Release Of Tumor Necrosis Factor Research Articles

Age-dependent effect between MARCO and TLR4 on PMMA particle phagocytosis by macrophages.

Progressive generation of total joint implant‐derived wear particles is one of the major risk factors in development of peri‐prosthetic osteolysis especially in the aging society. It is commonly accepted that macrophages predominantly drive the inflammatory response to wear debris particles. Among various surface receptors that activate the macrophages to phagocytize particles, it is believed that the Toll‐like receptor 4 (TLR4) and the scavenger macrophage receptor with collagenous structure (MARCO) play key roles in recognition of wear debris particles. However, a strong body of evidence indicates an age‐dependent diminished function of human TLRs. Thus, we hypothesized that the MARCO receptor may be more engaged than TLRs in the phagocytosis of wear debris particles which in turn up‐regulate production of pro‐inflammatory cytokines from aged macrophages. We demonstrated that peritoneal macrophages isolated from aged mice show elevated expression of MARCO receptor compared to that from young mice. In contrast the expression of TLR4 was significantly decreased on the surface of aged macrophages. Furthermore, using anti‐MARCO and anti‐TLR4 neutralizing mAbs, we demonstrated the age‐dependent pathogenic role of MARCO, but not TLR4, receptor in promoting poly‐methyl methacrylate (PMMA) bone cement particles phagocytosis by macrophages leading to the release of pro‐inflammatory cytokines migration inhibitory factor and tumour necrosis factor in vitro. These data also suggest that the approach to neutralize MARCO may lead to the development of therapeutic regimen for the prevention of particle‐induced osteolysis in aged patients.

Micronized, Heat-Treated Lactobacillus plantarum LM1004 Alleviates Cyclophosphamide-Induced Immune Suppression.

The present study investigated the immunomodulatory activity and associated mechanisms of heat-treated Lactobacillus plantarum LM1004 (HT-LM1004) in a cyclophosphamide (CTX)-induced mouse model of immunosuppression. HT-LM1004 induced phagocytic activity and nitric oxide production in RAW264.7 macrophages and stimulated the release of tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-2, and IL-12p70. In mice with CTX-induced immunosuppression, oral HT-LM1004 administration restored thymus and spleen indices, including spleen weight. Consistent with the in vitro results, HT-LM1004 increased TNF-α, IFN-γ, IL-2, and IL-12p70 levels in mice after 14 days of treatment and enhanced the natural killer (NK) cell activity of splenocytes from mice with CTX-induced immunosuppression against YAC-1 lymphoma cells. The method of HT-LM1004 generation influenced this activity: L. plantarum LM1004 grown in a membrane bioreactor, which reduced the size of the cells to <1.0 μm through physical stress (micronization), promoted NK cell cytotoxicity to a greater extent than LM1004 subjected to heat treatment alone. These findings indicate that HT-LM1004 without or with micronization can reverse CTX-induced immunosuppression without adverse side effects by potentiating NK cell function.

Mast cell stabilizing effect of a geranyl acetophenone in dengue virus infection using in vitro model of DENV3-induced RBL-2H3 cells.

Mast cells (MCs), a type of immune effector cell, have recently become recognized for their ability to cause vascular leakage during dengue virus (DENV) infection. Although MC stabilizers have been reported to attenuate DENV induced infection in animal studies, there are limited in vitro studies on the use of MC stabilizers against DENV induced MC degranulation. 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA) has been reported to be a potential MC stabilizer by inhibiting IgE-mediated MC activation in both cellular and animal models. The present study aims to establish an in vitro model of DENV3-induced RBL-2H3 cells using ketotifen fumarate as a control drug, as well as to determine the effect of tHGA on the release of MC mediators upon DENV infection. Our results demonstrated that the optimal multiplicities of infection (MOI) were 0.4 × 10−2 and 0.8 × 10−2 focus forming units (FFU)/cell. Ketotifen fumarate was proven to attenuate DENV3-induced RBL-2H3 cells degranulation in this in vitro model. In contrast, tHGA was unable to attenuate the release of both β-hexosaminidase and tumor necrosis factor (TNF)-α. Nonetheless, our study has successfully established an in vitro model of DENV3-induced RBL-2H3 cells, which might be useful for the screening of potential MC stabilizers for anti-dengue therapies.

LPS-neutralizing peptides reduce outer membrane vesicle-induced inflammatory responses

Outer membrane vesicles (OMVs) are secreted by Gram-negative bacteria and induce a stronger inflammatory response than pure LPS. After endocytosis of OMVs by macrophages, lipopolysaccharide (LPS) is released from early endosomes to activate its intracellular receptors followed by non-canonical inflammasome activation and pyroptosis, which are critically involved in sepsis development. Previously, we could show that the synthetic anti-endotoxin peptide Pep19-2.5 neutralizes inflammatory responses induced by intracellular LPS. Here, we aimed to investigate whether Pep19-2.5 is able to suppress cytoplasmic LPS-induced inflammation under more physiological conditions by using OMVs which naturally transfer LPS to the cytosol. Isothermal titration calorimetry revealed an exothermic reaction between Pep19-2.5 and Escherichia coli OMVs and the Limulus Amebocyte Lysate assay indicated a strong endotoxin blocking activity. In THP-1 macrophages and primary human macrophages Pep19-2.5 and polymyxin B reduced interleukin (IL)-1β and tumor necrosis factor (TNF) release as well as pyroptosis induced by OMVs, while the Toll-like receptor 4 signaling inhibitor TAK-242 suppressed OMV-induced TNF and IL-1β secretion, but not pyroptosis. Internalization of Pep19-2.5 was at least partially mediated by the P2X7 receptor in macrophages but not in monocytes. Additionally, a cell-dependent difference in the neutralization efficiency of Pep19-2.5 became evident in macrophages and monocytes, indicating a critical role for peptide-mediated IL-1β secretion via the P2X7 receptor. In conclusion, we provide evidence that LPS-neutralizing peptides inhibit OMV-induced activation of the inflammasome/IL-1 axis and give new insights into the mechanism of peptide-mediated neutralization of cytoplasmic LPS suggesting an essential and cell-type specific role for the P2X7 receptor.

Intraovarian regulation of luteolysis

The corpus luteum is a transient gland, which is only functional for 17-18 days in the cyclic cow or for up to 200 days in the pregnant cow. Regression of the corpus luteum is essential for normal cyclicity as it allows the development of a new ovulatory follicle, whereas prevention of luteolysis is necessary for the maintenance of pregnancy. Evidence acquired over the past three decades indicated that PGF2 alpha is the luteolytic hormone in ruminants. Nevertheless, the detailed mechanisms of PGF2 alpha action are just beginning to be clarified. A pivotal role for an endothelial cell product endothelin 1 (ET-1) has been documented in PGF2 alpha-induced luteal regression. ET-1 inhibited progesterone production by luteal cells in a dose-dependent manner via selective ET-1 binding sites (ETA). The inhibitory action of PGF2 alpha on progesterone secretion (in vivo and in vitro) was blocked by a selective ETA receptor antagonist. This implied that ET-1 (through ETA receptors present on steroidogenic cells) may have mediated the inhibitory effect of PGF2 alpha. The involvement of ET-1 in luteal regression was also suggested by the observation that the highest concentrations of ET-1 coincide with uterine PGF2 alpha surges. Furthermore, PGF2 alpha administration upregulated ET-1 expression within the corpus luteum. Later stages of luteal regression, which involve programmed cell death (PCD), are presumably mediated by immune cells. ET-1 may also be involved in this process by promoting leukocyte migration and stimulating macrophages to release tumour necrosis factor alpha (TNF alpha). The TNF alpha receptor type 1 (p55) is present on luteal cells (endothelial and steroidogenic cells) and could initiate PCD and the structural demise of the corpus luteum.

Soursop fruit extract mitigates scopolamine-induced amnesia and oxidative stress via activating cholinergic and Nrf2/HO-1 pathways.

Current therapeutic interventions for memory loss are inadequate and are associated with numerous adverse effects. There is an urgent need for new alternative agents for the treatment of memory loss and related disorders. Here, we investigated the potential neuroprotective role of soursop fruit extract (SSFE) in scopolamine (SCO)-induced amnesia and oxidative damage in the hippocampus of rats. Thirty-five rats were randomly allocated into 5 groups: control, SCO, SSFE, SCO, SSFE+SCO and N-acetylcysteine (NAC) + SCO. SCO-treatment increased acetylcholine esterase activity and decreased hippocampal levels of acetylcholine, serotonin, dopamine, norepinephrine, and histamine. The level of ATP increased. SCO-treated rats showed a disturbance in oxidative status, which was evident through the increase in malondialdehyde, and nitrites/nitrates and a decrease in cellular antioxidant molecules including glutathione, superoxide dismutase, catalase, glutathione reductase, and glutathione peroxidase. A disturbance was also observed via downregulation of the nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 defense pathways. SCO-treatment enhances a neuroinflammatory state, as indicated by the release of tumor necrosis factor- α and interleukin-1β and increased inducible nitric oxide synthase and mRNA expression. SCO-treatment decreased the expression of the anti-apoptotic protein, B cell lymphoma 2 and increased the expression of the pro-apoptotic protein, Bcl-2 associated X protein, caspase-3 and cytochrome c in hippocampal neurons. SSFE pretreatment markedly ameliorated hippocampal changes. Our findings revealed that SSFE exerts its potential anti-amnestic effect mainly through the activation of the cholinergic system and Nrf2/HO-1 pathway.

Isorhynchophylline exerts anti-inflammatory and anti-oxidative activities in LPS-stimulated murine alveolar macrophages

AimsExcessive inflammatory response and oxidative stress are considered as important pathogenic factors in the development of acute lung injury. Isorhynchophylline (IRN), a tetracyclic oxindole alkaloid isolated from Uncaria rhynchophylla, possesses anti-inflammatory and anti-oxidant activities. Our study aimed to investigate the effects and potential mechanisms of IRN on lipopolysaccharide (LPS)-stimulated murine alveolar macrophage cell lines MH-S and NR8383. Main methodsCCK-8 assay was used to evaluate the cytotoxicity of IRN and LPS. Inflammatory response was assessed by detecting the mRNA expressions and release of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, and plasminogen activator inhibitor-1 (PAI-1) using qRT-PCR and ELISA. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were examined by qRT-PCR and western blot. Oxidative stress was evaluated by detecting malondialdehyde (MDA) level and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT). The changes of the toll like receptor (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor protein 3 (NLRP3) inflammasome pathway was detected by western blot. Key findingsTreatment with LPS or IRN for 24 h showed no cytotoxicity on MH-S and NR8383 cells. IRN pretreatment inhibited LPS-induced production of inflammatory cytokines, expressions of iNOS and COX-2, and oxidative stress in murine alveolar macrophages. Additionally, IRN inhibited LPS-induced activation of TLR4/NF-κB/NLRP3 inflammasome pathway in MH-S cells. Mechanistically, inhibition of TLR4/NF-κB/NLRP3 inflammasome pathway by si-TLR4 suppressed LPS-induced inflammation and oxidative stress in murine alveolar macrophages. SignificanceIRN exerted anti-inflammatory and anti-oxidant effects on LPS-stimulated murine alveolar macrophages via inhibition of the TLR4/NF-κB/NLRP3 inflammasome pathway.

Transient Receptor Potential Vanilloid 1 Modulates Central Inflammation in Multiple Sclerosis.

Introduction: Disease course of multiple sclerosis (MS) is negatively influenced by proinflammatory molecules released by activated T and B lymphocytes and local immune cells. The endovanilloid system plays different physiological functions, and preclinical data suggest that transient receptor potential vanilloid type 1 (TRPV1) could modulate neuroinflammation in this disorder.Methods: The effect of TRPV1 activation on the release of two main proinflammatory cytokines, tumor necrosis factor (TNF) and interleukin (IL)-6, was explored in activated microglial cells. Furthermore, in a group of 132 MS patients, the association between the cerebrospinal fluid (CSF) levels of TNF and IL-6 and a single nucleotide polymorphisms (SNP) influencing TRPV1 protein expression and function (rs222747) was assessed.Results: In in vitro experiments, TRPV1 stimulation by capsaicin significantly reduced TNF and IL-6 release by activated microglial cells. Moreover, the anti-inflammatory effect of TRPV1 activation was confirmed by another TRPV1 agonist, the resiniferatoxin (RTX), whose effects were significantly inhibited by the TRPV1 antagonist, 5-iodoresiniferatoxin (5-IRTX). Vice versa, BV2 pre-treatment with 5-IRTX increased the inflammatory response induced by LPS. Moreover, in MS patients, a significant association emerged between TRPV1 SNP rs222747 and CSF TNF levels. In particular, the presence of a G allele, known to result in increased TRPV1 protein expression and function, was associated to lower CSF levels of TNF.Conclusions: Our results indicate that TRPV1 influences central inflammation in MS by regulating cytokine release by activated microglial cells. The modulation of the endovanilloid system may represent a useful approach to contrast neuroinflammation in MS.

Nigella damascena L. essential oil and its main constituents, damascenine and β-elemene modulate inflammatory response of human neutrophils ex vivo

Nigella damascena L belongs to Ranunculaceae family and is mentioned in Eastern traditional medicine for the treatment of high temperatures, regulation of menstruation or catarrhal affections. The anti-inflammatory activity of compounds present in the essential oil obtained from seeds of this plant can be found in literature, however no studies on immunomodulatory activity are provided. Hence, in this work anti-inflammatory activity of N. damascena seed essential oil as well as damascenine and main compound β-elemene was evaluated on ex-vivo lipopolysaccharide (LPS)–stimulated human neutrophils. For isolation of damascenine fast and efficient protocol was elaborated using high performance countercurrent chromatography technique for the first time. Also detailed spectroscopic characteristic of damascenine was provided for the first time. Damascenine was separated from the essential oil in a mixture of petroleum ether/acetonitrile/acetone (2:1.5:0.5 v/v/v) in reversed phase mode in 12 min with 99.47% purity. Essential oil, damascenine and β-elemene presented immunomodulatory activity evaluated in LPS–stimulated neutrophils ex vivo. All studied samples significantly inhibited release of interleukin 1 beta (IL-1β) and interleukin 8 (IL-8). What is more, damascenine and β-elemene decreased matrix metallopeptidase 9 (MMP-9) production similar to dexamethasone. The release of tumor necrosis factor (TNF-α) was also inhibited in all range of concentrations, however the activity was weaker then activity of dexametasone. The previously reported anti-inflammatory activity of damascenine and β-elemene investigated in murine models was confirmed in our study on human neuthrophils suggesting their possible strong inhibitory effect on inflammatory response progression.

Clinopodium chinense Attenuates Palmitic Acid-Induced Vascular Endothelial Inflammation and Insulin Resistance through TLR4-Mediated NF- B and MAPK Pathways.

Elevated palmitic acid (PA) levels are associated with the development of inflammation, insulin resistance (IR) and endothelial dysfunction. Clinopodium chinense (Benth.) O. Kuntze has been shown to lower blood glucose and attenuate high glucose-induced vascular endothelial cells injury. In the present study we investigated the effects of ethyl acetate extract of C. chinense (CCE) on PA-induced inflammation and IR in the vascular endothelium and its molecular mechanism. We found that CCE significantly inhibited PA-induced toll-like receptor 4 (TLR4) expression in human umbilical vein endothelial cells (HUVECs). Consequently, this led to the inhibition of the following downstream adapted proteins myeloid differentiation primary response gene 88, Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon- and TNF receptor-associated factor 6. Moreover, CCE inhibited the phosphorylation of Ikappa B kinase , nuclear factor kappa-B (NF- B), c-Jun N-terminal kinase, extracellular regulated protein kinases, p38-mitogen-activated protein kinase (MAPK) and subsequently suppressed the release of tumor necrosis factor- , interleukin-1 (IL-1 ) and IL-6. CCE also inhibited IRS-1 serine phosphorylation and ameliorated insulin-mediated tyrosine phosphorylation of IRS-1. Moreover, CCE restored serine/threonine kinase and endothelial nitric oxide synthase (eNOS) activation and thus increased insulin-mediated nitric oxide (NO) production in PA-treated HUVECs. This led to reverse insulin mediated endothelium-dependent relaxation, eNOS phosphorylation and NO production in PA-treated rat thoracic aortas. These results suggest that CCE can significantly inhibit the inflammatory response and alleviate impaired insulin signaling in the vascular endothelium by suppressing TLR4-mediated NF- B and MAPK pathways. Therefore, CCE can be considered as a potential therapeutic candidate for endothelial dysfunction associated with IR and diabetes.

Effects of Ultrasonic Dispersion Energy on the Preparation of Amorphous SiO2 Nanomaterials for In Vitro Toxicity Testing

Synthetic amorphous silica (SAS) constitute a large group of industrial nanomaterials (NM). Based on their different production processes, SAS can be distinguished as precipitated, fumed, gel and colloidal. The biological activity of SAS, e.g., cytotoxicity or inflammatory potential in the lungs is low but has been shown to depend on the particle size, at least for colloidal silica. Therefore, the preparation of suspensions from highly aggregated or agglomerated SAS powder materials is critical. Here we analyzed the influence of ultrasonic dispersion energy on the biologic activity of SAS using NR8383 alveolar macrophage (AM) assay. Fully characterized SAS (7 precipitated, 3 fumed, 3 gel, and 1 colloidal) were dispersed in H2O by stirring and filtering through a 5 µm filter. Aqueous suspensions were sonicated with low or high ultrasonic dispersion (USD) energy of 18 or 270 kJ/mL, respectively. A dose range of 11.25–90 µg/mL was administered to the AM under protein-free conditions to detect particle-cell interactions without the attenuating effect of proteins that typically occur in vivo. The release of lactate dehydrogenase (LDH), glucuronidase (GLU), and tumor necrosis factor α (TNF) were measured after 16 h. Hydrogen peroxide (H2O2) production was assayed after 90 min. The overall pattern of the in vitro response to SAS (12/14) was clearly dose-dependent, except for two SAS which showed very low bioactivity. High USD energy gradually decreased the particle size of precipitated, fumed, and gel SAS whereas the low adverse effect concentrations (LOECs) remained unchanged. Nevertheless, the comparison of dose-response curves revealed slight, but uniform shifts in EC50 values (LDH, and partially GLU) for precipitated SAS (6/7), gel SAS (2/3), and fumed SAS (3/3). Release of TNF changed inconsistently with higher ultrasonic dispersion (USD) energy whereas the induction of H2O2 was diminished in all cases. Electron microscopy and energy dispersive X-ray analysis showed an uptake of SAS into endosomes, lysosomes, endoplasmic reticulum, and different types of phagosomes. The possible effects of different uptake routes are discussed. The study shows that the effect of increased USD energy on the in vitro bioactivity of SAS is surprisingly small. As the in vitro response of AM to different SAS is highly uniform, the production process per se is of minor relevance for toxicity.

Hypoxia potentiates monocyte-derived dendritic cells for release of tumor necrosis factor α via MAP3K8.

Dendritic cells (DCs) constantly sample peripheral tissues for antigens, which are subsequently ingested to derive peptides for presentation to T cells in lymph nodes. To do so, DCs have to traverse many different tissues with varying oxygen tensions. Additionally, DCs are often exposed to low oxygen tensions in tumors, where vascularization is lacking, as well as in inflammatory foci, where oxygen is rapidly consumed by inflammatory cells during the respiratory burst. DCs respond to oxygen levels to tailor immune responses to such low-oxygen environments. In the present study, we identified a mechanism of hypoxia-mediated potentiation of release of tumor necrosis factor α (TNF-α), a pro-inflammatory cytokine with important roles in both anti-cancer immunity and autoimmune disease. We show in human monocyte-derived DCs (moDCs) that this potentiation is controlled exclusively via the p38/mitogen-activated protein kinase (MAPK) pathway. We identified MAPK kinase kinase 8 (MAP3K8) as a target gene of hypoxia-induced factor (HIF), a transcription factor controlled by oxygen tension, upstream of the p38/MAPK pathway. Hypoxia increased expression of MAP3K8 concomitant with the potentiation of TNF-α secretion. This potentiation was no longer observed upon siRNA silencing of MAP3K8 or with a small molecule inhibitor of this kinase, and this also decreased p38/MAPK phosphorylation. However, expression of DC maturation markers CD83, CD86, and HLA-DR were not changed by hypoxia. Since DCs play an important role in controlling T-cell activation and differentiation, our results provide novel insight in understanding T-cell responses in inflammation, cancer, autoimmune disease and other diseases where hypoxia is involved.

Neurochemical alterations following the exposure to di-n-butyl phthalate in rats.

Due to its ability to cross blood brain barrier and placenta, dibutyl phthalate (di-n-butyl phthalate, DBP) is expected to cause severe side effects to the central nervous system of animals and humans. A little data is available about the potential DBP neurotoxicity; therefore, this work was designed to investigate the brain tissue injury induced by DBP exposure. Forty Wister albino rats were allocated randomly into 4 groups (10 rats each). Group 1 served as control and the rats administered with physiological saline (0.9% NaCl) orally for 12weeks. Groups 2, 3 and 4 were orally treated with DPB (100, 250 and 500mg/kg) respectively for 12weeks. DBP-intoxicated rats showed a disturbance in the oxidative status in cerebral cortex, striatum and brainstem, as represented by the elevated oxidants [malondialdehyde (MDA), nitric oxide (NO), 8-hydroxy-2-deoxyguanosine (8-OHdG)] and the decreased antioxidant molecules [reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR)]. DBP also enhanced a pro-inflammatory state through increasing the release of tumor necrosis factor- α (TNF-α) and interleukin-1β (IL-1β). The increase of these cytokines was associated with the increase of pro-apoptotic proteins [Bcl-2 associated X protein (Bax) and caspase-3] and the decrease of the anti-apoptotic protein, B cell lymphoma 2 (Bcl-2). In addition, the levels of norepinephrine (NE), dopamine (DA) and acetylcholine esterase (AChE) activity were decreased. This was accompanied by the alterations in the major excitatory and inhibitory amino acids neurotransmitterslevels. The present findings indicated that DBP could exert its neuronal damage through oxidative stress, DNA oxidation, neuroinflammation, activation of apoptotic proteins and altering the monoaminergic, cholinergic and amino acids transmission.

Disaccharide-Based Anionic Amphiphiles as Potent Inhibitors of Lipopolysaccharide-Induced Inflammation.

Despite significant advances made in the last decade in the understanding of molecular mechanisms of sepsis and in the development of clinically relevant therapies, sepsis remains the leading cause of mortality in intensive care units with increasing incidence worldwide. Toll-like receptor 4 (TLR4)-a transmembrane pattern-recognition receptor responsible for propagating the immediate immune response to Gram-negative bacterial infection-plays a central role in the pathogenesis of sepsis and chronic inflammation-related disorders. TLR4 is complexed with the lipopolysaccharide (LPS)-sensing protein myeloid differentiation-2 (MD-2) which represents a preferred target for establishing new anti-inflammatory treatment strategies. Herein we report the development, facile synthesis, and biological evaluation of novel disaccharide-based TLR4⋅MD-2 antagonists with potent anti-endotoxic activity at micromolar concentrations. A series of synthetic anionic glycolipids entailing amide-linked β-ketoacyl lipid residues was prepared in a straightforward manner by using a single orthogonally protected nonreducing diglucosamine scaffold. Suppression of the LPS-induced release of interleukin-6 and tumor necrosis factor was monitored and confirmed in human immune cells (MNC and THP1) and mouse macrophages. Structure-activity relationship studies and molecular dynamics simulations revealed the structural basis for the high-affinity interaction between anionic glycolipids and MD-2, and highlighted two compounds as leads for the development of potential anti-inflammatory therapeutics.

Obesity and Inflammatory Bowel Disease: Outcomes of Patients in a Pro-inflammatory State on Biologic Therapy for Crohnʼs Disease and Ulcerative Colitis

Introduction: The prevalence of both inflammatory bowel disease (IBD) and obesity are increasing worldwide, and it is estimated that 15-40% of patients with IBD are obese. Adipose tissue is known to cause release of cytokines and tumor necrosis factor (TNF)-alpha implicated in both luminal and systemic inflammatory responses in patients with IBD. Currently, the effect of obesity on IBD and clinical outcomes remains under-described. Our aims are to characterize a cohort of patients with IBD on biologic therapy and to examine the effect of obesity (BMI > 30) on rates of liver function test abnormalities, development of malignancy, infections, and mortality. Methods: This is a retrospective analysis conducted at a tertiary care, academic-affiliated medical center with a center for inflammatory bowel disease. Patients with Crohn's disease or ulcerative colitis and on biologic therapy were identified using ICD-9 and 10 codes. Baseline anthropologic, serologic, and clinical data was obtained. Results: In a cohort of 72 patients, the mean age was 48, and 40% were female. Mean BMI was 28kg/m2. 59 patients (82%) had Crohn's disease. The mean years of disease duration was 17, and 20 patients (53%) also carried a diagnosis of NAFLD/NASH. 52 patients in the cohort were on biologic therapy, with 24 (46%) on infliximab, 18 (35%) adalimumab, 4 (8%) ustekinumab, 4 (8%) certolizumab pegol, and 2 (4%) vedolizumab. A higher proportion of obese vs non-obese patients on biologics had liver function test (LFT) abnormalities (77% vs 38%, P=0.019). There was no difference in peak alanine aminotransferase (ALT) levels between the two groups, 78 U vs 73 U, P=0.784 (ref range ALT 0-40 U). No difference was noted in the proportion of obese vs non-obese patients in developing malignancy (3% vs 0%, P=0.505), infection (23% vs 8%, P=0.226), and there were no deaths in either group. A higher proportion of patients that were obese were on infliximab (77% vs 30%, P=0.057). All obese patients were on an anti-TNF (infliximab or adalimumab). No mortality was noted in either group. Conclusion: Obese patients have higher risk for LFT abnormalities while on biologic therapy. This may be due to pre-existing hepatic inflammation from fatty liver disease. Moreover, obese patients did not develop more complications when on biologic therapy (e.g. malignancy or infection) compared to nonobese patients. Nonetheless, lifestyle modification and weight loss should be encouraged and be a part of routine IBD care in this population.

Actinofuranones D-I from a Lichen-Associated Actinomycetes, Streptomyces gramineus, and Their Anti-Inflammatory Effects.

Six new metabolites, actinofuranones D-I (compounds 1–6), were isolated together with three known compounds—JBIR-108 (7), E-975 (8), and E-492 (9)—from a fermentation broth of Streptomyces gramineus derived from the lichen Leptogium trichophorum. The structures of the new compounds 1–6 were established using comprehensive NMR spectroscopic data analysis, as well as UV, IR, and MS data. The anti-inflammatory activity of these isolated compounds were evaluated by examining their ability to inhibit nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophage cells. Compounds 4, 5, 8, and 9 attenuated the production of NO due to the suppression of the expression of nitric oxide synthase (iNOS) in LPS-induced RAW 264.7 cells. Moreover, 4, 5, 8, and 9 also inhibited LPS-induced release of proinflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α).

Suppressing Irf2bp2 expressions accelerates metabolic syndrome-associated brain injury and hepatic dyslipidemia

Increasing studies indicate that high fat diet (HFD) induces neuroinflammation in animal models with obesity, yet the pathology of it is unclear. Interferon Regulatory Factor 2 Binding Protein 2 (Irf2bp2) is a key regulator of macrophage polarization, playing an essential role in regulating inflammatory response. This study investigated the effects of Irf2bp2 on HFD-induced brain injury, and explored the possible molecular mechanisms using wild type (WT) and Irf2bp2 knockout (KO) mice. The results indicated that HFD-increased body weight of mice was further elevated by Irf2bp2-knockout. In addition, Irf2bp2-deletion accelerated HFD-induced metabolic syndrome, as evidenced by the promoted fasting glucose and insulin levels. In the results of behavioral measurements, Irf2bp2-knockout intensified cognitive deficit in HFD-fed mice by using Y-maze, passive avoidance, and morris water maze (MWM) tests. Further, Irf2bp2-deficiency accelerated the activation of astrocytes and microglia cells, as evidenced by the promoted expressions of glial fibrillary acidic protein (GFAP) and Iba-1 in hippocampus and hypothalamus of HFD-fed mice. HFD for 16 weeks induced oxidative stress in serum and brain of mice, as proved by the up-regulated malondialdehyde (MDA) levels and down-regulated superoxide dismutase (SOD) activity, which were significantly enhanced due to Irf2bp2 knockout. Moreover, HFD-triggered systematic and central nervous inflammation by increasing the release of interleukin 1β (IL-1β) and tumor necrosis factor (TNF)-α, accompanied with elevated p-nuclear factor-κB (NF-κB) expressions. Notably, HFD-induced inflammation was significantly exacerbated by Irf2bp2 deletion. Intriguingly, HFD-induced dyslipidemia in liver of mice was further aggravated by Irf2bp2 suppression. Our in vitro results verified the effects of Irf2bp2-inhibition on the promotion of inflammatory response in BV2 cells and lipid dysfunction in primary hepatocytes. Therefore, the findings above suggested that inhibiting Irf2bp2 expression provided a potential therapeutic approach for the prevention of metabolic syndrome-associated central nervous injury.

Aryl hydrocarbon receptor activation by benzo(a)pyrene inhibits proliferation of myeloid precursor cells and alters the differentiation state as well as the functional phenotype of murine bone marrow-derived macrophages

Intensive research during the past decade has highlighted the impact of the regulatory function of the aryl hydrocarbon receptor (AhR) in immunity. In this study, we focused on the influence of AhR activation on the differentiation of murine bone marrow-derived myeloid precursor cells into mature macrophages. Our results show that the activation of AhR by subtoxic doses of the AhR ligand benzo(a)pyrene (BaP) impaired the proliferation of bone marrow cells (BMCs) whereas the proportion of resulting adherent cells was not affected. Flow cytometric analysis revealed that the number of mature bone marrow-derived macrophages (BMMs) was significantly decreased by AhR activation. However, expression of the murine macrophage marker F4/80, the major histocompatibility complex class II (MHC-II) and the Fcγ receptor I (FcγRI/CD64) were upregulated on BaP-exposed BMMs in an AhR-dependent manner. Analysis of cytokine secretion after BMM activation with heat-killed (hk) salmonellae showed that BaP exposure resulted in suppressed secretion of interleukin (IL)-1β, IL-6 and the chemokine CXC motif ligand 1 (CXCL1). In contrast, the release of tumor necrosis factor (TNF)-α and IL-10 was increased following BaP exposure. In addition, the production of antimicrobial nitric oxide (NO) was increased AhR-dependently. Bacterial stimulation of BaP exposed BMMs also induced the expression of MHC-II and CD64, while the expression of F4/80 was dramatically decreased. In summary, this study demonstrates for the first time that sustained exposure over 6 days of bone marrow-derived myeloid precursors to subtoxic doses of BaP critically interferes with differentiation and activation of BMMs. We could convincingly show that AhR-induced gene regulation is crucial for homeostasis of pro- and anti-inflammatory cytokines during macrophage activation.

Comparison of Allergic Parameters between Whey Protein Concentrate and Its Hydrolysate in Rat Basophilic Leukemia (RBL)-2H3 Cells.

This study was conducted to compare the anti-allergic effects of a whey protein concentrate (WPC) and WPC hydrolysate. WPC hydrolysate was prepared using enzymatic digestion for 8 h with trypsin and α-chymotrypsin, after which it was freeze-dried. The allergic parameters assessed in rat basophilic leukemia (RBL)-2H3 cells were degranulation and release of β-hexosaminidase, release of tumor necrosis factor (TNF)-α, and changes in the expression of IL-1β, IL-4, and IL-10 by real time polymerase chain reaction (PCR). During preparation of the WPC hydrolysate, hydrolysis increased rapidly from 0 to 10 min and then gradually increased slowly from 1 h onwards, achieving a final degree of hydrolysis of 78.50%. The SDS-PAGE analysis revealed a reduction in the intensity of several protein bands in the WPC hydrolysate compared to the WPC. IgE-induced β-hexosaminidase release from RBL-2H3 cells was decreased to a higher degree following treatment with the hydrolysate compared to WPC treatment. W500 (500 µg/mL WPC) showed the least inhibition of β-hexosaminidase release, but there was no significant difference between W500 and W1000 (1,000 µg/mL) (p<0.05). H1000 (1,000 µg/mL WPC hydrolysate) inhibited β-hexosaminidase release by 39%. Compared to the control, treatment with H1000 decreased TNF-α secretion to 11.87 pg/mL. The gene expression levels of IL-1β, IL-4, and IL-13 were all significantly decreased in hydrolysate (p<0.05). In the case of IL-1β and IL-4, the expression levels in W1000 treated cells were decreased by 73.67% and 65%, respectively, and that of IL-13 was decreased by 66.43% compared to the control.

Isolation of spilanthol from Acmella oleracea based on Green Chemistry and evaluation of its in vitro anti-inflammatory activity

Acmella oleracea (jambu) is a South American plant whose pharmacological properties include antimalarial, anesthetic, and anti-inflammatory activities. However, the compounds contributing to the anti-inflammatory effect have received little attention. In this study, the active compound spilanthol was isolated using a Green Chemistry approach and presented significant anti-inflammatory activity in a lipopolysaccharide-activated human neutrophil model. First, the aerial parts of jambu were extracted and fractionated using supercritical carbon dioxide. The spilanthol-enriched fractions obtained were then subjected to further purification by flash chromatography, yielding spilanthol with 97% purity. Use of enzyme-linked immunosorbent assay (ELISA) revealed a reduction in the release of Interleukin-8 and Tumor Necrosis Factor - alpha by leukocytes exposed to spilanthol. In conclusion, this new approach enabled spilanthol to be obtained at high purity, in a fairly rapid procedure, while the results of the in vitro anti-inflammatory activity study indicated that the compound could be a promising new therapeutic agent.