Serum amyloid A expression in liver promotes synovial macrophage activation and chronic arthritis via NFAT5.

Nuclear factor of activated T-cells 5 (NFAT5), an osmo-sensitive transcription factor, can be activated by isotonic stimuli, such as infection. It remains unclear, however, whether NFAT5 is required for damage-associated molecular pattern-triggered (DAMP-triggered) inflammation and immunity. Here, we found that several DAMPs increased NFAT5 expression in macrophages. In particular, serum amyloid A (SAA), primarily generated by the liver, substantially upregulated NFAT5 expression and activity through TLR2/4-JNK signalling pathway. Moreover, the SAA-TLR2/4-NFAT5 axis promoted migration and chemotaxis of macrophages in an IL-6- and chemokine ligand 2-dependent (CCL2-dependent) manner in vitro. Intraarticular injection of SAA markedly accelerated macrophage infiltration and arthritis progression in mice. By contrast, genetic ablation of NFAT5 or TLR2/4 rescued the pathology induced by SAA, confirming the SAA-TLR2/4-NFAT5 axis in vivo. Myeloid-specific depletion of NFAT5 also attenuated SAA-accelerated arthritis. Of note, inflammatory arthritis in mice strikingly induced SAA overexpression in the liver. Conversely, forced overexpression of the SAA gene in the liver accelerated joint damage, indicating that the liver contributes to bolstering chronic inflammation at remote sites by secreting SAA. Collectively, this study underscores the importance of the SAA-TLR2/4-NFAT5 axis in innate immunity, suggesting that acute phase reactant SAA mediates mutual interactions between liver and joints and ultimately aggravates chronic arthritis by enhancing macrophage activation.

Effect of colostrum on the acute-phase response in neonatal dairy calves.

The core part of the mammal innate immune system is the acute-phase response (APR), during which acute-phase proteins (APP) are synthesized. Colostrum contains immunomodulating factors such as proinflammatory cytokines and APP in large quantities. We looked at proinflammatory cytokines [IL-1ß, IL-6, and tumor necrosis factor-α (TNF-α)] and APP [serum amyloid A (SAA) and haptoglobin (Hp)] in colostrum and in calves' serum. The aim of this study was to evaluate the effects of colostrum on the calves' systemic APR and the associations of the calves' serum APR with short- and long-term weight gain (at the age of 1, 3, and 9 mo). A total of 143 female dairy calves were studied during their first 3 wk of life. The calves were separated from their mothers immediately after birth and bottle-fed 3 L of quality-controlled colostrum once within 2 h after birth. Serum samples were collected once a week during the first 3 wk of life (a total of 1-3 samples per calf). Mean sampling age (±standard deviation) was 4.3 (±2.0) d in the first week, 11.0 (±2.0) d in the second week, and 18.0 (±2.0) d in the third week. Linear regression models were used to study associations of colostrum APP and cytokine concentration with serum APR markers and for studying associations of colostrum and serum APR markers with calves' average daily weight gain (ADWG). Mixed linear regression models were used to compare serum concentrations of APR markers by study weeks. The colostrum IL-6 concentrations were positively associated with serum IL-6 in the first 3 wk of life. Colostrum IL-1ß was positively associated with calves' serum IL-1ß during the first week of life, and colostrum TNF-α was positively associated with calves' serum TNF-α during the first 2 wk of life. Serum IL-1ß concentrations differed over the 3 wk, being the highest during the first week and the lowest during the second week. For IL-6, the concentration during the first week was the highest, and for TNF-α, a steady decline in the concentration was observed. Serum SAA concentrations were elevated during the first 2 wk of life and subsequently declined during the third week. Albumin concentrations were lowest in the first week, whereas Hp concentrations were highest during the second week. Serum concentrations of SAA, Hp, IL-6, and TNF-α during the second week were negatively associated with ADWG at 9 mo of age. The SAA concentrations during the third week of age had a negative association with 9-mo ADWG. Serum Hp concentrations in the third week were negatively associated with 3-mo ADWG. The results of our study suggest that colostrum cytokines influence calf serum cytokine concentrations. Thus, they influence the newborn calves' adaptation to the environment and the development of their immune system. Factors that activate an APR during the second and third week of life have a long-term influence on calves' development.

Epidemiology and clinical characteristics of 43 COVID-19 patients in Weifang, China.

BACKGROUND: To investigate the epidemiology and clinical characteristics of patients infected with coronavirus disease 2019 (COVID-19) in Weifang, China. METHODS: The demographic data of 43 COVID-19 patients identified in Weifang were used to investigate whether they had traveled to epidemic areas and whether they had close contact with confirmed cases. On admission, patients' symptoms and results of laboratory tests and imaging were analyzed. RESULTS: Among the 43 COVID-19 patients. including 9 third generation infected cases, 16 (37.2%) were imported, who infected the rest. Most cases were middle-aged with approximate sex ratio. A "super spreader", Mr. Zhang made it necessary to quarantine 69 medical personnel. Mr. Zhang directly infected six individuals who, in turn, infected another six individuals. Another patient, Mr. Wang, spread the infection to his five family members at a family gathering. Subsequently, the daughter infected her husband. The most common COVID-19 symptoms were fever, weakness, dry cough, and cough sputum. In most patients, white blood cell counts were not elevated and lymphocyte counts were decreased. Elevated C-reactive protein and serum amyloid A protein (SAA) levels were commonly observed. There was no death among the patients or infection among the medical staff. CONCLUSIONS: The infection by the COVID-19 in Weifang was mostly the result of close contact with imported cases. These circumstances underscore the need to comprehensively strengthen the management for patients to prevent and control the spread of the virus.

Dietary Intake of Rosmarinic Acid Increases Serum Inhibitory Activity in Amyloid A Aggregation and Suppresses Deposition in the Organs of Mice.

Serum amyloid A (SAA) is one of the most important precursor amyloid proteins and plays a vital step in AA amyloidosis, although the underlying aggregation mechanism has not been elucidated. Since SAA aggregation is a key step in this pathogenesis, inhibitors are useful to prevent and treat AA amyloidosis, serving as tools to investigate the pathogenic mechanism. In this study, we showed that rosmarinic acid (RA), which is a well-known inhibitor of the aggregation of amyloid ß (Aß), displayed inhibitory activity against SAA aggregation in vitro using a microliter-scale high-throughput screening (MSHTS) system with quantum-dot nanoprobes. Therefore, we evaluated the amyloid aggregation inhibitory activity of blood and the deposition of SAA in organs by feeding mice with Melissa officinalis extract (ME) containing RA as an active substance. Interestingly, the inhibitory activity of ME-fed mice sera for SAA and Aß aggregation, measured with the MSHTS system, was higher than that of the control group. The amount of amyloid deposition in the organs of ME-fed mice was lower than that in the control group, suggesting that the SAA aggregation inhibitory activity of serum is associated with SAA deposition. These results suggest that dietary intake of RA-containing ME enhanced amyloid aggregation inhibitory activity of blood and suppressed SAA deposition in organs. This study also demonstrated that the MSHTS system could be applied to in vitro screening and to monitor comprehensive activity of metabolized foods adsorbed by blood.

Oral exposure of pigs to the mycotoxin deoxynivalenol does not modulate the hepatic albumin synthesis during a LPS-induced acute-phase reaction.

The sensitivity of pigs to deoxynivalenol (DON) might be influenced by systemic inflammation (SI) which impacts liver. Besides following acute-phase proteins, our aim was to investigate both the hepatic fractional albumin (ALB) synthesis rate (FSR) and the ALB concentration as indicators of ALB metabolism in presence and absence of SI induced by LPS via pre- or post-hepatic venous route. Each infusion group was pre-conditioned either with a control diet (CON, 0.12 mg DON/kg diet) or with a DON-contaminated diet (DON, 4.59 mg DON/kg diet) for 4 wk. A depression of ALB FSR was observed 195 min after LPS challenge, independent of feeding group or LPS application route, which was not paralleled by a down-regulated ALB mRNA expression but by a reduced availability of free cysteine. The drop in ALB FSR only partly explained the plasma ALB concentrations which were more depressed in the DON-pre-exposed groups, suggesting that ALB levels are influenced by further mechanisms. The abundances of haptoglobin, C-reactive protein, serum amyloid A, pig major acute-phase protein, fibrinogen and LPS-binding protein mRNA were up-regulated upon LPS stimulation but not accompanied by increases in the plasma concentrations of these proteins, pointing at an imbalance between synthesis and consumption.

Effect of meloxicam and lidocaine administered alone or in combination on indicators of pain and distress during and after knife castration in weaned beef calves.

To assess the effect of meloxicam and lidocaine on indicators of pain associated with castration, forty-eight Angus crossbred beef calves (304 ± 40.5 kg of BW, 7-8 months of age) were used in a 28 day experiment. The experiment consisted of a 2 × 2 factorial design where main factors included provision of analgesia and local anaesthesia. Analgesia consisted of: no-meloxicam (N; n = 24) single s.c. administration of lactated ringer's solution and meloxicam (M; n = 24) single dose of 0.5 mg/kg of s.c. meloxicam. Local anesthesia consisted of: no-lidocaine (R; n = 24) ring block administration of lactated ringer's solution or lidociane (L; n = 24) ring block administration of lidocaine. To yield the following treatments: no meloxicam + no lidocaine (N-R; n = 12), no meloxicam + lidocaine (N-L; n = 12), meloxicam + no lidocaine (M-R; n = 12) and meloxicam + lidocaine (M-L; n = 12). Salivary cortisol concentrations were lower (lidocaine × time effect; P < 0.01) in L calves than R calves 0.5 and 1 hours after castration, while concentrations were lower (meloxicam × time effect; P = 0.02) in M calves than N calves at 2, 4 and 48 hours. The serum amyloid-A concentrations were greater (lidocaine × time effect; P < 0.01) in R calves than L calves on days 1, 3, 21 and 28 after castration. Haptoglobin concentrations were greater (meloxicam × time effect; P = 0.01) in N calves than M calves 24 and 48 hours after castration. Lower (lidocaine effect; P < 0.01) visual analog scale (VAS) scores, leg movement frequencies and head movement distance were observed in L calves than R calves at the time of castration. Escape behaviour during castration was lower (lidocaine effect; P < 0.05) in L calves than R calves based on data captured with accelerometer and head gate devices. Scrotal circumference had a triple interaction (lidocaine × meloxicam × time; P = 0.03), where M-R calves had greater scrotal circumference than M-L calves 28 d after castration, but no differences were observed between both groups and N-R and N-L calves. No differences (P > 0.05) were observed for average daily gain (ADG), weights or feeding behaviour. Overall, both lidocaine and meloxicam reduced physiological and behavioural indicators of pain. Although there was only one meloxicam × lidocaine interaction, lidocaine and meloxicam reduced physiological and behavioural parameters at different time points, which could be more effective at mitigating pain than either drug on its own.

Acute phase response in organic lambs associated with colostrum serum amyloid A, weight gain, and Cryptosporidium and Giardia infections.

In neonatal period, lamb's immune system goes through rapid adaptation to the extra-uterine environment. Success of this process can influence the animal's future performance and, thus, the quantitative assessment of it would greatly benefit sheep producers. The current study was conducted to investigate the acute phase response (APR) (measured through serum amyloid A (SAA), haptoglobin (Hp) and albumin (ALB)) in relation to later life growth (measured at 122 days of age), and naturally occurring Cryptosporidium and Giardia infections in neonatal lambs grown in organic farm. Serum (n = 692) and faecal (n = 141) samples were collected from 269 lambs in their first 3 weeks of life. The ewes' colostrum (n = 181) SAA concentrations were positively associated with the lambs' serum SAA and Hp concentrations at 2 to 4 days of age. Hp and ALB concentrations at the second week of age were positively associated with the growth rate at 122 days of age. Lamb serum globulin (GLOB) concentrations and Cryptosporidium-positive faecal samples were negatively associated at the second and third weeks of life. These findings suggest the importance of interactions between the immune system and environmental factors at the second week of the lambs' lives and its association with future performance.

Use of live yeast and mannan-oligosaccharides in grain-based diets for cattle: Ruminal parameters, nutrient digestibility, and inflammatory response.

The objective of this study was to evaluate the effects of diet supplementation with live yeast (Saccharomyces cerevisiae) and mannan-oligosaccharides (MOS) on ruminal parameters, nutrient digestibility, and the inflammatory response in cattle fed grain-based diets. Three Holstein steers (body weight of 497±3 kg) with ruminal and duodenal cannulas were assigned to a 3 × 3 Latin square design. The animals were kept in individual pens and fed a diet containing 5% sugarcane bagasse and 95% concentrate (906.5 g/kg ground corn). Diet treatments were Control (without additive), Yeast (1.5 g/kg DM live yeast Saccharomyces cerevisiae, NCYC 996) and MOS (1.5 g/kg DM MOS, ß-glucans and mannan). Dry matter intake, ruminal, intestinal, and total digestibility of nutrients were not affected by the treatments. The ruminal concentration of isobutyric acid increased in animals fed on diets supplemented with Yeast and MOS, whereas isovaleric acid increased with Yeast and decreased with MOS supplementation. Dietary supplementation with Yeast and MOS increased pH and decreased ammonia concentration in the rumen. Lipopolysaccharide (LPS) concentrations in the rumen and duodenal fluid were not influenced by the additives. However, both Yeast and MOS decreased the plasma levels of LPS and serum amyloid A (SAA). In conclusion, when high-concentrate diets fed to beef cattle are supplemented with either Yeast or MOS, ruminal pH is increased, LPS translocation into the blood stream is decreased, and blood SAA concentration is decreased. These factors reduce the inflammation caused by consumption of grain-based diets, and either supplement could be used to improve the rumen environment in beef cattle susceptible to ruminal subacute acidosis.

Recombinant Epinephelus lanceolatus serum amyloid A as a feed additive: Effects on immune gene expression and resistance to Vibrio alginolyticus infection in Epinephelus lanceolatus.

Recombinant Epinephelus lanceolatus serum amyloid A (rElSAA) exhibits strong immunostimulant activity and enhances phagocytic clearance of bacteria by macrophages. However, the effects of dietary rElSAA supplementation on growth performance, immunomodulation and disease resistance in giant grouper have not been previously evaluated. To test whether oral administration of rElSAA affects growth, fish were fed with 0, 0.88, 4.4 or 22 mg/kg rElSAA-containing diet for 28 days. No statistically significant differences in body weight were observed between groups. Next, we tested whether oral administration of rElSAA may enhance disease resistance. Fish were fed with 0, 0.88, 4.4 or 22 mg/kg rElSAA-containing diet for 3, 7, 14, 21 or 28 days, followed by challenge with Vibrio alginolyticus. Survival was then monitored for 4 days. Fish that were fed with rElSAA-containing diet for 28 days showed significantly improved survival after infection. In addition, the expression levels of immune defense-associated genes in hepatic tissue were assessed by quantitative real-time polymerase chain reaction before and after V. alginolyticus infection. Oral administration of rElSAA increased the expression level of toll-like receptor 5, whereas the expression levels of CC chemokine 1, SAA and C reactive protein were decreased. Thus, the data suggest that rElSAA may enhance host immunity by attenuating regulatory T cell-mediated suppression of inflammation. Together, our results demonstrate that rElSAA is a promising candidate as a feed additive for giant grouper, which may effectively enhance disease resistance after being administered for several weeks.

Eucalyptus robusta leaves methanolic extract suppresses inflammatory mediators by specifically targeting TLR4/TLR9, MPO, COX2, iNOS and inflammatory cytokines in experimentally-induced endometritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Bacterial endometritis is one of the major causes of reproductive disorders including infertility in farm animals. Antibiotics are generally used for treatment of such disorders but now a days residues of antibiotics are of great public health concern, therefore, phytoremediation is being considered as an alternative to use of antibiotics. AIM OF THE STUDY: Present study was undertaken to investigate the efficacy of Eucalyptus robusta leaves methanolic extract against endometritis along with the possible mechanism of action especially targeting inflammatory biomarkers. MATERIALS AND METHODS: Bacterial endometritis was produced using clinical isolates of E. coli and Staphyloccocus aureus from bovines (cows and buffaloes) endometritis cases. After seven days of inoculation of the mixed bacterial culture, endometritis was confirmed based on the presence of visible pus and edema, thinning of endometrial lining and presence of large number of polymorphonuclear cells and bacterial load in uterine flushing. Female Wistar rats were divided in to five groups namely control, sham-operated, endometritis, endometritis plus Eucalyptus leaves extract and endometritis plus cefixime. Serum specific inflammatory biomarkers (interleukin-1ß, interleukin-10, tumor necrosis factor-α, intercellular adhesion molecule-1, serum amyloid A) and myleoperoxidase, toll like receptors-4 and -9, inducible nitric oxide synthase, nitric oxide, cyclooxygenase 1 and 2 were estimated in uterine tissues using ELISA kits. RESULTS: Interleukin-10, serum amyloid A, myleoperoxidase, toll like receptors-4 and-9, cyclooxygenase-2, inducible nitric oxide synthase and nitric oxide were significantly increased while non significant increase in interleukin-1ß, cycloxygenase-1 and intercellular adhesion molecule-1 were observed but level of tumor necrosis factor-α was found decreased in rats of endometritis group. Histopathological lesions in uterus showed efficient induction of endometritis by presence of inflammatory cells which are lessened effectively after treatment with Eucalyptus leaves extract. Eucalyptus robusta leaves extract produced curative and protective effect against endometritis and results were comparable to or even better than cefixime. CONCLUSIONS: Eucalyptus robusta leaves extract possess promising antibacterial activity and efficacy against experimental endometritis and, therefore, holds promising potential for development of effective formulation for treatment of endometritis in animals.

Short-Term Postoperative Cognitive Dysfunction and Inflammatory Response in Patients Undergoing Cytoreductive Surgery and Hyperthermic Intraperitoneal Chemotherapy: A Pilot Study.

OBJECTIVES: To assess the association between short-term postoperative cognitive dysfuction (POCD) and inflammtory response in patients undergoing cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). DESIGN: A prospective cohort study. SETTING: University medical centre. PARTICIPANTS: Fifty-one adult patients who had undergone CRS-HIPEC and twenty control participants. MEASUREMENTS: The inflammatory marker levels in plasma and cognitive function were measured. RESULTS: Twenty (39.2%, 20/51) patients developed POCD at 1 w after CRS-HIPEC. The patients with POCD had higher serum interleukin 1ß (IL-1ß), serum amyloid A (SAA), S100 calcium-binding protein ß (S-100ß), and high mobility group box-1 protein (HMGB-1) levels at 1 and 24 h postoperatively than patients without POCD. There was an association between POCD and the maximum IL-1ß and S-100ß concentrations in serum, which remained following adjustment for age and FBS. CONCLUSION: In this pilot study, perioperative inflammatory marker levels increase significantly after CRS-HIPEC in adult patients, and such elevations are associated with the development of short-term cognitive dysfunction after this complex surgery. These results suggested the need for a larger RCT to replicate and confirm these findings.

Role of myeloperoxidase in abdominal aortic aneurysm formation: mitigation by taurine.

Oxidative stress plays a fundamental role in abdominal aortic aneurysm (AAA) formation. Activated polymorphonuclear leukocytes (or neutrophils) are associated with AAA and express myeloperoxidase (MPO), which promotes inflammation, matrix degradation, and other pathological features of AAA, including enhanced oxidative stress through generation of reactive oxygen species. Both plasma and aortic MPO levels are elevated in patients with AAA, but the role of MPO in AAA pathogenesis has, heretofore, never been investigated. Here, we show that MPO gene deletion attenuates AAA formation in two animal models: ANG II infusion in apolipoprotein E-deficient mice and elastase perfusion in C57BL/6 mice. Oral administration of taurine [1% or 4% (wt/vol) in drinking water], an amino acid known to react rapidly with MPO-generated oxidants like hypochlorous acid, also prevented AAA formation in the ANG II and elastase models as well as the CaCl2 application model of AAA formation while reducing aortic peroxidase activity and aortic protein-bound dityrosine levels, an oxidative cross link formed by MPO. Both MPO gene deletion and taurine supplementation blunted aortic macrophage accumulation, elastin fragmentation, and matrix metalloproteinase activation, key features of AAA pathogenesis. Moreover, MPO gene deletion and taurine administration significantly attenuated the induction of serum amyloid A, which promotes ANG II-induced AAAs. These data implicate MPO in AAA pathogenesis and suggest that studies exploring whether taurine can serve as a potential therapeutic for the prevention or treatment of AAA in patients merit consideration.NEW & NOTEWORTHY Neutrophils are abundant in abdominal aortic aneurysm (AAA), and myeloperoxidase (MPO), prominently expressed in neutrophils, is associated with AAA in humans. This study demonstrates that MPO gene deletion or supplementation with the natural product taurine, which can scavenge MPO-generated oxidants, can prevent AAA formation, suggesting an attractive potential therapeutic strategy for AAA.

Immunohistochemical Profiling of Corneas With Fuchs Endothelial Corneal Dystrophy.

PURPOSE: Fuchs endothelial corneal dystrophy (FECD) is the leading indication for endothelial keratoplasty. Further insight into its pathophysiology is needed to develop alternative therapies. METHODS: Sixteen genes from a previous microarray expression experiment (FECD vs. normal) were validated using immunohistochemistry on paraffin-embedded corneas (n = 6 FECD, n = 6 normal). The results were quantified manually and semiautomatically. RESULTS: A higher percentage of corneal endothelial cells stained for alpha-smooth muscle actin (αSMA), cytokeratin 7, and superoxide dismutase 3 in FECD versus normal [odds ratios (ORs) of 60.90, 41.70, and 15.16, respectively, P < 0.001]. Dot-like staining for major histocompatibility complex, class II, DR alpha was present in FECD, but not in normal. Higher percentages of stromal cells in FECD versus normal stained for αSMA (OR = 864.26, P < 0.001), brain-derived neurotrophic factor (BDNF, OR = 6.34, P = 0.005), fibroblast growth factor 7 (FGF-7, OR = 2.76, P = 0.011), FGF-9 (OR = 5.97, P < 0.001), receptor FGFR-3 (OR = 13.90, P = < 0.001), and serum amyloid A1 (OR = 3.45, P = 0.023). Higher percentages of corneal epithelial cells stained for αSMA (OR = 2.20, P = 0.006) and BDNF (OR = 3.94, P < 0.001) in FECD versus normal. CONCLUSIONS: These results support a role for epithelial-mesenchymal transition (αSMA), oxidative stress (superoxide dismutase 3), and major histocompatibility complex, class II, DR alpha cells with dendritic morphology in the pathophysiology of FECD. Furthermore, corneal stromal cells express trophic molecules (BDNF and FGFs) and markers of chronic inflammation (serum amyloid A1) in FECD.

Effects of 4-Pyridone-3-carboxamide-1ß-D-ribonucleoside on adenine nucleotide catabolism in the aortic wall; Implications for atherosclerosis in ApoE-/-LDLR-/- mice.

4-Pyridone-3-carboxamide-1-beta-D-ribonucleoside (4PYR) is an endogenously produced nucleoside that had been identified as a substrate for intracellular phosphorylation to form intracellular nucleotides. Previous studies demonstrated that 4PYR adversely affects metabolism of endothelial cells that is known risk factor for atherosclerosis. The purpose of this study was to evaluate effects of 4PYR on the progression of atherosclerosis and changes in extracellular nucleotides degradation on the surface of the vessel wall in the murine model. METHODS: Two month old ApoE-/-LDLR-/- mice were subcutaneously injected with 4PYR (4P) twice per day for one month or with saline in controls (C). Then, at the age of eight month hydrolysis rates of ATP, AMP and adenosine were evaluated in the intact aorta sections by HPLC based assays. Oil Red O (ORO) staining that indicates lipid deposition was quantified spectrophotometrically after extraction from the vessel. Serum amyloid A (SAA) content was analyzed with ELISA. RESULTS: Adenosine deamination rate (activity of eADA) increased from 8.7±1.4 nmol/min/cm2 in C to 16.0±2.6 nmol/min/cm2 in 4P (p<0.05). AMP dephosphorylation rate (activity of e5NT) and ATP hydrolysis rate (activity of eNTPD) were not different between C and 4P. ORO staining in the aorta of 4P mice increased by 75% as compared to C (p<0.01) while SAA content was similar in both groups. CONCLUSIONS: This data demonstrated that prolonged exposure to 4PYR of ApoE-/-LDLR-/- mice results in sustained elevation of vascular eADA activity and increased ORO staining indicating endothelial impairment and accelerated atherosclerosis.

Resolvin E1 attenuates atherosclerosis in absence of cholesterol-lowering effects and on top of atorvastatin.

BACKGROUND AND AIMS: Besides LDL-cholesterol, local vascular inflammation plays a key role in atherogenesis. Efficient therapies to treat the inflammatory component of the disease have not been established. The discovery of specialized inflammation-resolving mediators, such as resolvins may provide new opportunities for treatment. This study examines whether the ω-3 fatty acid eicosapentaenoic acid-derived resolvin E1 (RvE1), can reduce atherosclerosis, when administered alone or in combination with a cholesterol-lowering statin. METHODS: ApoE*3Leiden mice were fed a hypercholesterolemic diet for 9 weeks and subsequently treated with RvE1-low (1 mg/kg/day), RvE1-high (5 mg/kg/day), atorvastatin (1.5 mg/kg/day) or the combination of atorvastatin and RvE1-low for the following 16 weeks. RESULTS: RvE1-low and RvE1-high reduced atherosclerotic lesion size to the same extent (-35%; p < 0.05), attenuated the formation of severe lesions, also seen as a proportional increase in the presence of mild lesions, but did not alter plasma cholesterol levels. Cholesterol-lowering atorvastatin reduced atherosclerosis (-27%, p < 0.05), and the combination of RvE1 and atorvastatin further attenuated lesion size (-51%, p < 0.01) and increased the content of mild lesions. RvE1 did not affect plasma SAA, E-selectin, VCAM-1 or MCP-1 but did reduce plasma EPHX4 and down-regulated the local expression of pro-atherogenic genes in the aortae, (e.g. Cd74, Cd44, Ccl2, Ccr5 and Adam17) and significantly inactivated IFN-γ (p < 0.001) and TNF-α (p < 0.001) signalling pathways. CONCLUSIONS: RvE1 attenuates atherogenesis both alone and on top of a statin. The local effects of RvE1 are demonstrated by the modulated aortic expression of genes involved in inflammatory and immune responses, without altering plasma cholesterol or circulating SAA.

Thymol and Carvacrol Affect Hybrid Tilapia through the Combination of Direct Stimulation and an Intestinal Microbiota-Mediated Effect: Insights from a Germ-Free Zebrafish Model.

BACKGROUND: Essential oils (EOs) are commonly used as animal feed additives. Information is lacking on the mechanisms driving the beneficial effects of EOs in animals, especially the role played by the intestinal microbiota of the host. OBJECTIVE: The purpose of this study was to clarify the relative contribution of direct effects of EOs on the physiology and immune system of tilapia and indirect effects mediated by the intestinal microbiota by using a germ-free zebrafish model. METHODS: Juvenile hybrid tilapia were fed a control diet or 1 of 4 treatment diets containing 60-800 mg Next Enhance 150 (NE) (an EO product containing equal levels of thymol and carvacrol)/kg for 6 wk. The key humoral and cellular innate immune parameters were evaluated after the feeding period. In another experiment, the gut microbiota of tilapia fed a control or an NE diet (200 mg/kg) for 2 wk were transferred to 3-d postfertilization (dpf) germ-free (GF) zebrafish, and the expression of genes involved in innate immunity and tight junctions was evaluated in zebrafish at 6 dpf. Lastly, NE was directly applied to 3-dpf GF zebrafish at 3 doses ranging from 0.2 to 20 mg/L, and the direct effect of NE on zebrafish was evaluated after 1 and 3 d. RESULTS: NE supplementation at 200 mg/kg enhanced phagocytosis activity of head kidney macrophages (×1.36) (P < 0.05) and plasma lysozyme activity (×1.69) of tilapia compared with the control (P < 0.001), indicating an immunostimulatory effect. Compared with those colonized with control microbiota, GF zebrafish colonized with NE microbiota showed attenuated induction of immune response marker genes serum amyloid a (Saa; ×0.62), interleukin 1ß (Il1ß; ×0.29), and interleukin 8 (Il8; ×0.62) (P < 0.05). NE treatment of GF zebrafish at 2 and 20 mg/L for 1 d upregulated the expression of Il1ß (×2.44) and Claudin1 (×1.38), respectively (P < 0.05), whereas at day 3 the expression of Occludin2 was higher (×3.30) in the 0.2-mg NE/L group compared with the GF control (P < 0.05). CONCLUSION: NE may affect the immunity of tilapia through a combination of factors, i.e., primarily through a direct effect on host tissue (immune-stimulating) but also an indirect effect mediated by microbial changes (immune-relieving).

Effects of the Dietary ω3:ω6 Fatty Acid Ratio on Body Fat and Inflammation in Zebrafish (Danio rerio).

The diets of populations in industrialized nations have shifted to dramatically increased consumption of ω6 polyunsaturated fatty acids (PUFA), with a corresponding decrease in the consumption of ω3 PUFA. This dietary shift may be related to observed increases in obesity, chronic inflammation, and comorbidities in the human population. We examined the effects of ω3:ω6 fatty acid ratios in the context of constant total dietary lipid on the growth, total body fat, and responses of key inflammatory markers in adult zebrafish (Danio rerio). Zebrafish were fed diets in which the ω3:ω6 PUFA ratios were representative of those in a purported ancestral diet (1:2) and more contemporary Western diets (1:5 and 1:8). After 5 mo, weight gain (fat free mass) of zebrafish was highest for those that received the 1:8 ratio treatment, but total body fat was lowest at this ratio. Measured by quantitative real-time RT-PCR, mRNA levels from liver samples of 3 chronic inflammatory response genes (C-reactive protein, serum amyloid A, and vitellogenin) were lowest at the 1:8 ratio. These data provide evidence of the ability to alter zebrafish growth and body composition through the quality of dietary lipid and support the application of this model to investigations of human health and disease related to fat metabolism.

The evaluation of inflammatory and oxidative stress biomarkers on coffee-diabetes association: results from the 10-year follow-up of the ATTICA Study (2002-2012).

BACKGROUND/OBJECTIVES: The purpose of this work was to investigate the association between coffee drinking and diabetes development and potential mediation by oxidative stress and inflammatory biomarkers. SUBJECTS/METHODS: In 2001-2002, a random sample of 1514 men (18-87 years old) and 1528 women (18-89 years old) were selected to participate in the ATTICA study (Athens metropolitan area, Greece). A validated food-frequency questionnaire was used to assess coffee drinking (abstention, casual, habitual) and other lifestyle and dietary factors. Evaluation of oxidative stress and inflammatory markers was also performed. During 2011-2012, the 10-year follow-up of the ATTICA study was carried out. The outcome of interest in this work was incidence of type 2 diabetes, defined according to American Diabetes Association criteria. RESULTS: During follow-up, 191 incident cases of diabetes were documented (incidence 13.4% in men and 12.4% in women). After various adjustments, individuals who consumed ⩾250 ml of coffee (≈1.5cup) had 54% lower odds of developing diabetes (95% confidence interval: 0.24, 0.90), as compared with abstainers. A dose-response linear trend between coffee drinking and diabetes incidence was also observed (P for trend=0.017). When controlling for several oxidative stress and inflammatory biomarkers, the inverse association between habitual coffee drinking and diabetes was found to be mediated by serum amyloid-A levels. CONCLUSIONS: This work highlights the significance of long-term habitual coffee drinking against diabetes onset. The anti-inflammatory effect of several coffee components may be responsible for this protection.

Acute perioperative-stress-induced increase of atherosclerotic plaque volume and vulnerability to rupture in apolipoprotein-E-deficient mice is amenable to statin treatment and IL-6 inhibition.

Myocardial infarction and stroke are frequent after surgical procedures and consume a considerable amount of benefit of surgical therapy. Perioperative stress, induced by surgery, is composed of hemodynamic and inflammatory reactions. The effects of perioperative stress on atherosclerotic plaques are ill-defined. Murine models to investigate the influence of perioperative stress on plaque stability and rupture are not available. We developed a model to investigate the influence of perioperative stress on plaque growth and stability by exposing apolipoprotein-E-deficient mice, fed a high cholesterol diet for 7 weeks, to a double hit consisting of 30 min of laparotomy combined with a substantial blood loss (approximately 20% of total blood volume; 400 µl). The innominate artery was harvested 72 h after the intervention. Control groups were sham and baseline controls. Interleukin-6 (IL-6) and serum amyloid A (SAA) plasma levels were determined. Plaque load, vascular smooth muscle cell (VSMC) and macrophage content were quantified. Plaque stability was assessed using the Stary score and frequency of signs of plaque rupture were assessed. High-dose atorvastatin (80 mg/kg body weight/day) was administered for 6 days starting 3 days prior to the double hit. A single dose of an IL-6-neutralizing antibody or the fusion protein gp130-Fc selectively targeting IL-6 trans-signaling was subcutaneously injected. IL-6 plasma levels increased, peaking at 6 h after the intervention. SAA levels peaked at 24 h (n=4, P<0.01). Plaque volume increased significantly with the double hit compared to sham (n=8, P<0.01). More plaques were scored as complex or bearing signs of rupture after the double hit compared to sham (n=5-8, P<0.05). Relative VSMC and macrophage content remained unchanged. IL-6-inhibition or atorvastatin, but not blocking of IL-6 trans-signaling, significantly decreased plaque volume and complexity (n=8, P<0.01). Using this model, researchers will be able to further investigate the pathophysiology of perioperative plaque stability, which can result in myocardial infarction, and, additionally, to test potential protective strategies.