Expression of IL-32 modulates NF-κB and p38 MAP kinase pathways in human esophageal cancer

BackgroundEsophageal cancer is the seventh leading cause of cancer death in males in USA, and there is a strong link has been demonstrated between inflammation and esophageal cancer, interleukin (IL)-32 is a recently described pro-inflammatory cytokine characterized by the induction of nuclear factor NF-κB activation, the p38MAPK also plays an important role in key cellular processes related to inflammation and cancer. We investigated whether the IL-32 expression may be involved in esophageal carcinogenesis through modulates the activity of NF-κB and p-p38 MAPK.MethodMalignant esophageal tissue and blood samples were obtained from 65 operated untreated patients, normal samples was obtained from 35 patients operated for other reasons as control. IL-32 expression visualized by immunohistochemistry, Real time RT–PCR for IL-32 mRNA expression, NF-κB phosphorylation and phosphorylated p38mapk were analyzed by immunoblotting, ELISA for further detection IL-32 and cytokines (TNF-α, IL-1β, IL-6 and IL-8) concentration in the patient’s sera.ResultsIL-32 expression was increased in immunohistochemical staining for malignant esophageal tissue and it’s correlated with the relative expression level of IL-32 mRNA P = 0.007, the P-NF-κB level elevated in tumor tissue compared with control and no difference in the total NF-κB level P = 0.003 while the IL-32 up-regulated the P-pNF-κB in the esophageal tumor P = 0.005. There is increase in p-p38MAPK activation underlying IL-32 expression in tumor P = 0.004, but no change in total p38 MAPK in malignant esophagus. The plasma level of IL-32 expression was increased in malignant esophageal patients P = 0.01, with increased in the levels of the cytokines TNF-α, IL-6, and IL-1β P<0.05.ConclusionsUnderstanding the pathway of IL-32 expression to stimulate the secretion cytokines via the activation of NF-κB and up-regulation of p-p38MAPK may or may not prove to be a therapeutic target, or a biomarker, and future studies will finally answer this hypothesis generated.     

Transforming growth factor-β and Th17 responses in resistance to primary murine schistosomiasis mansoni

Discovery of the T-helper (Th) 17 cell lineage and functions in immune responses of mouse and man prompted us to investigate the role of transforming growth factor-beta (TGF-β) and interleukin (IL)-17 in innate resistance to murine schistosomiasis mansoni. Schistosoma mansoni-infected BALB/c and C57BL/6 mice were administered with recombinant TGF-β or mouse monoclonal antibody to TGF-β to evaluate the impact of this cytokine on host immune responses against lung-stage schistosomula, and subsequent effects on adult worm parameters. Developing schistosomula elicited increase in peripheral blood mononuclear cells (PBMC) mRNA expression and/or plasma levels of IL-4, IL-17, and interferon-gamma (IFN-γ), cytokines known to antagonize each other, resulting in impaired Th1/Th2, and Th17 immune responses and parasite evasion. Mice treated with TGF-β showed elevated PBMC mRNA expression of IL-6, IL-17, TGF-β, and TNF-α mRNA and increased IL-23 and IL-17 or TGF-β plasma levels, associated with significantly (P < 0.02–<0.0001) lower S. mansoni adult worm burden compared to controls in both mouse strains, thus suggesting that TGF-β led to heightened Th17 responses that mediated resistance to the infection. Mice treated with antibody to TGF-β showed increase in PBMC mRNA expression and plasma levels of IL-4, IL-12p70, and IFN-γ, and significantly (P < 0.02 and <0.0001) reduced worm burden and liver worm egg counts than untreated mice, indicating that Th1/Th2 immune responses were potentiated, resulting in significant innate resistance to schistosomiasis. The implications of these observations for schistosome immune evasion and vaccination were discussed.     

Severe preeclampsia: Association of genes polymorphisms and maternal cytokines production in Brazilian population

IntroductionPreeclampsia (PE) is a multi-system disorder of pregnancy characterized by hypertension and proteinuria. Healthy pregnancy is associated with a controlled inflammatory process, which is exacerbated in PE in response to excessive placental stimuli. Gene expression levels can affect inflammation and immune regulation. It is known that differences in cytokine allele frequencies amongst populations may contribute to difference in the incidence of several diseases.ObjectiveThe aim of this study was to investigate the frequency of TNF-α, IL-6, IFN-γ and IL-10 genes polymorphisms and their relationship with the cytokines plasma levels in PE.MethodsA total of 281 women were included in this study; 116 with severe PE, 107 normotensive pregnant and 58 non-pregnant women. Cytokine genotyping was carried out by the polymerase chain reaction. The analyzed polymorphisms were: TNF-α (−308 G → A), IL-10 (−1082 G → A), IL-6 (−174 G → C), and IFN-γ (+874 A → T). Cytokine plasma levels were measured by Cytometric Bead Array method.ResultsA higher frequency of the IFN-γ (+874) T/T genotype in severe PE comparing to normotensive pregnant women was found (P < 0.001). TNF-α, IL-6 and IFN-γ plasma levels were higher in PE women compared to non-pregnant women (P < 0.001; P < 0.001; P = 0.004). IL-6 and IFN-γ levels were also higher in PE women compared to normotensive pregnant (P < 0.001; P = 0.010). IL-10 levels were higher in normotensive pregnant women compared to PE (P < 0.001). IFN-γ and IL-6 genes polymorphisms influenced the genic expression in PE and normotensive pregnant women, respectively.ConclusionsThese results suggest that IFN-γ seems to play a role in PE occurrence.     

The relationship between interleukin-6 in saliva,venous and capillary plasma,at rest and in response to exercise

IL-6 plays a mechanistic role in conditions such as metabolic syndrome, chronic fatigue syndrome and clinical depression and also plays a major role in inflammatory and immune responses to exercise. The purpose of this study was to investigate the levels of resting and post exercise IL-6 when measured in venous plasma, saliva and capillary plasma. Five male and five females completed 2 separate exercise trials, both of which involved standardized exercise sessions on a cycle ergometer. Venous blood and saliva samples were taken immediately before and after Trial A, venous and capillary blood samples were taken immediately before and after Trial B. IL-6 values were obtained using a high-sensitivity enzyme-linked immunosorbent assay (ELISA). In Trial A venous plasma IL-6 increased significantly from 0.4 ± 0.14 pg/ml to 0.99 ± 0.29 pg/ml (P < 0.01) while there was no increase in salivary IL-6. Venous plasma and salivary IL-6 responses were not correlated at rest, post exercise or when expressed as an exercise induced change. In Trial B venous and capillary plasma IL-6 increased significantly (venous: 0.22 ± 0.18 to 0.74 ± 0.28 pg/ml (P ⩽ 0.01); capillary: 0.37 ± 0.22 to 1.08 ± 0.30 pg/ml (P < 0.01). Venous and capillary plasma responses did not correlate at rest (r = 0.59, P = 0.07) but did correlate post exercise (r = 0.79, P ⩾ 0.001) and when expressed as an exercise induced change (r = 0.71, P = 0.02). Saliva does not appear to reflect systemic IL-6 responses, either at rest or in response to exercise. Conversely, capillary plasma responses are reflective of systemic IL-6 responses to exercise.     

Wide-spectrum profile of inflammatory mediators in the plasma and scales of patients with psoriatic disease

Psoriasis is a chronic recurrent inflammatory disorder of the skin. Clinical subtypes include psoriasis vulgaris (PV), psoriatic arthropathy, and erythrodermic psoriasis. Aim of this study was to analyse relevant inflammatory mediators in the plasma of patients with distinct subtypes of active psoriasis, and in the scales of mild-to-moderate PV patients, and correlation to disease severity. Compared to healthy controls (n = 10), patients affected by very severe forms of psoriasis (n = 30) were characterized by increased plasma levels of IL-4, IL-6, MCP-1, VEGF and in particular PDGFbb. Each group with severe psoriasis had distinct characteristic features of plasma cytokine profile. Mild-to-moderate PV patients (n = 35) showed higher levels of IL-4, IL-6, IL-10, and IL-13 when compared to healthy controls. No correlation was found between PV severity assessed by PASI (Psoriasis Area and Severity Index) and levels of these mediators. By contrast, disease severity correlated to scale levels of IP-10. For the first time, we found exaggerated circulating levels of the pro-angiogenic PDGFbb and VEGF in severe psoriasis. Evidence that the severity of skin symptoms correlated exclusively with scale levels of IP-10, but not with any up-regulated inflammatory mediator in plasma, suggests that distinct skin-independent processes contribute to the circulating cytokine profile in psoriasis.     

Distribution and interspecific correlation of root biomass density in an arid Elaeagnus angustifolia–Achnatherum splendens community

Main objective of this study was to determine the interspecific relationships between two dominant species in terms of root distribution in a typical arid tree-herbage (Elaeagnus angustifolia–Achnatherum splendens) community at Xidatan, Pingluo County, Ningxia Autonomous Region, Northwest China. Eight concentric zones (namely, Z1–Z8) were set from the bases of E. angustifolia individuals to the open lands and five soil profiles were excavated in each zone. Each soil profile was divided into five layers at the depths of 0–10 cm, 10–30 cm, 30–60 cm, 60–100 cm and 100–150 cm. Roots were collected for each species, and soil water content (SWC) and soil bulk density (SBD) were measured for each layer. We found noteworthy roots layer separation in the sub-canopy zones (Z1–Z4). The soil layers with highest fine root biomass density (FRBD) of A. splendens was primarily in the 0–10 cm, which were significantly shallower than those of E. angustifolia; whereas in the inter-canopy zones (Z5–Z8), inconsistent separation, or even overlapping of highest-FRBD-layers emerged between the two dominant species. Correlation analyses showed that negative correlations of FRBD between the two species mainly occurred in those soil layers with relatively higher FRBD and lower SWC. In contrast, positive correlations corresponded with relatively lower FRBD and higher SWC.     

The roles of aerobic exercise training and suppression IL-6 gene expression by RNA interference in the development of insulin resistance

ObjectiveTo demonstrate the hypothesis that aerobic exercise training inhibits the development of insulin resistance through IL-6 and probe into the possible molecular mechanism about it.MethodsRats were raised with high-fat diets for 8 weeks to develop insulin resistance, and glucose infusion rates (GIRs) were determined by hyperinsulinemic–euglycemic clamping to confirm the development of insulin resistance. Aerobic exercise training (the speed and duration time in the first week were respectively 16 m/min and 50 min, and speed increased 1 m/min and duration time increased 5 min every week following it) and/or IL-6shRNA plasmid injection (rats received IL-6shRNA injection via the tail vein every two weeks) were adopted during the development of insulin resistance. The serum IL-6, leptin, adiponectin, fasting blood glucose, fasting serum insulin, GIR, IL-6 gene expression levels, p-p38 in various tissues and p-STAT3/t-STAT3 ratio in the liver were measured.ResultsRats fed with high-fat diets for 8 weeks were developed insulin resistance and the IL-6mRNA levels of IL-6shRNA injection groups in various tissues were significantly lower than those of control group (P < 0.05), respectively. The development of insulin resistance in exercise rats significantly decreased, however, compared with that, the GIR of exercise rats injected by IL-6shRNA was lower (P < 0.05). The IL-6mRNA levels were highest in the fat tissue and lowest in the skeletal muscles in all the rats. The serum adiponectin levels decreased (P < 0.05) following the development of insulin resistance, and it increased (P < 0.05) when the rats were intervened by aerobic exercise training for 8 weeks at the same time. However, there were not significant differences when serum leptin concentrations were compared (P > 0.05). The p-p38 significantly increased in the rats fed with high-fat diets, however, p-p38 of the exercise high-fat diets rats in the liver and fat tissues significantly decreased than that (P < 0.05). The changes of p-p38 in exercise rats injected by IL-6shRNA were irregular. The activation of STAT3 in the liver significantly increased (P < 0.05) following the development of insulin resistance, and it decreased (P < 0.05) when the rats were intervened by aerobic exercise training for 8 weeks at the same time, and the gene silencing of IL-6 did not have effects on the activation of STAT3 in the liver (P > 0.05).ConclusionsIn conclusion, aerobic exercise training prevented the development of insulin resistance through IL-6 to a certain degree. The gene expression and secretion of IL-6 could inhibit the development of insulin resistance. The mechanism of the effects were possibly related with elevating the levels of serum adiponectin, and/or inhibiting the activation of STAT3 in the liver and p38MAPK in the skeletal muscles, liver and fat tissues.     

Th1/Th2/Th17/Treg cytokine imbalance in systemic lupus erythematosus (SLE) patients: Correlation with disease activity

AimImbalance of T-helper-cell (TH) subsets (TH1/TH2/TH17) and regulatory T-cells (Tregs) is suggested to contribute to the pathogenesis of Systemic lupus erythematosus (SLE). Therefore, we evaluated their cytokine secretion profile in SLE patients and their possible association with disease activity.MethodsSixty SLE patients, 24 rheumatoid arthritis (RA) patients and 24 healthy volunteers were included in this study. Demographic, clinical, disease activity and serological data were prospectively assessed. Plasma cytokines levels of TH1 (IL-12, IFN-γ), TH2 (IL-4, IL-6, IL-10), TH17 (IL-17, IL-23) and Treg (IL-10 and TGF-β) were measured by enzyme linked immunosorbent assays (ELISA).ResultsSLE patients were found to have significantly higher levels of IL-17 (p < 0.001), IL-6 (p < 0.01), IL-12 (p < 0.001) and IL-10 (p < 0.05) but comparable levels of IL-23 and IL-4 and slight reduction (but statistically insignificant) of TGF-β levels compared to controls. IL-6, IL-10 and IL-17 were significantly increased (p < 0.05) with disease activity. The RA group exhibited significantly higher levels of plasma IL-4 (p < 0.01), IL-6 (p < 0.05), IL-17 (p < 0.001), IL-23 (p < 0.01) and TGF-β (p < 0.5) and lower IFN-γ (p < 0.001) and IL-10 (p < 0.01) than those of healthy subjects.ConclusionOur study showed a distinct profile of cytokine imbalance in SLE patients. Reduction in IFN-γ (TH1) and TGF-β1 (Treg) with the elevation in IL-6 and IL-17 (TH17) could imply skewing of T-cells toward TH17 cells. Breaking TH17/Treg balance in peripheral blood may play an important role in the development of SLE and could be responsible for an increased pro-inflammatory response especially in the active form of the disease.     

The −251A>T polymorphism of interleukin-8 is associated with longer mechanical ventilation and hospital staying after coronary surgery

Background: Cardiac surgery is associated with inflammatory responses that are known to affect its outcome. The present study was designed to define whether post-operative release of interleukin (IL)-6, 8 and tumor necrosis factor-alpha (TNF-α) is related to the presence of a certain allele in functional polymorphism and its relationship to clinical outcome after off-pump coronary artery bypass (OPCAB). Methods: One hundred and forty-five patients undergoing first time elective OPCAB were genotyped for IL-6(−174G>C), IL-8(−251A>T) and TNF-α(−308G>A) polymorphisms using polymerase chain reaction (PCR) and gene sequencing. Cytokine levels were measured in plasma samples taken before the operation and 4, 24 and 72 h postoperatively by suspension array system. Results: Levels of IL-6 and IL-8 increased significantly after OPCAB. Patients with IL-6−174GG and IL-8−251AA genotypes had higher post-operative circulating levels of IL-6 and IL-8, respectively. Logistic regression showed that IL-8−251AA genotype was an independent risk factor of ventilation time more than 1 day (OR = 11.80, 95% CI: 1.87–74.48) and hospital staying more than 14 days (OR = 38.00, 95% CI: 4.15–347.87) after surgery. Conclusions: OPCAB results in post-operative inflammatory responses. Genetic backgrounds alter the extent of inflammatory response and might relate to clinical outcome of OPCAB.     

Potential of Typha angustifolia for phytoremediation of heavy metals from aqueous solution of phenol and melanoidin

Typha angustifolia was evaluated for various heavy metals (Cu, Pb, Ni, Fe, Mn, and Zn) bioremediation potential from aqueous solution containing variable concentrations of phenol (100–800 mg l^?1) and melanoidin (2500–8500 Co–Pt) at 20, 40, and 60 days. The concentration of phenol (200–400 mg l^?1) along with melanoidin 2500 Co–Pt showed optimum for phytoremediation of tested heavy metals, while, higher concentrations of melanoidin (5600–8500 Co–Pt) showed toxic effect on T. angustifolia along with phenol. Phenol and melanoidin showed adverse effect on T. angustifolia of up to 20 days incubation, but this leads to induction of peroxidase and ascorbic acid activity to cope with adverse conditions. Subsequently, as pollutants were decreased along with plant growth, peroxidase and ascorbic acid also declined. However, with reduction of peroxidase, catalase level was increased. The Cu, Zn, and Ni were accumulated at maximum in all tested conditions. The TEM observations of T. angustifolia showed clotted deposition of metals and shrinkage of cell in root, breakdown of spongy and palisade parenchyma of leaves at higher concentration of phenol (100 mg l^?1) and melanoidin (5500 Co–Pt). Thus, this study concluded that T. angustifolia could be a potential phytoremediator for heavy metals from metal, melanoidin, and phenol containing industrial wastewater at optimized condition.     

Vocal fold fibroblasts immunoregulate activated macrophage phenotype

Recent evidence suggests that fibroblasts play a critical role in regulating inflammation during wound healing because they express several inflammatory mediators in response to bacteria. The objective of this study was to analyze the effects of lipopolysaccharide (LPS) on the immunomodulatory properties of vocal fold fibroblasts (VFFs) derived from polyps, scar and normal tissue co-cultured with macrophages, to provide insight into their interactions during the inflammatory process. Fibroblasts were co-cultured with CD14+ monocytes and after 7 days, wells were treated with LPS for 24 and 72 h. Culture supernatants were collected and concentrations of TNF-α, IL-6, IL-8, IL-10, IL-12, IL-1β and MCP-1 were quantified by ELISA. Normal VFF and CD14+ monocultures were used as controls. Twenty-four hours after LPS activation, macrophages co-cultured with polyp VFF had significantly increased expression of TNF-α, IL-1β, IL-12 and IL-10 compared to controls (p < 0.0001). In contrast, macrophages co-cultured with scar VFF had significantly lower expression of TNF-α, IL-1β and IL-12 with significantly higher IL-10 compared to control (p < 0.0001). After 72 h, macrophages co-cultured with polyp VFF increased expression of TNF-α, IL-1β, IL-10, IL-6, IL-8, MCP-1 and TGF-β (p < 0.01) and macrophages co-cultured with scar VFF significantly decreased their expression of IL-1β and IL-12 compared to control (p < 0.0001). Scar VFF at both time points produced significantly lower levels of IL-8, MCP-1, IL-6 and TGF-β compared to controls (p < 0.05). Based on our findings, VFF and macrophages secrete several inflammatory mediators that modify their diverse functions. Polyp and scar VFF may play a role in regulating abnormal inflammatory responses, which could result in excessive ECM deposition that disrupts the function of the vocal folds.     

Circulating IL-6 concentrations and associated anthropometric and metabolic parameters in South Asian men and women in comparison to European whites

ObjectiveTo determine any ethnic differences in circulating interleukin (IL)-6 concentrations among SAs and Europeans, and to assess their relationship with body composition and insulin resistance measures.MethodsBody composition was assessed among 80 SA and European men and women using anthropometry, dual-energy X-ray absorptiometry and abdominal CT scan. Oral glucose tolerance tests with insulin response were performed to assess insulin resistance measures. IL-6 levels were measured by high sensitivity ELISA.ResultsMedian IL-6 values were higher in SA compared with European women: 1.94 mg/l versus 1.51 mg/l, p = 0.041, but not so in men (1.56 mg/l versus 1.57 mg/l). Only measures of obesity, in particular percentage fat area (r = 0.6, p = 0.003), were positively correlated with IL-6 in SAs. Differences in body fat percentage (visceral and total) could explain up to 30% of the IL-6 difference between Asian and European women.ConclusionSA women have elevated circulating IL-6 levels, in part due to greater visceral and percent fat levels compared with European women. This observation may in part explain why Asians are at elevated cardiovascular disease risk. Future studies should address the effects of lifestyle factors (physical activity, diet) on plasma IL-6 concentrations in SA women.     

Insulin mimetic impact of Catechin isolated from Cassia fistula on the glucose oxidation and molecular mechanisms of glucose uptake on Streptozotocin-induced diabetic Wistar rats

Diabetes mellitus is the most common and serious metabolic disorder among people all over the world. Many plants have successfully been used to overcome this problem. Cassia fistula, an ethnomedicnal plant, is widely used in Indian medicine to treat diabetes. Methanol extract of stem of plant, reduced the blood glucose levels in Streptozotocin-induced diabetic rats. Bioassay guided fractionation was followed to isolate Catechin from methanol extract. Catechin was administered to Streptozotocin (60 mg/kg b.w.)-induced diabetic male Wistar rats at different doses (5, 10, 20 mg/kg b.w.) for 6 weeks to assess its effect on fasting plasma glucose. The plasma glucose was significantly (p<0.05) reduced when compared to the control. Oral administration of Catechin (20 mg/kg b.w.) markedly increased tissue glycogen, and ¹⁴C-glucose oxidation without any change in plasma insulin and C-peptide. Catechin restored the altered Glucokinase, glucose-6 Phosphatase, Glycogen Synthase and Glycogen Phosphorylase levels to near normal. GLUT4 mRNA and protein expression were enhanced after Catechin treatment. The results of this experimental study indicated that Catechin possesses hypo-glycemic, Glucose oxidizing and insulin mimetic activities and hence it could be used as a drug for treating diabetes.     

The hypoglycemic activity of Euclea undulata Thunb. var. myrtina (Ebenaceae) root bark evaluated in a streptozotocin–nicotinamide induced type 2 diabetes rat model

The hypoglycemic activity of a crude acetone extract of the root bark of Euclea undulata var. myrtina was evaluated in a streptozotocin–nicotinamide induced type 2 diabetes rat model after positive results were obtained by in vitro screening of glucose utilization by C2C12 myocytes, 3T3-L1 preadipocytes and Chang liver cells and alpha-glucosidase inhibition. Thirty male Wistar rats were used for the experiment. Type 2 diabetes was induced by a single intraperitoneal injection of streptozotocin and administration of nicotinamide 15 min after. Animals exhibiting fasting glucose levels of 140–200 mg/dl after 7 days were screened as type 2 diabetes. Extract was administered for 21 days orally at a concentration of 50 mg/kg and 100 mg/kg respectively. Glibenclamide (1 mg/kg) was used as positive control. On day 21, blood lipid profiles and body weight were determined by using standard enzymatic colorimetric kits before the rats were sacrificed by cervical decapitation. The crude acetone extract of E. undulata root bark at a concentration of 100 mg/kg body weight significantly lowered fasting blood glucose levels as well as elevated cholesterol and triglyceride levels to near normal without any weight gain. The results indicate that the crude acetone root bark extract of E. undulata exhibit antidiabetic activity in type 2 induced diabetic rats. It confirms the in vitro screening results as well as its use in the treatment of diabetes by traditional healers and herbalists in southern Africa.     

Madecassoside attenuates inflammatory response on collagen-induced arthritis in DBA/1 mice

Madecassoside (MA), a triterpenoid product isolated from Centella asiatica, has been described to exhibit antioxidant and anti-inflammatory activities. The present study was undertaken to determine whether madecassoside (MA) is efficacious against collagen-induced arthritis (CIA) in mice and its possible mechanisms. DBA/1J mice were immunized with bovine type II collagen and treated with MA (3, 10 and 30 mg/kg d, i.g.) from days 21 to 42 after immunization. Arthritis was evaluated by hind paw swelling, polyarthritis index, and histological examination. In vitro proliferation of spleen cells was examined using 3-[4,5-dimethylthylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT) assay. Plasma levels of cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10) and the expression of prostaglandin E₂ (PGE₂), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in synovial tissues were also determined. The results showed that comparing with untreated CIA mice, treated with MA dose-dependently suppressed the clinical arthritis score and joints tissues pathological damage, reduced the proliferation of spleen cells, plasma levels of TNF-α and IL-6, synovial tissues PGE₂ production and COX-2 protein expression, however, the expression of COX-1 in symovial tissues did not change and the plasma levels of IL-10 were increased. These results suggest that MA can effectively alleviate inflammatory response on CIA, and anti-inflammatory effects of MA can be attributed, at least partially, to the inhibition of pro-inflammatory mediators, including COX-2 expression, PGE₂ production, TNF-α and IL-6 levels and the up-regulation anti-inflammatory molecule IL-10.     

Interleukin-6 and its receptor in the development of in vitro fertilized ovine embryos

To study the effects of interleukin-6 (IL-6) and its receptor (IL-6R) at each stage in the early development of in vitro-fertilized (IVF) sheep embryos, the mRNA and protein expression levels of IL-6 and IL-6R were evaluated along with their localization, through real-time PCR, protein blotting, and immunohistochemistry. Specific patterns of IL-6 and IL-6R expression were recorded at both the mRNA and protein level at each stage of early ovine embryonic development. IL-6 was distributed primarily on the cytoplasm, however IL-6R was concentrated much more on the cell membrane. The role of IL-6 and IL-6R in the early ovine embryonic development was studied via the in vitro addition of recombinant human IL-6, at different concentrations and RNA interference. The addition of the recombinant IL-6 increased the embryonic IL-6 mRNA level, and the addition of 10 ng/mL IL-6 significantly increased the embryo cleavage rate (p < 0.05). IL-6 RNA interference effectively reduced the IL-6 protein level, but did not affect the rate of development of the IVF ovine embryos. This study provides a practical basis for further indepth studies on the mechanisms involved during early ovine embryonic development.     

Effect of Astragalus polysaccharides on expression of TNF-α, IL-1β and NFATc4 in a rat model of experimental colitis

AimAstragalus membranaceus is a Chinese medicinal herb and has been shown to improve hapten-induced experimental colitis. One of its major components is polysaccharides. We investigated the effect of Astragalus polysaccharides (APS) on expression of TNF-α, IL-1β and NFATc4 in a rat model of experimental colitis.MethodsThe experimental colitis model was induced by TNBS. Forty five rats were divided into five groups (n = 9): Normal control group, receiving ethanol vehicle with no TNBS during induction and IP saline injection during treatment; TNBS colitis model group (TNBS + IP saline), receiving only IP saline vehicle treatment; APS low dose group (TNBS + L-APS), receiving APS 100 mg/kg; APS high dose group (TNBS + H-APS), receiving APS 200 mg/kg; and positive control group (TNBS + Dexm), receiving dexamethasone 0.3 mg/kg. The clinical features, macroscopic and microscopic scores were assessed. The expressions of TNF-α, IL-1β and NFATc4 were measured by real-time PCR and ELISA assays.ResultsCompared to normal control rats, TNBS + IP saline had significant weight loss, increased macroscopic and microscopic scores, higher disease activity index (DAI) up-regulation of TNF-α, IL-1β and NFATc4 mRNA expression and up-regulation of TNF-α and IL-1β protein expression. Compared to TNBS + IP saline, treatment with APS or dexamethasone significantly reduced DAI, partially but significantly prevented TNBS colitis-induced weight loss and improved both macroscopic and microscopic scores; high dose APS or dexamethasone significantly down-regulated TNF-α and IL-1β expressions (both mRNA and protein) and up-regulated NFATc4 mRNA and protein expression. The effect of high dose APS and dexamethasone is comparable.ConclusionsAPS significantly improved experimental TNBS-induced colitis in rats through regulation of TNF-α, IL-1β and NFATc4 expression.     

Aneuploidy assessed by DNA index influences the effect of iron status on plasma and/or supernatant cytokine levels and progression of cells through the cell cycle in a mouse model

Aneuploidy, a condition associated with altered chromosome number, hence DNA index, is frequently seen in many diseases including cancers and affects immunity. Iron, an essential nutrient for humans, modulates the immune function and the proliferation of normal and cancer cells. To determine whether impaired immunity seen in iron-deficient subjects may be related to aneuploidy, we measured spleen cell DNA index, percent of cells in different phases of the cell cycle, plasma and/or supernatant IL-2, IL-10, IL-12, and interferon-gamma in control, pair-fed, iron-deficient, and iron-replete mice (N = 20–22/group). The test and control diets differed only in iron content (0.09 mmol/kg versus 0.9 mmol/kg) and were fed for 68 days. Mean levels of hemoglobin and liver iron stores of iron-deficient and iron-replete mice were 40–60% lower than those of control and pair-fed mice (P < 0.05). Mean plasma levels of IL-10, interferon-gamma and percent of cells in S + G2/M phases were lower in mice with than in those without aneuploidy (P < 0.05). Lowest plasma IL-12 and interferon-gamma concentrations were observed in iron-deficient mice with aneuploidy. Mean percents of cultures with aneuploidy and DNA indexes were higher in iron-deficient and iron-replete than in control and pair-fed mice likely due to delayed cell division (P < 0.05). Aneuploidy decreased the concentration of IL-2 and interferon-gamma in baseline cultures while it increased that of interferon-gamma in anti-CD3 treated cultures. Aneuploidic indexes negatively correlated with cytokine levels, percents of cells in S + G2/M phases and indicators of iron status (P < 0.05). Although chromosome cytogenetics was not performed, for the first time, we report that increased aneuploidy rate may modulate the immune function during iron-deficiency.     

Activation of foal neutrophils at different ages by CpG oligodeoxynucleotides and Rhodococcus equi

Toll-like receptor 9 (TLR9) activation stimulates protective immune responses against intracellular pathogens by phagocytes, including neutrophils. This study examined TLR9-mediated neutrophil activation in neonatal foals. Unmethylated CpGs, ligands for TLR9, were used to stimulate equine neutrophils, either purified or in contact with other peripheral blood leukocytes. Rhodococcus equi was used as another stimulus in parallel. TLR9 mRNA was constitutively expressed at a similar level in purified equine neutrophils across different ages from birth to adulthood, and expression was not affected by either CpG or R. equi. Purified foal neutrophils were directly sensitive to CpG stimulation, reflected by enhanced reactive oxygen species generation following fMLP stimulation, and by expressing significantly (P < 0.05) greater mRNA of IFN-γ, IL-8, IL-12p35, and significantly (P < 0.05) decreased TNF-α mRNA. In comparison, purified foal neutrophils stimulated by R. equi showed significantly (P < 0.05) increased mRNA production of IL-6, IL-8, IL-23p19, and TNF-α. Neutrophils co-cultured with other leukocytes expressed a distinct profile of cytokine mRNA than purified neutrophils in response to CpG stimulation, whereas the profile was very similar following R. equi stimulation irrespective of neutrophil purity. When co-cultured with other leukocytes, foal neutrophils were significantly (P < 0.05) activated at birth by B-class CpGs and produced IL-6, IL-8, IL-12p40, and IL-23p19 at similar magnitudes to those at 2 months of age. In foal neutrophils at birth, R. equi significantly (P < 0.05) induced all cytokines stimulated by CpGs (except IL-12p40), as well as TNF-α. Our results indicate that foal neutrophils were sensitive to CpG or R. equi activation as early as at birth, and that B-class CpGs enhanced foal neutrophil functions in vitro.     

Tumor Necrosis Factor-α, Interleukins-12(p40), 6, and 10 levels in cerebrospinal fluid and outcome prediction in Ossimi sheep with encephalitic listeriosis

Encephalitic listeriosis in sheep is a life-threatening disease. However, little is known about the cytokine response and their predictive value in this disease. The aim of present study was to assess the prognostic significance of Tumor Necrosis Factor-α (TNF-α), Interleukin-12(p40) (IL-12 p40), Interleukin-6 (IL-6), and Interleukin 10 (IL-10) levels in cerebrospinal fluid (CSF) in sheep with encephalitic listeriosis. Fifty-nine ewes in 14 flocks were diagnosed clinically as having listeriosis. CSF was collected and subjected to bacteriological examination and estimation of selected cytokines. Twenty-eight ewes were confirmed to be infected with Listeria monocytogenes. Based on antimicrobial sensitivity test, sheep were treated and the outcome was recorded as survivors (n = 10) and non-survivors (n = 18). Cutoff points for CSF cytokines were determined by Receiver operating characteristic analysis (ROC). Association between levels of CSF cytokines and outcome of listeriosis was assessed by logistic regression. TNF-α, IL-6 and IL-12(p40) levels as well as TNF-α/IL-10 ratio were significantly higher in non-survivors than survivors (p = 0.002, 0.0021, 0.0033, and 0.001, respectively). However, IL-10 level was significantly lower in non-survivors than survivors (p = 0.0058). ROC analysis revealed that IL-6 and TNF-α/IL-10 ratio had the highest AUC values (0.98, 0.984, respectively). Final multivariate logistic regression model showed that TNF-α/IL-10 ratio was the only variable that has predictive value for mortality in diseased sheep (p: 0.001; OR: 7.2; 95% CI: 5.7–9.8). TNF-α showed a positive correlation with IL-12β (r = 0.917) and IL-6 (r = 0.965). IL-12 (p40) showed also a positive correlation with IL-6 (r = 0.906). However, IL-10 showed a negative correlation with TNF-α (r = −0.915), IL-12(p40) (r = −0.790), and IL-6 (r = −0.902). In conclusion, TNF-α/IL-10 ratio may provide predictive information about outcome of encephalitic listeriosis in sheep.