin, as well as serum lipopolysaccharide (LPS) level in mice, were found to be improved following MD-FMT intervention. Furthermore, FMT reversed HFD-induced gut dysbiosis and increased the abundance of beneficial bacteria such as Blautia and Akkermansia. NAFLD patients and healthy controls showed distinct gut microbiota. Likewise, HFD altered gut microbiota in mice compared to NCD-fed controls. MD-FMT restored gut dysbiosis in HFD-fed mice and attenuated liver steatosis, and should be considered as an effective treatment option for NAFLD.

Efficacy of a synthetic peptide Chlamydia pecorum major outer membrane protein vaccine in a wild koala (Phascolarctos cinereus) population

Chlamydiosis is a significant disease affecting Eastern Australian koala (Phascolarctos cinereus) populations, impacting individual animal welfare and fecundity and therefore influencing population dynamics. The aim of this study was to investigate the effect of a synthetic peptide vaccine based on 4 components of the Chlamydia pecorum major outer membrane protein (MOMP), over an 18-month period in a koala population severely impacted by chlamydiosis. Wild koalas were recruited into a vaccination or a placebo treatment group on a random allocation, then followed through a period of 18 months, with recapture at 6 monthly intervals. Vaccination did not alter clinical disease expression or chlamydial shedding from the ocular or urogenital sites. Vaccination did not stimulate a significant plasma anti-MOMP IgG response, when compared to the placebo group. There was no significant effect of vaccination on IFN-γ and IL-17A mRNA expression of peripheral blood lymphocytes when stimulated with rMOMP. We have demonstrated that a synthetic peptide vaccination against chlamydiosis is not an effective management tool in a koala population with a high prevalence of C. pecorum infection and related disease. The lack of antigenic response found in this study suggests that further research utilising a larger, full-length antigen is an avenue worth investigation if we are to consider vaccination as a part of a management strategy in diseased koala populations.

Inhibitory effect of Eimeria maxima IFN-γ inhibitory molecules on the immune function of T cell subsets in chickens

It has been reported that infection of chicken coccidian could inhibit the production of Th1 cytokine IFN-γ, thereby evading clearance by the host immune system. The present study aimed to have a further investigation into the effects of Eimeria maxima IFN-γ inhibitory molecules (EmHPSP-2 and EmHPSP-3) on the immune function of chicken peripheral blood mononuclear cells (PBMC) and various T cell subsets. First, separated PBMC or sorted T cell subsets were used for incubation with recombinant proteins of EmHPSP-2 (rEmHPSP-2) and EmHPSP-3 (rEmHPSP-3). Subsequently, the effects of rEmHPSP-2 and rEmHPSP-3 on proliferative capacity, nitric oxide (NO) release and mRNA levels of cytokines of the above cells were detected. The sorting purity of CD8+, CD4+ CD25-, CD4+, and CD4+ CD25+ T cells was 93.01, 88.88, 87.04, and 81.26%, respectively. The NO release of PBMC was significantly inhibited by rEmHPSP-2 and rEmHPSP-3. The proliferation of PBMC and CD4+ T cells was significantly inhibited by rEmHPSP-2 and rEmHPSP-3, whereas CD8+, CD4+ CD25-, and CD4+ CD25+ T cells was significantly promoted by the 2 proteins. The 2 proteins significantly downregulated interferon-gamma (IFN-γ) mRNA level, upregulated the transcriptional levels of interleukin-10 (IL-10) and transforming growth factor-beta1 (TGF-β1) in PBMC. IFN-γ and IL-2 transcriptional levels were markedly inhibited in CD8+ T cells. IFN-γ transcriptional level was significantly inhibited, but IL-4 was promoted by rEmHPSP-2 and rEmHPSP-3 in CD4+ CD25- T cells. Meanwhile, the inhibitory effects of rEmHPSP-2 and rEmHPSP-3 on the transcriptional levels of IFN-γ and IL-2 were more obvious in CD4+ T cells containing CD25+ cells compared with the CD25+ cells depletion group. It was found that IL-10, TGF-β1, and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) mRNA levels were significantly upregulated upon stimulation of chicken CD4+ CD25+ T cells by proteins. This study is not only of great significance to clarify the immune evasion mechanism of chicken coccidia, but also provides candidate antigen molecules for development of a novel vaccine against chicken coccidiosis.

Essential oil from Artemisia argyi alleviated liver disease in zebrafish (Danio rerio) via the gut-liver axis.

The popularity of intensive fish farming has led to the emergence of fish diseases characterized by hepatobiliary syndrome. Artemisia argyi (A. argyi) essential oils have anti-inflammatory and anti-oxidant effects. However, their alleviating effects and mechanism on liver disease in fish are still unclear. Thus, adult zebrafish were used to construct an animal model to observe histopathological damages, determine biochemical parameters and expression of inflammatory cytokines and mRNAs in the PPAR-γ/NF-κB pathway, and conduct 16S sequencing of intestinal microbiota. The results found that after treatment with A. argyi essential oil, the histopathological damage caused by ethanol was relieved; the CAT, SOD, and GSH levels were remarkably elevated, while the MDA level was obviously lowered (P<0.05); the expression levels of IL-10 and IFN-γ mRNAs were enhanced, but the levels of IL-1β, IL-6, PPAR-γ, NF-κB, and TNF-α mRNAs were reduced (P<0.05) relative to the EtOH group. A. argyi essential oil remarkably attenuated the damage to intestinal tissue structure, and elevated the levels of Muc2, ZO-1, Claudin-1, and Occludin mRNA (P<0.05). Sequencing of the gut flora showed that A. argyi essential oil significantly altered the composition of gut microbes compared with the EtOH group. In addition, KEGG and COG analyses also showed significant (P<0.05) changes in acetate cycling metabolism in the EtOH group, catechol 2, 3-dioxygenase and nitroreductase were significantly increased (P<0.001), and lipid metabolism and terpenoid synthesis were significantly elevated (P<0.001) in A. argyi essential oil group. The results indicate that A. argyi essential oil could effectively relieve ethanol-caused histopathological damage of livers by modulating the composition of gut microbiota, thus inhibiting the level of IL-1β and mRNAs in the PPAR-γ/NF-κB pathway, increasing the IL-10 level, reducing the oxidative stress. This may offer a rationale for further research on the rationality of A. argyi as a substitute for feed antibiotics in aquaculture.

Performance of T-Track® SARS-CoV-2, an Innovative Dual Marker RT-qPCR-Based Whole-Blood Assay for the Detection of SARS-CoV-2-Reactive T Cells.

T-cell immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a central role in the control of the virus. In this study, we evaluated the performance of T-Track® SARS-CoV-2, a novel CE-marked quantitative reverse transcription-polymerase chain reaction (RT-qPCR) assay, which relies on the combined evaluation of IFNG and CXCL10 mRNA levels in response to the S1 and NP SARS-CoV-2 antigens, in 335 participants with or without a history of SARS-CoV-2 infection and vaccination, respectively. Of the 62 convalescent donors, 100% responded to S1 and 88.7% to NP antigens. In comparison, of the 68 naïve donors, 4.4% were reactive to S1 and 19.1% to NP. Convalescent donors <50 and ≥50 years of age demonstrated a 100% S1 reactivity and an 89.1% and 87.5% NP reactivity, respectively. T-cell responses by T-Track® SARS-CoV-2 and IgG serology by recomLine SARS-CoV-2 IgG according to the time from the last immunisation (by vaccination or viral infection) were comparable. Both assays showed a persistent cellular and humoral response for at least 36 weeks post immunisation in vaccinated and convalescent donors. Our results demonstrate the very good performance of the T-Track® SARS-CoV-2 molecular assay and suggest that it might be suitable to monitor the SARS-CoV-2-specific T-cell response in COVID-19 vaccinations trials and cross-reactivity studies.

Identification of potential SLA-I-specific T-cell epitopes within the structural proteins of porcine deltacoronavirus

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that mainly threatens newborn piglets and poses a potential broad cross-species transmission risk. The antigenic epitopes of PDCoV are currently unidentified, and no information about T cell epitopes is available. Here, T-cell epitopes of PDCoV structural proteins were predicted using computational methods. 17 epitope peptides were synthesized and then screened using ELIspot, intracellular cytokine staining (ICS), and RT-qPCR detection of IFN-γ mRNA to evaluate their ability to elicit interferon-gamma (IFN-γ) responses in peripheral blood mononuclear cells (PBMCs) from PDCoV-challenged pigs. Five peptides (M1, M2, M3, N6, and S4) elicited high levels of IFN-γ and were investigated further as potential T-cell epitope candidates. All five peptides were cytotoxic T lymphocyte (CTL) epitopes, and two peptides (M3, N6) were recognized simultaneously by CD8 + and CD4 + T cells. A multi-epitope peptide combining the five epitopes (designated “5T”) was synthesized and its immune response and protection efficacy was evaluated in a piglet model. ELISpot assay results indicated that 5T induces robust epitope-specific cellular immune responses. Four epitopes (M1, M2, N6, S4) elicited IFN-γ responses in 5T-vaccinated piglets. No obvious protection efficacy was detected in piglets vaccinated with 5T alone. Our results provide valuable information concerning PDCoV-related antigenic epitopes and will be useful in the design of epitope-based vaccines.

Proinflammatory and Immunomodulatory Gene and Protein Expression Patterns in Spinal Cord and Spleen Following Acute and Chronic High Thoracic Injury.

In addition to paralysis and loss of sensation, high-level spinal cord injury (SCI) causes sympathetic dysfunction that can lead to autonomic dysreflexia (AD) and chronic immune suppression involving splenic leukopenia. Evidence has shown that treatment with either gabapentin or blockade of TNFα mitigates maladaptive plasticity and the underlying hemodynamic dysfunction, spleen atrophy, and immune dysfunction associated with AD. Because significant improvements long term was noted following treatments only during acute stages of recovery, we sought to systematically examine changes in proinflammatory and immunomodulatory cytokines to ascertain the reason. Adult female Wistar rats underwent complete T4 spinal transection before euthanasia at systematic intervals from 3 days to 8 weeks after injury. Using qRT-PCR and meso scale discovery (MSD) assays, the gene and protein expression of TNFα and IFNγ in the spleen, upper thoracic (T4-9) and lumbosacral (L5-S6) spinal cords were analyzed. We found that spleen atrophy occurs in a biphasic manner compared to naïve controls, with significant decreases in the spleen mass noted at 3 days and 8 weeks after injury. Splenic TNFα mRNA and protein levels did not change significantly over time, while IFNγ gene expression dipped acutely with trends for increased protein levels at more chronic time points. TNFα protein increased significantly only in thoracic spinal cord segments from 3 to 14 days post-injury. IFNγ mRNA and protein levels remained unelevated in injured spinal cords over time, with trends for increased protein levels at 2 and 8 weeks in the lumbosacral segments. Novel temporal-spatial cytokine expression profiles reveal that TNFα protein levels are increased solely in upper thoracic segments after high thoracic SCI, while IFNγ remains unaltered. Splenic leukopenia and latent systemic immunosuppression are not associated with altered TNFα or IFNγ expression in the spleen or spinal cord.

Potent In Vitro and In Vivo Effects of Stachys lavandulifolia Methanolic Extract against Toxoplasma gondii Infection.

MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the in vitro effect of the SLME on T. gondii tachyzoites. Totally, 72 male BALB/c mice (40 mice for in vivo evaluation of SLME and 32 mice for its toxicity effects on liver and kidney serum enzymes) were used for the present investigation. At first, 40 mice were orally pre-treated with the SLME at doses of 25, 50, and 75 mg/kg/day for two weeks. Mice were checked daily, and the rate of survival and the mean number of tachyzoites were recorded. Liver lipid peroxidation (LPO) and nitric oxide (NO) levels, the effects on kidney and liver function, as well as the expression level of the proinflammatory cytokines such as interleukin-1β (IL-1β) and interferon-γ (IFN-γ), were studied by the quantitative real-time PCR. Flow cytometry analysis was performed on the effects of SLME on the detection of apoptotic and necrotic cells in T. gondii tachyzoites. The SLME at the concentrations 75 and 150 µg/mL completely killed the tachyzoites after 2 hr of incubation. SLME at 25, 50, and 75 mg/kg/day increased the survival rate of infected mice by the sixth, seventh, and eighth days, respectively. SLME also significantly (p < 0.05) decreased the LPO and NO levels and upregulated the IL-1β and IFN-γ mRNA gene expression levels, whereas no considerable change was observed in the serum level of kidney and liver enzymes. Flow cytometry analysis revealed the prompted early and late apoptosis after exposure to T. gondii tachyzoites with various concentrations of SLME. We found the relevant in vitro anti-Toxoplasma effects of SLME against T. gondii. Moreover, the results confirmed the promising in vivo prophylactic effects of SLME. SLME provokes the innate immune system, induces apoptosis, modulates the proinflammatory cytokines, and inhibits hepatic injury in infected mice. With all these descriptions, further surveys are required to support these findings and elucidate this plant's possible mechanisms of action.

Low-dose rosmarinic acid and thymoquinone accelerate wound healing in retinal pigment epithelial cells.

Thymoquinone (TQ) and rosmarinic acid (RA) are two biologically active compounds found in plants and that possess remarkable anti-oxidant and anti-inflammatory properties. The present study aimed to investigate the potential protective effects of RA and TQ, which have known anti-inflammatory and anti-oxidant effects, on retinal damage by establishing a wound healing model for retinal pigment epithelial cells (ARPE-19). To this end, IC50 doses of RA and TQ in ARPE-19 cells were calculated by MTT assay. Both agents were administered at IC50, IC50/2 and IC50/4 doses for wound healing assay, and wound closure percentages were analyzed. Since the best wound healing was found at IC50/4 dose (low dose) for both agents, other biochemical and molecular analyses were planned to be performed using these doses. Following low dose RA and TQ treatments, the cells were lysed and TGF-β1 and MMP-9 levels were analyzed by ELISA technique from the cell lysates obtained. In addition, the mRNA expression levels of TLR3, IFN-γ and VEGF were calculated by RT-PCR technique. Low dose of RA and TQ dramatically increased wound healing. RA may have achieved this by increasing levels of MMP-9 and TLR-3. In contrast, the mRNA expression level of VEGF remained unchanged. TQ accelerated wound healing by increasing both the protein levels of TGF-β1 and MMP-9. Furthermore, low dose of TQ decreased both TLR3 and IFN-γ mRNA expression levels. Low doses of RA and TQ were clearly demonstrated to have protective properties against possible damage to retinal pigment epithelial cells.

SteE regulation of Th1/Th2 cytokines expression in chickens during S. Pullorum infection

Nowadays, timely monitoring of zoonotic agents, including salmonellosis, which are caused by various serovars of the family Salmonella, is relevant. Attention should be paid to the study of cytokine levels in combination with immunological studies. This helps clarify the pathogenesis of infectious diseases and develop preventive measures. The main purpose of study was to detect the process of regulating Th1/Th2 cytokines expression in chickens infected with salmonellosis. The field strain of S. Pullorum CVCC 530 was used in the research. The steE deletion mutant (ΔsteE) and steE-complemented ΔsteE:steE (ΔsteE+steE) strains were constructed in the WT strain using the λ-Red recombination method. Chickens were orally infected with WT, ΔsteE, and ΔsteE+steE strains (1×109 CFU/individual). The effect of steE on the host immune response remains unknown. Compared with the group infected with the WT or ΔsteE+steE strain, IL-12 and IFN-γ mRNA transcript levels were significantly higher, while IL-10 mRNA expression was significantly reduced in the liver and bursa infected with the ΔsteE strain; IL-4 showed a dramatically reduced transcription level, but IL-18 mRNA expression was significantly increased in the ΔsteE strain – spleen, cecum, and heart; IL-10 mRNA expression was significantly reduced in the spleen and cecum infected with the ΔsteE strain. These results suggest that steE may regulate the Th1/Th2 cytokine response balance in chickens infected with S. Pullorum and provide new insights into the pathogenesis of salmonellosis for the treatment of persistent infection

Distinct roles of IL-18 and IL-1β in murine model of macrophage activation syndrome

IL-18 and IL-1β play a central role in the pathogenesis of systemic juvenile idiopathic arthritis and its life-threatening complication, macrophage activation syndrome (MAS). This study aimed to clarify the role of IL-18 and IL-1β in the pathogenesis of MAS. We developed a mouse model to evaluate the role of each cytokine with Toll-like receptor 9 stimulation after continuous infusion with IL-18, IL-1β, and a combination of both for 7 days. The symptoms and laboratory findings were compared among the IL-18, IL-1β, and combination (IL-18+IL-1β) groups. Body weight was significantly decreased in the IL-1β and combination groups. Splenomegaly was observed in all groups, whereas hepatomegaly was noted in the IL-18 group only. Decreased T-cell numbers, anemia, and thrombocytopenia were observed in the combination group. IFN-γ, CXCL9, and IL-12A mRNA levels were upregulated and IL-10 mRNA levels in the spleen were downregulated in the IL-18 group. Hepatomegaly and splenomegaly in the IL-18 group were observed in a dose-dependent manner. TNF-α, CXCL9, and IL-12A mRNA levels were upregulated only in those mice with extremely elevated plasma IL-18 levels. IL-18 and IL-1β have distinct roles in the pathogenesis of MAS. Dual blockade of IL-18 and IL-1β might be necessary to treat MAS.