Suppression of the TNF‐alpha level is mediated by Gan‐Lu‐Yin (traditional Chinese medicine) in human oral cancer cells through the NF‐kappa B,AKT, and ERK‐dependent pathways

Oral cancer is one of the major causes of deaths in the male population of Taiwan. Gan‐Lu‐Yin (GLY) is used for an adjuvant treatment of Traditional Chinese Medicine in clinical patients. In this study, we investigated the molecular mechanisms in oral cancer cell lines after exposure to GLY. The cytometric bead‐based array (CBA) method was used for the examining and analyzing of tumor necrosis factor‐alpha (TNF‐α) secretion level. TNF‐α mRNA expression was determined by real‐time PCR analysis. Nuclear factor kappa B (NF‐κB) activity and other relative proteins were determined by NF‐κB promoter assay, Western blotting, electrophoretic mobility shift assay (EMSA), and immuno‐staining analyses. GLY decreased the secretion of TNF–α from the oral cancer CAL 27 cells. Furthermore, 2000 μg/mL of GLY significantly suppressed TNF‐α mRNA expression of CAL 27 cells in a time‐dependent manner. GLY reduced the levels of proteins, including nuclear NF‐κB (p65 and p50), p‐IKK (ser176), p‐IκB, p‐AKT, p‐ERK, and nuclear Egr‐1 in a time and dose‐dependent manner. GLY also suppressed the NF‐κB activity and translocation in CAL 27 cells. We suggest that GLY might promote the cure of oral cancer through decreasing the level of TNF‐α cytokine, and these actions were mediated partially through the NF‐κB, AKT, and ERK‐dependent pathways in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1196–1205, 2016.     

Bis‐(3‐hydroxyphenyl) diselenide inhibits LPS‐stimulated iNOS and COX‐2 expression in RAW 264.7 macrophage cells through the NF‐kB inactivation

Objectives Previously, we reported that diaryl diselenide compounds have strong inhibitory effects on lipopolysaccharide (LPS)‐induced nitric oxide (NO) production in macrophages. In this study, we investigated the molecular mechanisms underlying NO suppression and prostaglandin E₂ (PGE₂) production by diaryl diselenide compounds, bis‐(2‐hydroxyphenyl) diselenide (DSE‐A), bis‐(3‐hydroxyphenyl) diselenide (DSE‐B), bis‐(4‐hydroxyphenyl) diselenide (DSE‐C), dipyridyl diselenide (DSE‐D) and diphenyl diselenide (DSE‐E). Methods The effect of these compounds on NO suppression and PGE₂ production was investigated in RAW 264.7 macrophages. Key findings Our data indicate that of the above, DSE‐B most potently inhibits NO and PGE₂ production, and that it also significantly reduces the releases of tumour necrosis factor (TNF)‐α, interleukin(IL)‐1β and IL‐6. Consistent with these observations, DSE‐B also reduced the protein levels of inducible NO synthase (iNOS) and cyclooxygenase‐2 (COX‐2), and the mRNA levels of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6. Furthermore, DSE‐B inhibited LPS‐induced nuclear factor‐κB (NF‐κB) activation, which was associated with the prevention of the inhibitor κB‐α (IκB‐α) degradation and a subsequent reduction in nuclear p65 protein levels. Conclusions Taken together, our data suggest that the anti‐inflammatory properties of DSE‐B are due to reduction in the expression of iNOS, COX‐2, TNF‐α, IL‐1β and IL‐6 through the down‐regulation of NF‐κB binding activity.     

The role of Syk/IĸB‐α/NF‐ĸB pathway activation in the reversal effect of BAY 61‐3606, a selective Syk inhibitor,on hypotension and inflammation in a rat model of zymosan‐induced non‐septic shock

Spleen tyrosine kinase (Syk), a non‐receptor tyrosine kinase, plays an important role in allergic diseases and inflammation. Syk triggers several intracellular signalling cascades including Toll‐like receptor signalling to activate inflammatory responses following fungal infection but the role of this enzyme in zymosan (ZYM)‐induced non‐septic shock and its impacts on hypotension and inflammation in rats is not well understood. This study was conducted to determine the effects of Syk inhibition on ZYM‐induced alterations in the expression and/or activities of Syk, inhibitor ?B (I?B)‐α, and nuclear factor‐?B (NF‐?B) p65. We also examined the effect of Syk inhibition on inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)‐2, and tumour necrosis factor (TNF)‐α, and activity of myeloperoxidase (MPO) that contribute to hypotension and inflammation. Administration of ZYM (500 mg/kg, ip) to male Wistar rats decreased blood pressure and increased heart rate. These changes were associated with increased expression and/or activities of Syk, NF‐κB p65, iNOS and COX‐2 and decreased expression of IκB‐α with enhanced levels of nitrite, nitrotyrosine, 6‐keto‐PGF_1α, and TNF‐α and activity of MPO in renal, cardiac and vascular tissues. ZYM administration also elevated serum and tissue nitrite levels. The selective Syk inhibitor BAY 61‐3606 (3 mg/kg, ip) given 1 hour after ZYM injection reversed all of these changes induced by ZYM. These results suggest that Syk/I?B‐α/NF‐?B pathway activation contributes to hypotension and inflammation caused by the production of vasodilator and proinflammatory mediators in the zymosan‐induced non‐septic shock model.     

Inflammatory cytokines tumour necrosis factor‐α and interleukin‐8 enhance airway smooth muscle contraction by increasing L‐type Ca2+ channel expression

Inflammation elevates intracellular calcium concentrations ([Ca²⁺]_i) in airway smooth muscle (ASM). The L‐type Ca²⁺ channel (L‐VDCC) plays an important role in regulating Ca²⁺ influx in ASM. However, the role of L‐VDCC in the inflammatory cytokine‐induced pathology of ASM remains unclear. In the present study, we used calcium imaging and isometric tension measurements to assess the role of L‐VDCC in agonist‐induced [Ca²⁺]_i rise and the associated contractions in mouse ASM, and we used immunoblotting to identify L‐VDCC protein expression levels in mouse ASM after exposure to tumour necrosis factor alpha (TNF‐α) or interleukin‐8 (IL‐8). Our results showed that high‐K⁺‐ or carbachol‐induced contractions of mouse ASM were significantly greater after pretreatment with TNF‐α or IL‐8 for 24 hours. Both verapamil and nifedipine, L‐VDCC inhibitors, abolished this increased contraction induced by TNF‐α or IL‐8 pretreatment. Moreover, TNF‐α treatment enhanced carbachol‐induced Ca²⁺ influx in ASM cells, and this effect was abrogated by verapamil. Additionally, immunoblotting results showed that preincubation of mouse ASM with TNF‐α or IL‐8 also enhanced L‐VDCC protein expression. On the basis of these findings, we concluded that proinflammatory cytokines, such as TNF‐α and IL‐8, increase the expression level of L‐VDCC, which in turn contributes to augmented agonist‐induced ASM contractions. This effect of inflammation on L‐VDCC expression in ASM may be associated with airway hyper‐responsiveness and involved in the development of asthma.     

Daphnetin inhibits TNF‐α and VEGF‐induced angiogenesis through inhibition of the IKKs/IκBα/NF‐κB,Src/FAK/ERK1/2 and Akt signalling pathways

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti‐angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti‐arthritic and anti‐inflammatory. The present study was performed to investigate the anti‐angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti‐angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)‐induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8 ng/mL)‐induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10 ng/mL) and VEGF‐induced HUVECs by extracting the mRNA and protein levels using RT‐qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor‐κB signalling together including TNF‐α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF‐κB‐p65 protein. Furthermore, western blotting revealed that DAP significantly down‐regulated the VEGF‐induced signalling such as c‐Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF‐stimulated HUVECs by the caspase‐3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi‐targeted medication for the anti‐angiogenesis and cancer therapy.     

Effects of thalidomide and 3‐phthalimido‐3‐(3,4‐dimethoxyphenyl)‐propanamide on bile duct obstruction‐induced cirrhosis in the rat

Tumor necrosis factor‐alpha (TNF‐α) plays a central role in cellular necrosis, apoptosis, organ failure, tissue damage, inflammation, and fibrosis. These processes, occurring in liver injury, may lead to cirrhosis. Thalidomide and its analogs have shown to be effective TNF‐α inhibitors. The aim of this work was to synthesize a thalidomide analog, the 3‐phthalimido‐3‐(3,4‐dimethoxyphenyl)‐propanamide (PDP) and to evaluate its hepatoprotective properties on bile duct obstruction‐induced cirrhosis. Synthesis, purification, and spectroscopic characterization of PDP were carried out. Thalidomide (200 mg/kg) or PDP (15 mg/kg) were administered to sham (Sh) or bile duct ligated (BDL) rats. The animals were sacrificed 28 days after treatments. Alkaline phosphatase (Alk. Phosph.), γ‐glutamyl transpeptidase (γ‐GTP) and alanine aminotransferase (ALT) enzyme activities, bilirubins, and TNF‐α concentrations were evaluated in plasma. Collagen was estimated by the liver hydroxyproline content and histopathology was performed. Both drugs showed partial amelioration of cirrhosis. However, the hepatoprotective effects of thalidomide were poor when compared to those afforded by PDP. While PDP improved the majority of the biochemical markers of liver injury and prevented partial but significantly collagen accumulation, thalidomide showed only modest beneficial effects on bilirubins and ALT. PDP was effective in preventing the increase in plasma TNF‐α levels, while thalidomide not only failed to inhibit TNF‐α, but increased it. Differences between thalidomide and PDP effectiveness may be due to their stability and different mechanism of action, as reported by others. Inhibition of proinflamatory cytokines is an interesting pharmacological aim to treat cirrhosis. Drug Dev. Res. 54:209–218, 2001. © 2002 Wiley‐Liss, Inc.     

Protocatechuic Acid Induces Angiogenesis through PI3K‐Akt‐eNOS‐VEGF Signalling Pathway

In this study, we sought to elucidate whether protocatechuic acid contributes to induce angiogenesis as well as its mechanisms. To this end, we examined the role of protocatechuic acid on human brain microvascular endothelial cell line (HBMEC) proliferation, invasion and tube formation in in vitro. For the study of mechanisms involved, the phosphoinositide 3 kinase (PI3K)‐Akt inhibitor LY294002, the endothelial nitric oxide synthase (eNOS) inhibitor L ‐NAME, vascular endothelial growth factor (VEGF), antagonist sFlt‐1 and VEGF receptor blocker SU‐1498 were used. Proliferation of HBMEC was tested by MTT. Scratch adhesion test was used to assess the ability of invasion. A Matrigel tube formation assay was performed to test capillary tube formation ability. PI3K‐Akt‐eNOS‐VEGF pathway activation in HBMEC was tested by Western blot. Our data suggested that protocatechuic acid induces angiogenesis in vitro by increasing proliferation, invasion and tube formation. VEGF expression was increasing by protocatechuic acid and counteracted by VEGF antagonist sFlt‐1, LY294002 and L‐NAME in HBMEC. Tube formation was increased by protocatechuic acid and counteracted by VEGF receptor blocker‐SU1498, LY294002 and L‐NAME. These data suggest that protocatechuic acid may be a candidate therapy for stroke recovery by promoting angiogenesis via a programmed PI3K/Akt/eNOS/VEGF signalling axis.     

Comparison of anti‐inflammatory activities of ruscogenin,a major steroidal sapogenin from Radix Ophiopogon japonicus,and Its succinylated derivative,RUS‐2HS

Ruscogenin (RUS), first isolated from Ruscus aculeatus, is also a major steroidal sapogenin of the traditional Chinese herb Radix Ophiopogon japonicus. It has robust anti‐inflammatory activities. In previous studies, a ruscogenin affinity column, derived from succinylated ruscogenin (RUS‐2HS), was used to purify an antibody of ruscogenin. A ruscogenin affinity column can also be used to explore its protein targets. However, until now there have been no related pharmacological reports about ruscogenin derivatives. Whether the activity groups of ruscogenin have been blocked during the derivation process remains unknown. The present study was performed to compare the anti‐inflammatory activities in vitro of RUS‐2HS and ruscogenin. Both compounds reduced tumor necrosis factor‐α (TNF‐α)‐induced adhesion of human pro‐myelocytic leukemia cells (HL‐60) to endothelial ECV304 cells with IC₅₀ values of 6.90 nM and 7.45 nM, respectively. They were also inhibited overexpression of ICAM‐1 in ECV304 cells at the mRNA level as evaluated by real‐time PCR and at the protein level evaluated by flow cytometry with similar potency. Such data demonstrate that the functional groups of ruscogenin were not blocked by derivation, suggesting further use of the ruscogenin affinity column for target investigation. Meanwhile, RUS‐2HS was found to have remarkable anti‐inflammatory activity for the first time, indicating it would be a new lead compound with improved bioavailability. Drug Dev Res 69: 196–202, 2008. © 2008 Wiley‐Liss, Inc.     

Anti‐contractile effects of perivascular adipose tissue in thoracic aorta from rats fed a high‐fat diet: role of aerobic exercise training

The aim of the present study was to evaluate the effects of aerobic exercise training on perivascular adipose tissue (PVAT) function in thoracic aorta from rats fed a high‐fat diet. Aortic vascular reactivity was performed in sedentary (SD), trained (TR), sedentary high‐fat diet (SD‐HF), and trained high‐fat diet (TR‐HF) male Wistar rats in the absence (PVAT?) or in the presence (PVAT+) of thoracic PVAT. We also measured circulatory concentrations of leptin and tumour necrosis factor alpha (TNF‐α), as well as the protein expressions of TNF‐α receptor 1 (TNFR1) and inducible nitric oxide synthase (iNOS) on PVAT. In the SD‐HF group, the body weight, epididymal fat pad, thoracic PVAT, circulatory triglycerides, insulin, leptin and TNF‐α were increased when compared with the SD group, whereas exercise training reduced these values in TR‐HF group. The relaxing response curves to acetylcholine and sodium nitroprusside were not modified by either intervention (high‐fat diet or exercise training) or the presence of PVAT. The presence of PVAT had an anti‐contractile effect in response to serotonin in all groups. In SD‐HF group, the increased magnitude of anti‐contractile effects was in parallel with an up‐regulation of iNOS protein expression in PVAT without alteration in TNFR1. Exercise training was effective in normalizing the vascular reactivity in rings PVAT+ and in reducing the iNOS protein expression. Exercise training prevented the PVAT–induced alteration in thoracic aorta from rats fed a high‐fat diet.     

Molecular pathways mediating differential responses to lipopolysaccharide between human and baboon arterial endothelial cells

1. Vascular inflammation plays a critical role in atherogenesis. Previously, we showed that baboon arterial endothelial cells (BAEC) were hyporesponsive to lipopolysaccharide (LPS) compared with human arterial endothelial cells (HAEC). 2. In the present study, we investigated mechanisms underlying differential responses between HAEC and BAEC to tumour necrosis factor (TNF)‐α and LPS. 3. Both HAEC and BAEC responded similarly to TNF‐α. However, BAEC showed retarded responses to LPS in expression of E‐selectin, intercellular adhesion molecule‐1, monocyte chemotactic protein‐1 and interleukin‐8 (P < 0.05). These changes were confirmed at the mRNA level. Tumour necrosis factor‐α activated nuclear factor‐κB members such as p50, p52, p65, c‐rel and RelB in both HAEC and BAEC. In contrast, LPS activated p50 and p65 only in HAEC. Using microarray assays, we found that TNF receptor‐associated factor 2 (TRAF‐2), TNF receptor superfamily, member 1A‐associated via death domain (TRADD) and nuclear factors such as nuclear factor of kappa in B‐cells inhibitor, α (NFKBIA) and nuclear factor of kappa in B‐cells inhibitor, β (NFKBIB) were upregulated by LPS only in HAEC. Although the baseline expression of Toll‐like receptor (TLR) 4 was low in both HAEC and BAEC, TNF‐α activated TLR4 expression in both cell types. Although LPS increased TLR4 expression only in HAEC, human and baboon peripheral blood mononuclear cells exhibited similar TLR4 expression and response to LPS. Transfecting BAEC with TLR4/myeloid differentiation protein‐2 overexpression vector conferred BAEC responsiveness to LPS. 4. The findings of the present study indicate that an altered TLR4 system may be responsible for the resistance of baboon endothelial cells to LPS. Given the importance of TLR4 in human immune responses and vascular diseases, the natural resistance of baboons to LPS/TLR4‐initiated inflammation could make the baboon a valuable animal model in which to study how inflammation affects atherogenesis.     

Cobalt oxide nanoparticles induced oxidative stress linked to activation of TNF‐α/caspase‐8/p38‐MAPK signaling in human leukemia cells

The purpose of this study was to determine the intracellular signaling transduction pathways involved in oxidative stress induced by nanoparticles in cancer cells. Activation of reactive oxygen species (ROS) has some therapeutic benefits in arresting the growth of cancer cells. Cobalt oxide nanoparticles (CoO NPs) are an interesting compound for oxidative cancer therapy. Our results showed that CoO NPs elicited a significant (P <0.05) amount of ROS in cancer cells. Co‐treatment with N‐aceyltine cystine (an inhibitor of ROS) had a protective role in cancer cell death induced by CoO NPs. In cultured cells, the elevated level of tumor necrosis factor‐alpha (TNF‐α) was noted after CoO NPs treatment. This TNF‐α persuaded activation of caspase‐8 followed by phosphorylation of p38 mitogen‐activated protein kinase and induced cell death. This study showed that CoO NPs induced oxidative stress and activated the signaling pathway of TNF‐α‐Caspase‐8‐p38‐Caspase‐3 to cancer cells. Copyright © 2015 John Wiley & Sons, Ltd.     

RNAi Silencing of IL‐1β and TNF‐α in the Treatment of Post‐traumatic Arthritis in Rabbits

Post‐traumatic arthritis is a secondary complication to severe joint trauma. With the disease progression, it may eventually lead to osteoarthritis in patients whose age is considerably younger than patients with traditional bone arthritis. The main objective of this study was to explore the feasibility of using lentiviral‐mediated RNA interference silencing of IL‐1β and TNF‐α to treat post‐traumatic arthritis in rabbits. About 48 New Zealand rabbits underwent bilateral knee joint surgery to stimulate traumatic arthritis. They were then randomly divided into four groups of 12 rabbits each. The histopathology of the cartilage was observed, and the changes were assessed by Mankin scoring. ELISA was used to detect the expression of IL‐1β and TNF‐α in the synovial fluid. (i) Compared with the control group, the transfection and co‐transfected groups displayed reduced cartilage damage and speed of degeneration. The co‐transfected group showed the greatest alleviation of symptoms. The Mankin score was statistically different (p < 0.01). (ii) Compared with the control group, the expression of IL‐1β or TNF‐α was reduced in the respective transfection groups (p < 0.01 in both groups) and IL‐1β and TNF‐α were reduced in the co‐transfected group (p < 0.01). The co‐transfected group showed the lowest expression of the three experimental groups of both IL‐1β and TNF‐α (p < 0.01). Lentivirus‐mediated RNA interference can knock down the expression of IL‐1β and TNF‐α in joint fluids and, in a synergistic effect when two siRNAs are co‐transfected, ease cartilage degeneration.     

EFFECTS OF ANTI-HYPERTENSIVE DRUGS ON PRODUCTION OF SOLUBLE FMS-LIKE TYROSINE KINASE 1 AND SOLUBLE ENDOGLIN FROM HUMAN NORMAL AND PRE-ECLAMPTIC PLACENTAS IN VITRO

• 1 Increases in soluble fms‐like tyrosine kinase 1 (sFlt‐1) and soluble endoglin (sEng) contribute to the pathogenesis of pre‐eclampsia. Soluble Flt‐1 binds to circulating free vascular endothelial growth factor and placenta growth factor and this is associated with endothelial dysfunction. Soluble endoglin, a transforming growth factor (TGF)‐β coreceptor, was reported to synergize with sFlt‐1 to amplify endothelial dysfunction by inhibiting TGF‐β1‐mediated vasorelaxation.
• 2 The aim of the present study was to examine whether the antihypertensive drugs clonidine (0.08–1.3 µg/mL), diazoxide (25–300 µg/mL), frusemide (60–1000 µg/mL) and hydralazine (6.3–100 µg/mL) have any effect on placental production of sFlt‐1 and sEng in placentas from normal and pre‐eclamptic pregnancies.
• 3 Explants were taken from non‐laboured term placentas of normal pregnancy (n = 5) and women with pre‐eclampsia (n = 5). Villous explants were cultured with increasing doses of antihypertensive drugs. Placental sFlt‐1 and sEng production was examined using ELISA.
• 4 Baseline sFlt‐1 production was higher in placentas from women with pre‐eclampsia than from normal pregnancy (4.5 ± 1.4 vs 3.2 ± 0.6 ng/mg of total protein, respectively; P < 0.001), as was sEng production (9.0 ± 2.3 vs 4.1 ± 0.6 ng/mg of total protein, respectively; P < 0.001). With the exception of frusemide, none of the antihypertensive drugs tested had any effect on sFlt‐1 and sEng production from placental explants of normal pregnancy and women with pre‐eclampsia. Increasing frusemide concentrations were correlated with increased sEng production in normal pregnancy (P < 0.005).
• 5 In conclusion, placental sFlt‐1 and sEng production was higher in pre‐eclampsia and antihypertensive drugs had no effect on placental production of sFlt‐1 and sEng in vitro.

     

Poly(ADP‐ribose)polymerase 1 inhibition protects against age‐dependent endothelial dysfunction

Age‐related endothelial dysfunction is closely associated with the local production of reactive oxygen species (ROS) within and in the vicinity of the vascular endothelium. Oxidant‐induced DNA damage can activate the nuclear enzyme poly(ADP‐ribose) polymerase 1 (PARP‐1), leading to endothelial dysfunction in various pathophysiological conditions. The present study aimed to investigate the role of PARP‐1 in age‐dependent changes in endothelial cell function and its underlying mechanism. Wild‐type (WT) and PARP‐1^?/? mice were divided into young (2 months) and old (12 months) groups. Isolated aortic rings were suspended to record isometric tension to assess endothelial function. Nitric oxide (NO) production and content in plasma were detected by spectrophotometry. Superoxide ( production was detected by dihydroethidium. Expression of PARP‐1, endothelial nitric oxide synthase (eNOS), induced nitric oxide synthase (iNOS), and arginase‐2 (Arg2) was assessed by western blot analysis. Endothelium‐dependent relaxation in response to acetylcholine was lost in old WT, but not PARP‐1^?/?, mice. Endothelium‐independent vasodilation was not impaired in aging mice. Production of was greater in aging WT mice than young or aging PARP‐1^?/? mice. eNOS expression was not affected by aging in WT or PARP‐1^?/? mice, but p‐eNOS expression decreased and iNOS and Arg2 levels were upregulated only in aging WT mice. In conclusion, PARP‐1 inhibition may protect against age‐dependent endothelial dysfunction, potentially by regulating NO bioavailability via iNOS. Inhibition of PARP‐1 may help in vascular aging prevention.     

Tributyltin and dibutyltin alter secretion of tumor necrosis factor alpha from human natural killer cells and a mixture of T cells and natural killer cells

Butyltins (BTs) have been in widespread use. Tributyltin (TBT) has been used as a biocide in a variety of applications and is found in human blood samples. Dibutyltin (DBT) has been used as a stabilizer in polyvinyl chloride plastics and as a de‐worming agent in poultry. DBT, like TBT, is found in human blood. Human natural killer (NK) cells are the earliest defense against tumors and viral infections and secrete the cytokine tumor necrosis factor‐alpha (TNF‐α). TNF‐α is an important regulator of adaptive and innate immune responses. TNF‐α promotes inflammation and an association between malignant transformation and inflammation has been established. Previously, we have shown that TBT and DBT were able to interfere with the ability of NK cells to lyse tumor target cells. Here we show that BTs alter cytokine secretion by NK cells as well as a mixture of T and NK lymphocytes (T/NK cells). We examined 24‐, 48‐h and 6‐day exposures to TBT (200–2.5 nM) and DBT (5–0.05 μM) on TNF‐α secretion by highly enriched human NK cells and T/NK cells. The results indicate that TBT (200–2.5 nM) decreased TNF‐α secretion from NK cells. In the T/NK cells, 200 nM TBT decreased secretion whereas 100–5 nM TBT increased secretion of TNF‐α. NK cells or T/NK cells exposed to higher concentrations of DBT showed decreased TNF‐α secretion whereas lower concentrations showed increased secretion. The effects of BTs on TNF‐α secretion are seen at concentrations present in human blood. Copyright © 2012 John Wiley & Sons, Ltd.     

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS IMPROVE HEPATIC STEATOSIS BY MODULATING EXPRESSION OF TUMOUR NECROSIS FACTOR-α, INTERLEUKIN-6 AND ADIPONECTIN RECEPTOR-2 IN RATS WITH TYPE 2 DIABETES

• 1 Angiotensin‐converting enzyme inhibitors (ACEI) are hypotensive drugs that have been shown to prevent Type 2 diabetes mellitus (T2DM) in high‐risk individuals. However, in T2DM, the effects of ACEI on hepatic steatosis are not known. The aim of the present study was to examine the effects of ACEI on changes in liver histology and hepatic mRNA expression of adipokines in rats with T2DM.
• 2 Thirty‐six rats were divided into a normal control group, a T2DM group and a fosinopril‐treated group. After six weeks of treatment with 5 mg/kg per day fosinopril, an ACEI, changes in liver histology, serum fasting glucose (FG), insulin, triglyceride (TG), total cholersterol (TC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumour necrosis factor (TNF)‐α, interleukin (IL)‐6, adiponectin were evaluated, as was hepatic TNF‐α, IL‐6 and adiponectin receptor‐2 (adipoR2) mRNA expression.
• 3 The degree of hepatic steatosis and inflammation, serum FG, insulin, TG, TC, ALT, TNF‐α and IL‐6 concentrations and hepatic TNF‐α and IL‐6 mRNA expression were significantly higher in rats with T2DM than in normal controls. Serum adiponectin concentrations and hepatic adipoR2 mRNA expression in rats with T2DM were significantly lower than in normal controls. Fosinopril significantly reduced the degree of hepatic steatosis, serum FG, insulin, ALT, TNF‐α and IL‐6 concentrations and hepatic TNF‐α and IL‐6 mRNA expression. Fosinopril significantly increased serum adiponectin concentrations and hepatic adipoR2 mRNA expression.
• 4 In conclusion, the ACEI improved insulin sensitivity and hepatic steatosis in rats with T2DM by increasing circulating adiponectin and hepatic adipoR2 levels, in addition to reducing pro‐inflammatory cytokine levels in the circulation and liver.

     

TNF‐α level affects etanercept clearance: TNF‐α concentration as a new correction factor of allometric scaling to predict individual etanercept clearances in patients with ankylosing spondylitis

Etanercept (ETN) is a widely used anti‐tumour necrosis factor‐α (TNF‐α) agent, which relieves the symptoms of ankylosing spondylitis (AS) by binding to TNF‐α to inhibit its inflammation effects. In this study, the effect of TNF‐α level on ETN clearance (CL) was investigated, and the TNF‐α concentration was initially set as a correction factor for allometric scaling to improve the predictions of individual ETN CLs. Individual ETN CLs and TNF‐α concentrations in healthy volunteers and patients with AS were determined by performing ETN pharmacokinetic studies in the two cohorts. Accordingly, individual ETN CLs in both healthy volunteers and patients with AS were predicted from data of two animal species using different methods, including simple allometric scaling, scaling with a correction factor of maximum life span potential or brain weight, and scaling with a correction factor of the TNF‐α concentration. The accuracies of such predictions were evaluated by the percentage errors. Consequently, increased TNF‐α concentration was shown to improve ETN CL, by comparing both ETN CLs and TNF‐α concentrations between healthy volunteers and patients with AS. More importantly, better predictions of individual ETN CLs were achieved in patients with AS using allometric scaling with TNF‐α concentration as the correction factor. In conclusion, in vivo levels of TNF‐α can affect ETN CL, and allometric scaling corrected with the TNF‐α concentration can be used to estimate the individual CLs of anti‐TNF‐α monoclonal antibodies based on preclinical data.     

5‐Amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives with in vitro immunomodulatory activities

Isoxazoles are an important class of compounds of potential therapeutic value. The aim of this study was to determine immunotropic effects of 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives on spontaneous and mitogen‐induced lymphocyte proliferation in young and old mice, cytokine production by peritoneal cells as well as possible mechanism of action in a model of Jurkat cells. Three‐month‐old and 13‐month‐old BALB/c mice were used as donors of the cells from a thymus, a spleen, mesenteric lymph nodes, and a peritoneal cavity. Spontaneous and concanavalin A or lipopolysaccharide (LPS)‐induced cell proliferation was measured by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide colorimetric method. IL‐1β and TNF‐α production induced by LPS in macrophage‐enriched peritoneal cell cultures was measured by enzyme‐linked immunoassay. 5‐amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide, 01K (4‐phenyl‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide), and 06K (4‐(4‐chlorophenyl)‐1‐(5‐amino‐3‐methylisoxazole‐4‐carbonyl)‐thiosemicarbazide) exhibited regulatory activity in the proliferation tests. Prevailing stimulatory activity of the hydrazide and inhibitory activity of 01K and 06K was observed. Those effects were connected with different influence of the compounds on signaling proteins expression in Jurkat cells. The regulatory effects of the compounds on IL‐1β production were more profound than those on TNF‐α. Differences in the compound activity in young versus old mice were mainly restricted to 01K. Immunosuppressive isoxazole leflunomide and a stimulatory RM‐11 (1,7‐dimethyl‐8‐oxo‐1,2H‐isoxazole [5,4‐e]triazepine) were applied as reference drugs.