IL-2 mRNA Research Articles

Abstract 510: Immune-regulatory Mechanism Of P210-complexed Nanoparticle Immunization For Atherosclerosis

Background: The use of nanoparticles for efficient delivery of therapeutic modalities in humans is rapidly increasing. We have previously reported that a nanovaccine consisting of apoB-100 peptide P210 complexed with amphiphilic micelles (P210-PAM) significantly reduces atherosclerosis in apoE-/- mice. In this study, we defined the mechanistic pathway for the atheroprotective effect of P210-PAM. Methods and Results: Seven-week male apoE-/- mice were immunized with P210-PAM or mouse serum albumin peptide amphiphile micelles (MSA-PAM) at a dose of 100ug per injection at 7, 10, and 12 weeks of age. One week after the second booster, mice were euthanized and the splenocytes were collected for immune profiling and immune assays. A second group of immunized mice were injected with thioglycolate to elicit peritoneal macrophages. Splenocytes from P210-PAM mice collected 1 week after the second booster had significantly increased CD4+CD25+FoxP3+ Treg cells and CD8+CTLA-4+ T cells compared to MSA-PAM mice (N=9 each; P≤0.05). Splenocytes from P210-PAM mice had reduced CD4+ T cell proliferation and CD8+CD107a+ T cells in response to P210 recall compared to MSA-PAM (N=8 and 9, respectively; P<0.05). No effects were observed with non-specific PMA/ionomycin stimulation indicating antigen-specificity. Splenic mRNA expression of IL-1β was unchanged but there was significant reduction in IL-1R1, IL-6, and IL-17a in P210-PAM mice (N=12 each; P<0.05). Flow cytometric analysis indicated that the reduced IL-1R1 expression was specific to F4/80+ macrophages (N=10 each; P<0.05). Macrophages enriched from the peritoneal exudate showed significantly reduced iNOS, IL-6, IL-12 and IL-10 mRNA expression in P210-PAM mice compared to MSA-PAM (N=4 each; P<0.05). Conclusion: The mechanism of the protective effect of P210-PAM nanoparticles in reducing the immune-inflammatory response in atherosclerosis is at the level of both adaptive T cell response as well as innate macrophage response. The use of P210-PAM in future investigations as potential clinical therapy for atherosclerosis is thus warranted.

Untargeted metabolomic profiling identifies disease-specific and outcome-related signatures in chronic rhinosinusitis

Although metabolomics provides novel insights into disease mechanisms and biomarkers, the metabolic alterations in local tissues affected by chronic rhinosinusitis (CRS) are unknown. This study aimed to determine the metabolomic profiles of sinonasal tissues associated with different types of CRS and their treatment outcomes. Untargeted metabolomic profiling was performed on sinonasal tissues obtained from patients with eosinophilic CRS with nasal polyps (CRSwNP), noneosinophilic CRSwNP or CRS without nasal polyps (CRSsNP), and controls. The messenger RNA (mRNA) levels of inflammatory cytokines in nasal tissues were detected by quantitative real-time reverse transcriptase PCR. Nasal polyp tissues were cultured exvivo and treated with glutathione. Distinct metabolomic profiles were observed for the CRS subtypes. Eosinophilic CRSwNP had profoundly enhanced unsaturated fatty acid oxidization, which correlated with mucosal eosinophil numbers and IL-5 mRNA levels. Noneosinophilic CRSwNP was characterized by uric acid accumulation. Increased uric acid levels were positively correlated with mucosal neutrophil numbers and IFN-γ, IL-17A, IL-1β, and IL-8 mRNA levels. Disrupted purine metabolism was specifically detected in CRSsNP. Reduced levels of amino acid metabolites were found in eosinophilic CRSwNP and CRSsNP, and were inversely associated with mucosal total inflammatory cell numbers and inflammatory cytokines. Compared to non-difficult-to-treat CRS, difficult-to-treat CRS had higher glutathione disulfide levels, which were positively correlated with IL-8 mRNA levels. Glutathione treatment reduced IL-8 mRNA expression in cultured nasal polyp tissues. Specific metabolic signatures are associated with different types of CRS, inflammatory patterns, and disease outcomes, which may provide novel insights into pathophysiologic mechanisms, subtype-specific biomarkers, and treatment targets of CRS.

HOIL1 regulates group 2 innate lymphoid cell numbers and type 2 inflammation in the small intestine

Patients with mutations in HOIL1 experience a complex immune disorder including intestinal inflammation. To investigate the role of HOIL1 in regulating intestinal inflammation, we employed a mouse model of partial HOIL1 deficiency. The ileum of HOIL1-deficient mice displayed features of type 2 inflammation including tuft cell and goblet cell hyperplasia, and elevated expression of Il13, Il5 and Il25 mRNA. Inflammation persisted in the absence of T and B cells, and bone marrow chimeric mice revealed a requirement for HOIL1 expression in radiation-resistant cells to regulate inflammation. Although disruption of IL-4 receptor alpha (IL4Rα) signaling on intestinal epithelial cells ameliorated tuft and goblet cell hyperplasia, expression of Il5 and Il13 mRNA remained elevated. KLRG1hi CD90lo group 2 innate lymphoid cells were increased independent of IL4Rα signaling, tuft cell hyperplasia and IL-25 induction. Antibiotic treatment dampened intestinal inflammation indicating commensal microbes as a contributing factor. We have identified a key role for HOIL1, a component of the Linear Ubiquitin Chain Assembly Complex, in regulating type 2 inflammation in the small intestine. Understanding the mechanism by which HOIL1 regulates type 2 inflammation will advance our understanding of intestinal homeostasis and inflammatory disorders and may lead to the identification of new targets for treatment.

Particulate Matter Exposure and the Changes in Immune Biomarkers: Effects of Biyeom-Go on the Nasal Mucosa of Patients with Allergic Rhinitis and a Particulate Matter-Treated Mouse Model

This study was to investigate the effects of Biyeom-go (BYG, an herbal formula) on immune biomarkers present in the nasal mucosa of patients with allergic rhinitis under exposure to particulate matter 2.5 (PM2.5), and on changes in goblet cells and immune biomarkers in mice under exposure to Korea diesel particulate matter (KDP20). Thirty patients showing characteristic allergic rhinitis symptoms were enrolled in Jeonju-si, Korea, and treated with BYG thrice a day for four weeks. Changes in the expression of immune biomarkers (interleukin 4 (IL-4), IL-5, IL-8, IL-13, IL-33, and thymic stromal lymphopoietin (TSLP) mRNA), total nasal symptom scores (TNSS), mini-rhinitis-specific quality of life questionnaire (RQLQ) results, and visual analog scale scores were evaluated after 4 weeks of treatment. Additionally, the difference in PM2.5 concentrations in the air in Jeonju-si, Korea (November, 2019 ∼ March, 2020), was analyzed to determine the change in TNSS. KDP20 (100 μg/mL) was exposed to C57BL/6 mice for 10 days; 0.05% Nasonex (a positive control, mometasone furoate), or BYG was administrated for 5 days twice a day. The expression of inflammatory factors was detected via qRT-PCR using nasopharynx tissue samples of mice. BYG treatment was found to be associated with significant improvement in total nasal symptoms, especially itching and sneezing (p < 0.0001), and mini-RQLQ after 4 weeks. IL-8 (p < 0.01), IL-33 (p < 0.01), and TSLP (p < 0.001) expression levels decreased after BYG treatment. In mice, administration of BYG reduced the number of goblet cells increased through KDP20 treatment. KDP20-induced immune biomarkers (IL-33, TSLP, tumor necrosis factor alpha, and IL-8) were also significantly downregulated in the nasopharynx tissue after BYG treatment. Therefore, BYG may show therapeutic effects against allergic rhinitis in humans, and it was confirmed that the expression of PM-induced inflammatory factors in mice was decreased via BYG treatment.

Compound probiotics alleviate cadmium-induced intestinal dysfunction and microbiota disorders in broilers

Cadmium (Cd), a common environmental pollutant, seriously threatens the health of intestine. This research aimed to investigate the effects of compound probiotics (CP) on intestinal dysfunction and cecal microbiota dysregulation induced by Cd in broilers. A total of 240 1-day-old Arbor Acre (AA) broilers were randomly assigned to four groups. After 120 days of feeding, the jejunum tissues and cecal contents were sampled for jejunum histopathological observation, the intestinal barrier and inflammatory factors related mRNA and proteins examinations, and intestinal microbiota analysis. The results showed that Cd could cause jejunal villus damage and inflammatory cells infiltration, down-regulate the mRNA levels of intestinal barrier related genes (ZO-1, ZO-2, ZO-3, Claudin1, Claudin3, Claudin4, Occludin, and E-cadherin) and inflammatory factor related genes (IL-1β, IL-18, IFN-γ, NF-κB), and the protein levels of Claudin1, ZO-1, Occludin, but up-regulate the Claudin2, IL-2, IL-4 and IL-10 mRNA levels. However, the addition of CP could effectively improve these changes. In addition, 16S rRNA gene sequencing analysis showed that compared with the Cd group, supplementation CP increased the abundance of Lactobacillales, Clostridiales, Firmicutes, together with regulations on the pathways responsible for energy metabolism, translation and amino acid metabolism. In conclusion, CP could improve intestinal barrier damage and intestinal microbiota disturbance induced by Cd.

Delivery of cisplatin and resiquimod in nanomicelles for the chemoimmunotherapy of ovarian cancer

BackgroundTo explore the effect and mechanism of delivery of cisplatin (CDDP) and resiquimod in nanomicelles for the chemoimmunotherapy of ovarian cancer in vivo and in vitro.MethodsPoly(l-glutamic acid)-graft-methoxypolyethylene glycols (PLG-g-mPEG) was used to carry cisplatin and resiquimod for the preparation of CDDP/resiquimod/PLG-g-mPEG. We determined the loading content (LC) and encapsulation efficiency (EE), and then observed the particle shape, particle size distribution and zeta potential. In this study, we recruited 30 healthy adult participants and isolated mononuclear cells, and they were randomly classified into a control group, a CDDP group, a resiquimod group, a CDDP/resiquimod/PLG-g-mPEG group and a IFN-γ + LPS group to identify macrophages markers in different polarization states. We conducted microprobe synchrotron radiation X-ray fluorescence (SRXRF) imaging to observe the cell uptake. Furthermore, we observed the effects of CDDP/resiquimod/PLG-g-mPEG on the growth and colony formation of SW626 cells and the expressions of apoptosis-associated genes and proteins. Tumor-bearing mouse models of ovarian cancer were prepared and randomized into a negative control group, a PLG-g-mPEG group, a CDDP group, a resiquimod group and a CDDP/resiquimod/PLG-g-mPEG group, so as to analyze the anti-cancer effect of CDDP/resiquimod/PLG-g-mPEG in vivo.ResultsThe LC and EE of CDDP/resiquimod/PLG-g-mPEG were 19.42% and 90.12%, respectively. Nanoparticles were uniform spherical in shape and closely arranged together, with a typical core–shell structure, and their average particle size and zeta potential were 82.36 nm and − 23.69 mV, respectively. When CDDP/resiquimod/PLG-g-mPEG group was compared with the control group, the positive expression rate of CD16 in the CDDP/resiquimod/PLG-g-mPEG group highly increased, whereas the positive expression rate of CD163 dramatically decreased. In the meantime, Arg1 and Mrc1 mRNA expressions significantly decreased whereas IL-12 and NOS2 mRNA expressions dramatically increased (P < 0.05). Elemental mapping of cells exhibited notable internalization of cisplatin delivered by CDDP/resiquimod/PLG-g-mPEG to cytoplasm. We compared the cell survival rate between the CDDP/resiquimod/PLG-g-mPEG group and the control group, the CDDP/resiquimod/PLG-g-mPEG group sharply reduced (P < 0.05). What’s more, the inhibitory effect got strengthened as the reaction time was prolonged, with the synergy coefficient of 0.31.ConclusionPLG-g-mPEG-loaded CDDP and resiquimod effectively achieves the targeted delivery of chemotherapy and immunotherapy, with a strong synergistic anti-cancer effect.

Cytokine and LL-37 gene expression levels in Bartonella spp. seropositive and seronegative patients of a rheumatology clinic

PurposeThe variation in the immune response to Bartonella spp. infection in humans remains unclear. The present study compares the expression of selected interleukins, cytokines and cathelicidin (LL-37) in rheumatology clinic patients suffering from musculoskeletal symptoms with healthy blood donors. The patients had previously been tested for the presence of Bartonella henselae antibodies. MethodsGene expression of LL-37, interleukin (IL)-2, IL-4, IL-6, IL-12, interferon-(IFN)-γ, and tumor necrosis factor (TNF-α)-α was determined in blood samples using quantitative Polymerase Chain Reaction (qPCR). Statistical analysis was prepared with STATISTICA. ResultsStatistically significant differences in the mRNA levels of the tested cytokines (IFN-γ, TNF-α, IL-2, IL-4, IL-6, IL-12; p<0.0001) were observed between the healthy controls and patients; however, no difference was observed for LL37 mRNA (p ​= ​0.1974). No significant differences in mRNA expression were observed between IgG in anti-Bartonella seropositive and seronegative individuals (p>0.05). The only significant differences between the Bartonella spp. DNA positive and negative patients, indicated by PCR, were observed for TNF-α and IL-12 mRNA (p ​= ​0.0045 and p ​= ​0.0255, respectively). ConclusionA broadly similar immune response to the tested cytokines was observed among the participants irrespective of anti-Bartonella spp. IgG seropositivity. However, the Bartonella DNA-positive participants demonstrated significantly lower expression of IL-12 and TNF-α mRNA; this may indicate that these bacteria have a suppressive influence on the immune system.

Prenatal Exposure of Rats to Staphylococcal Enterotoxin B Alters the Expression of Th1 and Th2 Cytokines via Decreased Methylation in the Spleen of Adult but not Neonatal Offspring.

The methylation of IFN-γ and IL-4 genes is regarded as an epigenetic regulation that maintains the Th1 or Th2 phenotype. To explore the influence of prenatal administration of the staphylococcal enterotoxin B (SEB) in pregnant rats, on the IFN-γ or IL-4 expression in the offspring spleen. The SEB or PBS was administered intravenously to pregnant rats on the embryo-day 16. After normal delivery, the spleens from the fifth-day neonates and adult offspring were isolated under anesthesia. Quantitative PCR, western blot, ELISA and MeDIP-qPCR were applied to determine the levels of the splenic IFN-γ or IL-4 mRNAs, their protein levels, and methylation status, respectively. Prenatal administration of the SEB in pregnant rats decreased the levels of the splenic IFN-γ and IL-4 proteins in neonates, but increased their mRNA levels. However, prenatal administration of the SEB significantly augmented both mRNA and protein levels of the IFN-γ and IL-4 in the adult spleen. In addition, the prenatal SEB administration decreased the methylation of the splenic IFN-γ and IL-4 in adult but not neonatal offspring. The prenatal administration of SEB in pregnant rats can cause a mixed Th1 and Th2 cytokines response in the offspring spleen, and alter the cytokine expression of the Th1 and Th2 via decreasing the methylation in adult but, not neonatal offspring spleen.

Effect of moxibustion with wheat-grain-sized moxa cones on uterine natural killer cells and deci-dualization in rats with thin endometrium

To observe the effect of moxibustion at "Shenshu"(BL23) and "Guanyuan" (CV4) on decidua-lization and uterine natural killer cells in rats with thin endometrium, so as to explore its mechanism underlying promotion of embryo implantation. Female SD rats were randomly divided into normal control, model and wheat-grain-sized moxa cone moxibustion (moxibustion) groups, with 14 rats in each group. The thin endometrium model was established by bilaterally intrauterine perfusion of 95% ethanol (first) and saline (later) during estrus. For rats of the moxibustion group, the ignited wheat-grain-sized moxa cones were applied to bilateral BL23 and CV4, with 7 moxa cones for each acupoint, once a day, continuously for 3 estrous cycles. Then the male and female rats were raised in the same cage. On the 5th day of pregnancy, the rats were killed under anesthesia and the uterus tissue was collected for measuring the endometrium thickness and the numbers of blood vessels and glands after H.E. staining, detecting the levels of the proportion of natural killer cells with flow cytometry. After the uterine natural killer cells were sorted by the immunomagnetic bead method, the expression levels of insulin-like growth factor binding protein (IGFBP-1), interferon(INF-γ), tumor necrosis factor(TNF-α), transforming growth factor(TGF-β), interleukin 4(IL-4) and IL-10 mRNAs were detected by using fluorescence quantitative real-time PCR. Compared with the normal control group, the endometrium thickness, number of glands and blood vessels, and the expression levels of IGFBP-1, TGF-β, IL-4 and IL-10 mRNAs were significantly decreased (P<0.01, P<0.05), while the expression levels of IFN-γ and TNF-α mRNAs were significantly increased (P<0.05，P<0.01) in the model group. In contrast to the model group, the endometrium thickness, number of glands and blood vessels, and the expression levels of IGFBP-1, TGF-β, IL-4 and IL-10 mRNAs were significantly increased (P<0.05, P<0.01), while the expression levels of IFN-γ and TNF-α mRNAs were considerably down-regulated (P<0.05, P<0.01) in the moxibustion group. No significant difference was found among the 3 groups in the proportion of natural killer cells in the endometrium (P>0.05). Moxibustion of BL23 and CV4 with wheat-grain-sized moxa cones can improve the degree of thin endometrial decidualization, which may be related with its functions in regulating the levels of cytokines secreted from natural killer cells in the uterus.

MiRNA-155 inhibition enhances porcine embryo preimplantation developmental competence by upregulating ZEB2 and downregulating ATF4

The in vitro embryo's competence is lower than in vivo counterparts and miRNAs found to affect the developmental competence, affecting pluripotency, stress level and apoptosis in embryos. We aimed to investigate the effect of miRNA-155 on parthenogenically activated early development porcine embryos at the preimplantation blastocyst stage. We designed miRNA-155 mimics and inhibitors and microinjected them into post-activated oocytes. The embryonic cleavage rate, blastocyst rate, and embryo development quality in terms of stress and apoptosis levels were investigated by confocal microscopy. Furthermore, we selected target genes, analyzed gene interaction and prediction networks, and compared the gene expression level in treatments and controls. miRNA-155 inhibition improved in vitro developmental competence by increasing cell numbers and reducing stress and apoptosis levels. The cleavage rate in the miRNA-155 inhibitor group was significantly higher (P < 0.05) than that in the miRNA-155 mimic group, but not in the control, whereas the blastocyst rate of the miRNA-155 inhibitor group was statistically significantly higher (P < 0.05) than in both control and miRNA-155 mimic groups. The relative gene expression level analysis showed downregulation of mRNAs related to stress and apoptosis, BAX, and the stress-induced autophagy gene ATF4, and TNF-ɑ in the miRNA-155 inhibitor group. Moreover, miRNA-155 inhibition showed upregulation of the relative expression of OCT4, ZEB2, BCL2, and IL-1 mRNA compared to control and mimic groups. However, the miRNA-155 mimic-injected group showed lower cleavage rates and blastocyst development rates than the other two groups. In conclusion, miRNA-155 inhibition in porcine in vitro embryos improved their preimplantation developmental competence and in vitro embryo production.

Human CD4+CD45RA+ T Cells Behavior after In Vitro Activation: Modulatory Role of Vasoactive Intestinal Peptide.

Naїve CD4+ T cells, which suffer different polarizing signals during T cell receptor activation, are responsible for an adequate immune response. In this study, we aimed to evaluate the behavior of human CD4+CD45RA+ T cells after in vitro activation by anti-CD3/CD28 bead stimulation for 14 days. We also wanted to check the role of the VIP system during this process. The metabolic biomarker Glut1 was increased, pointing to an increase in glucose requirement whereas Hif-1α expression was higher in resting than in activated cells. Expression of Th1 markers increased at the beginning of activation, whereas Th17-associated biomarkers augmented after that, showing a pathogenic Th17 profile with a possible plasticity to Th17/1. Foxp3 mRNA expression augmented from day 4, but no parallel increases were observed in IL-10, IL-2, or TGFβ mRNA expression, meaning that these potential differentiated Treg could not be functional. Both VIP receptors were located on the plasma membrane, and expression of VPAC2 receptor increased significantly with respect to the VPAC1 receptor from day 4 of CD4+CD45RA+ T activation, pointing to a shift in VPAC receptors. VIP decreased IFNγ and IL-23R expression during the activation, suggesting a feasible modulation of Th17/1 plasticity and Th17 stabilization through both VPAC receptors. These novel results show that, without polarizing conditions, CD4+CD45RA+ T cells differentiate mainly to a pathogenic Th17 subset and an unpaired Treg subset after several days of activation. Moreover, they confirm the important immunomodulatory role of VIP, also on naїve Th cells, stressing the importance of this neuropeptide on lymphocyte responses in different pathological or non-pathological situations.

Circadian Pharmacological Effects of Paeoniflorin on Mice With Urticaria-like Lesions.

Paeoniflorin (PF) is a monoterpene glucoside with various biological properties, and it suppresses allergic and inflammatory responses in a rat model of urticaria-like lesions (UL). In the present study, we treated OVA-induced mice presenting UL with PF at four circadian time points (ZT22, ZT04, ZT10, and ZT16) to determine the optimal administration time of PF. The pharmacological effects of PF were assessed by analyzing the scratching behavior; histopathological features; allergic responses such as immunoglobulin E (IgE), leukotriene B4 (LTB4), and histamine (HIS) release; inflammatory cell infiltration [mast cell tryptase (MCT) and eosinophil protein X (EPX)]; and mRNA levels of inflammatory cytokines such as interleukin (IL)-12, IL-6, interferon-γ (IFN-γ), and IL-4. It was demonstrated that PF significantly alleviated scratching behavior and histopathological features, and ZT10 dosing was the most effective time point in remission of the condition among the four circadian time points. Moreover, PF decreased the serum levels of IgE, LTB4, and HIS, and PF administration at ZT10 produced relatively superior effectiveness. PF treatment, especially dosing at ZT10, significantly reduced the number of mast cells and granules and diminished the infiltration of MCT and EPX in the skin tissues of mice with UL. Furthermore, the oral administration of PF effectively decreased the inflammatory cytokine levels of IL-12 mRNA. In conclusion, different administration times of PF affected its efficacy in mice with UL. ZT10 administration demonstrated relatively superior effectiveness, and it might be the optimal administration time for the treatment of urticaria.

Utilization of Treg Cells in Solid Organ Transplantation.

Organ transplants have been a life-saving form of treatment for many patients who are facing end stage organ failure due to conditions such as diabetes, hypertension, various congenital diseases, idiopathic diseases, traumas, and other end-organ failure. While organ transplants have been monumental in treatment for these conditions, the ten year survival and long-term outcome for these patients is poor. After receiving the transplant, patients receive a multi-drug regimen of immunosuppressants. These drugs include cyclosporine, mTOR inhibitors, corticosteroids, and antibodies. Polyclonal antibodies, which inhibit the recipient’s B lymphocytes, and antibodies targeting host cytokine inhibitors which prevent activation of B cells by T cells. Use of these drugs suppresses the immune system and increases the risk of opportunistic pathogen infections, tumors, and further damage to the transplanted organs and vasculature. Many regulatory mechanisms are present in organs to prevent the development of autoimmune disease, and Tregs are central to these mechanisms. Tregs secrete suppressive cytokines such as IL-10, TGF-B, and IL-35 to suppress T cells. Additionally, Tregs can bind to target cells to induce cell cycle arrest and apoptosis and can inhibit induction of IL-2 mRNA in target T cells. Tregs also interact with CTLA-4 and CD80/CD86 on antigen presenting cells (APCs) to prevent their binding to CD28 present on T cells. Due to their various immunosuppressive capabilities, Tregs are being examined as a possible treatment for patients that receive organ transplants to minimize rejection and prevent the negative outcomes. Several studies in which participants were given Tregs after undergoing organ transplantations were reviewed to determine the efficacy and safety of using Tregs in solid organ transplantation to prevent adverse outcomes.

MiR9 inhibits 6-mer HA-induced cytokine production and apoptosis in human chondrocytes by reducing NF-kB activation

The present study aimed to investigate the expression of miR9 and its correlation with cytokines, proteolytic enzymes and apoptosis in an experimental model of 6-mer HA induced inflammation in human chondrocytes.Human articular chondrocytes, transfected with a miR-9 mimic and miR-9 inhibitor, were stimulated with 6-mer HA in presence/absence of a specific NF-kB inhibitor.6-mer HA induced a significant increase of TLR-4, CD44, IL-8, IL-18, MMP-9, ADAMTS-5, BAX and BCL-2 mRNAs expression and the related proteins, as well as NF-kB activation, associated with a significant up regulation of miR-9. In chondrocytes transfected with the miR-9 mimic before 6-mer HA treatment we found a decrease of such inflammatory cytokines, metalloproteases and pro-apoptotic molecules, while we found them increased in chondrocytes transfected with the miR9 inhibitor before 6-mer HA stimulation. The activities of TLR-4 and CD44, up regulated by 6-mer HA, were not modified by miR9 mimic/inhibitor, while the NF-kB activation was significantly affected.We suggested that the up regulation of miR9, induced by 6-mer HA, could be a cellular attempt to limit cell damage during inflammation.

Alterations in T-Cell Transcription Factors and Cytokine Gene Expression in Late-Onset Alzheimer's Disease.

Late-onset Alzheimer's disease (LOAD) is associated with many environmental and genetic factors. The effect of systemic inflammation on the pathogenesis of neurodegenerative diseases such as AD has been strongly suggested. T helper cells (Th) are one of the important components of the immune system and can easily infiltrate the brain in pathological conditions. The development of each Th-subset depends on the production of unique cytokines and their main regulator. This study aimed to compare the mRNA levels of Th-related genes derived from peripheral blood mononuclear cells of LOAD patients with control. Also, the identification of the most important Th1/Th2 genes and downstream pathways that may be involved in the pathogenesis of AD was followed by computational approaches. This study involved 30 patients with LOAD and 30 non-demented controls. The relative expression of T-cell cytokines (IFN-γ, TNF-α, IL-4, and IL-5) and transcription factors (T-bet and GATA-3) were assessed using Real-time PCR. Additionally, protein-protein interaction (PPI) was investigated by gene network construction. A significant decrease at T-bet, IFN-γ, TNF-α, and GATA-3 mRNA levels was detected in the LOAD group, compared to the controls. However, there was no significant difference in IL-4 or IL-5 mRNA levels. Network analysis revealed a list of the highly connected protein (hubs) related to mitogen-activated protein kinase (MAPK) signaling and Th17 cell differentiation pathways. The findings point to a molecular dysregulation in Th-related genes, which can promising in the early diagnosis or targeted interventions of AD. Furthermore, the PPI analysis showed that upstream off-target stimulation may involve MAPK cascade activation and Th17 axis induction.

Peripheral NF-\u03baB dysregulation in people with schizophrenia drives inflammation: putative anti-inflammatory functions of NF-\u03baB kinases

Elevations in plasma levels of pro-inflammatory cytokines and C-reactive protein (CRP) in patient blood have been associated with impairments in cognitive abilities and more severe psychiatric symptoms in people with schizophrenia. The transcription factor nuclear factor kappa B (NF-κB) regulates the gene expression of pro-inflammatory factors whose protein products trigger CRP release. NF-κB activation pathway mRNAs are increased in the brain in schizophrenia and are strongly related to neuroinflammation. Thus, it is likely that this central immune regulator is also dysregulated in the blood and associated with cytokine and CRP levels. We measured levels of six pro-inflammatory cytokine mRNAs and 18 mRNAs encoding NF-κB pathway members in peripheral blood leukocytes from 87 people with schizophrenia and 83 healthy control subjects. We then assessed the relationships between the alterations in NF-κB pathway genes, pro-inflammatory cytokine and CRP levels, psychiatric symptoms and cognition in people with schizophrenia. IL-1β and IFN-γ mRNAs were increased in patients compared to controls (both p < 0.001), while IL-6, IL-8, IL-18, and TNF-α mRNAs did not differ. Recursive two-step cluster analysis revealed that high levels of IL-1β mRNA and high levels of plasma CRP defined ‘high inflammation’ individuals in our cohort, and a higher proportion of people with schizophrenia were identified as displaying ‘high inflammation’ compared to controls using this method (p = 0.03). Overall, leukocyte expression of the NF-κB-activating receptors, TLR4 and TNFR2, and the NF-κB subunit, RelB, was increased in people with schizophrenia compared to healthy control subjects (all p < 0.01), while NF-κB-inducing kinase mRNAs IKKβ and NIK were downregulated in patients (all p < 0.05). We found that elevations in TLR4 and RelB appear more related to inflammatory status than to a diagnosis of schizophrenia, but changes in TNFR2 occur in both the high and low inflammation patients (but were exaggerated in high inflammation patients). Further, decreased leukocyte expression of IKKβ and NIK mRNAs was unique to high inflammation patients, which may represent schizophrenia-specific dysregulation of NF-κB that gives rise to peripheral inflammation in a subset of patients.

Intranasal curcumin and dexamethasone combination ameliorates inflammasome (NLRP3) activation in lipopolysachharide exposed asthma exacerbations

The inflammasome NOD-like receptor (NLR) family, the pyrin domain containing 3 (NLRP3) is closely associated with exacerbation of asthma as endotoxin (lipopolysaccharide, LPS) is one of its activators present in the environment. Present study is undertaken to investigate anti-inflammatory effects of a well known phytochemical, curcumin, which might regulate LPS exposed asthma exacerbations by modulating NLRP3 activation if given through intranasal route. Balb/c mice were sensitized with intraperitoneal injection of OVA (Ovalbumin; 100 μg of OVA with alum) from day 1 to 8 and exposed to LPS with 1% OVA aerosol from day 9 to 15. LPS (0.1 μg) was given an hour before sensitization and OVA-aerosol challenge. Significant decrease in inflammatory cell recruitment and restoration of structural changes in lungs, alterations in mRNA and protein expressions of TLR-4, NF-κB, NLRP3, Caspase-1, IL-1β, MMP-9, IL-5 and IL-17 in intranasal curcumin alone and corticosteroid combined pretreatment group.

Pathophysiological Role of TLR4 in Chronic Relapsing Itch Induced by Subcutaneous Capsaicin Injection in Neonatal Rats.

Despite the high prevalence of chronic dermatitis and the accompanied intractable itch, therapeutics that specifically target itching have low efficacy. Increasing evidence suggests that TLRs contribute to immune activation and neural sensitization; however, their roles in chronic itch remain elusive. Here, we show that the RBL-2H3 mast cell line expresses TLR4 and that treatment with a TLR4 antagonist opposes the LPS dependent increase in mRNA levels of Th2 and innate cytokines. The pathological role of TLR4 activation in itching was studied in neonate rats that developed chronic itch due to neuronal damage after receiving subcutaneous capsaicin injections. Treatment with a TLR4 antagonist protected these rats with chronic itch against scratching behavior and chronic dermatitis. TLR4 antagonist treatment also restored the density of cutaneous nerve fibers and inhibited the histopathological changes that are associated with mast cell activation after capsaicin injection. Additionally, the expression of IL-1β, IL-4, IL-5, IL-10, and IL-13 mRNA in the lesional skin decreased after TLR4 antagonist treatment. Based on these data, we propose that inhibiting TLR4 alleviated itch in a rat model of chronic relapsing itch, and the reduction in the itch was associated with TLR4 signaling in mast cells and nerve fibers.

Efficacy of dietary quercetin supplementation with high-energy diet model in broilers: implications on zootechnical parameters, serum biochemistry, antioxidant status, patho-morphology and gene expression studies

Context Currently, fat supplementation is one of the commonest strategies in poultry production for early economic return. However, it might cause various unidentified metabolic alterations in chickens. Flavonoid compound quercetin has potential to modulate oxidative stress and lipid metabolism. Aim The study was designed to evaluate the effect of dietary quercetin supplementation in broilers challenged with high-energy diet (HED). Methods In total, 192 days old Vencobb-400 broiler chicks were randomly allocated to four dietary treatments with four replicates (12 birds/replicate) per treatment. Four treatments included basal diet without any supplementation (T1), basal diet + quercetin (1 g/kg; T2), HED supplemented with vegetable oil [34 g/kg (2–3 weeks), 35 g/kg (4–6 weeks); T3], HED supplemented with vegetable oil [34 g/kg (2–3 weeks), 35 g/kg (4–6 weeks)] + quercetin (1 g/kg; T4) for the age of 2–3 (starter) and 4–6 (finisher) weeks. Key results Improvement was observed in the growth performance and feed conversion ratio in broilers with the dietary treatments (P ≤ 0.05). HED-supplemented group showed increased levels of metabolic stress, which was explained by the elevated concentrations of cortisol and malondialdehyde, and reduced serum/liver superoxide dismutase and glutathione peroxidase activity. Moreover, lipotoxicity was found due to the accumulation of fat, and lipid peroxidation caused various injuries to the vital organs such as liver and kidney, which were manifested by histopathological findings, and also upregulated the hepatic inflammatory interleukin (IL)-1β, IL-6, and tumour necrosis factor-α mRNA expression in the HED group. HED in combination with quercetin attenuates the altered serum metabolic markers, lipid peroxidation with subsequent rising in endogenous enzyme activity. In addition, it exhibited lipolytic action by lysis of accumulated fat and ameliorated the pathomorphic alteration in vital organs and downregulation of hepatic IL-1, IL-6 and TNF-α mRNA expression. Conclusions High dietary energy exhibits metabolic alteration and injuries to the vital organs. It is concluded that quercetin has potential to protect against the adverse effects induced by consumption of high-energy diet in broilers. Implications The supplementation of quercetin as an antioxidant seems to be beneficial for poultry production as an herbal feed additive for better performance, production, and health status.

The relationship between ghrelin and inflammation in diabetic rat stomach

Ghrelin is a hormone that regulates the digestive system, as well as has immunomodulating effects. The aim of this study is to explain effects of ghrelin on inflammation and oxidative stress parameters in the stomach. Male Sprague Dawley rats 8-10 weeks old (n = 21) were randomly divided into three groups as control, type 2 diabetes (T2DM) and diabetes and given exogenous ghrelin (T2DM+Gh). The daily feed and water intake of the animals were measured. The levels of interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), and interleukin-10 (IL-10) mRNA in tissues were analyzed using RT-PCR technique. Ghrelin and nuclear factor-κβ (NF-κβ) peptides were detected by immunohistochemistry. T2DM group had a significant increase in water intake when compared to control group. T2DM group had significantly higher levels of IL-6, IL-1, and IL-10 mRNA expression than control group. IL-1β and IL-10 mRNA expression were significantly lower in T2DM+Gh group than in T2DM group. In T2DM group, NF-κβ was higher than in control group, but it was lower in T2DM+Gh group. In terms of oxidative stress, there were non-significant changes. According to our findings, exogenous ghrelin intake was found to be highly effective in reducing inflammation in stomach tissue with type 2 diabetes (Tab. 1, Fig. 3, Ref. 33). Text in PDF www.elis.sk Keywords: ghrelin, rat, type 2 diabetes, stomach, inflammation.