TNF-alpha mRNA Expression Levels Research Articles

Treatment of Ischemic Stroke by Atorvastatin-Loaded PEGylated Liposome.

There is insufficient evidence on the effect of nanoparticles, particularly liposomes loaded with a statin, on acute ischemic stroke. We investigated the impact of atorvastatin-loaded PEG (polyethylene glycol) conjugated liposomes (LipoStatin) on the outcomes in rats with cerebral ischemia-reperfusion. PEGylated liposome loaded with atorvastatin was developed as a nanoparticle to specifically accumulate in an ischemic region and release the drug to ameliorate the harmful effects of the stroke. LipoStatin was administered to rats with transient middle cerebral artery occlusion through the tail vein immediately after reperfusion (LipoStatin group). LipoStatin efficiently accumulated at the cerebral ischemic injury site of the rat. The LipoStatin group showed a significantly reduced infarct volume (p < 0.01) in brain micro-MR imaging and improved neurological function recovery compared to the control group (p < 0.05). In addition, markedly improved brain metabolism using fluorine-18 fluorodeoxyglucose micro-PET/CT imaging was demonstrated in the LipoStatin group compared with the control group (p < 0.01). Mechanistically, as a result of evaluation through IL-1 beta, TNF-alpha, ICAM-1, and Iba-1 mRNA expression levels at 5days after cerebral ischemia, LipoStatin showed significant anti-inflammatory effects. Protein expression of occludin, JAM-A, Caveolin-1, and eNOS by western blot at 3days and fluorescent images at 7days showed considerable recovery of blood-brain barrier breakdown and endothelial dysfunction. PEGylated LipoStatin can be more effectively delivered to the ischemic brain and may have significant neuroprotective effects. Thus, PEGylated LipoStatin can be further developed as a promising targeted therapy for ischemic stroke and other major vascular diseases.

Dietary soybean β‐conglycinin suppresses growth performance and inconsistently triggers apoptosis in the intestine of juvenile grass carp ( Ctenopharyngodon idella ) in association with ROS‐mediated MAPK signalling

The current study aimed to investigate the effects of dietary soybean beta-conglycinin on growth performance and intestine apoptosis in juvenile grass carp (Ctenopharyngodon idella). For fish fed with the 80 g beta-conglycinin/kg diet for 7 weeks, the specific growth rate and feed intake were decreased. In the proximal intestine, dietary beta-conglycinin did not induce DNA fragmentation, tended to decrease the reactive oxygen species (ROS) content, and decreased ROS-generating enzyme (NADPH oxidase [NOX]) activity. Subsequently, in the mid-intestine, dietary beta-conglycinin caused DNA fragmentation, tended to increase the ROS content, increased caspase-3, caspase-8 and caspase-9 activities, upregulated the mRNA levels of proapoptotic molecules (apoptotic protease-activating factor-1 [Apaf1] and Bcl-2-associated X protein [BAX]) and mitogen-activated protein kinase (MAPK)-related signal molecules (Jun N-terminal kinase (JNK) and p38 MAPK) and increased the protein levels of p38 MAPK and phospho-p38 MAPK. Moreover, in the distal intestine, dietary beta-conglycinin induced DNA fragmentation, elevated NOX activity and the ROS content and increased caspase-3, caspase-8 and caspase-9 activities, death ligand (TNF-alpha) mRNA expression level, and p38 MAPK and phospho-p38 MAPK protein levels. In summary, dietary soybean beta-conglycinin suppressed fish growth and inconsistently caused apoptosis among the different intestinal segments which was partially associated with ROS-mediated MAPK signalling.

The role of endothelin-1 and endothelin receptor antagonists in allergic rhinitis inflammation: ovalbumin-induced rat model

Desloratadine is a biologically active metabolite of loratadine which is indicated for the treatment of allergic rhinitis. Bosentan is a dual endothelin receptor antagonist used to treatment of pulmonary artery hypertension (PAH). In this study, we aimed to determine the role of endothelins in allergic rhinitis (AR) and the effects of endothelin receptor antagonists in AR rat models through comparison with desloratadine. In total, 20 adult Sprague-Dawley rats were used in this study. An ovalbumin-induced allergic rhinitis model was formed in three study groups except for the control group. Bosentan (100 mg/kg/day) was given to the bosentan-treated group for 7 days and desloratadine (10 mg/kg/day) was administered to the antihistaminic-treated group for 7 days. Nasal symptom scorings and histopathological examinations of the nasal tissues were carried out. Serum IgE levels and ET-1 and TNF-alpha mRNA expression levels were analysed. Between group comparisons for nasal symptoms, histopathological analysis, and molecular analyses were performed with a one-way ANOVA and Duncans multiple comparison tests. Significance was accepted at p smaller than 0.05. Bosentan inhibited nasal symptom more significantly than desloratadine. The IgE level, ET-1 and TNF-alpha mRNA expression levels statistically increased in the allergic rhinitis group when compared to other groups. Conversely, the bosentan-treatment group showed a significant recovery from the same parameters. The deterioration in histopathological parameters reached the highest levels in the allergic rhinitis group. The histopathological findings were close to those of the control group in the bosentan and antihistaminic-treated group. ET-1 is one of the mediators that impact AR development and ET-1 antagonists can be useful for symptom control and for decreasing allergic inflammation in AR patients.

The role of endothelin-1 and endothelin receptor antagonists in allergic rhinitis inflammation: ovalbumin-induced rat model.

Desloratadine is a biologically active metabolite of loratadine which is indicated for the treatment of allergic rhinitis. Bosentan is a dual endothelin receptor antagonist used to treatment of pulmonary artery hypertension (PAH). In this study, we aimed to determine the role of endothelins in allergic rhinitis (AR) and the effects of endothelin receptor antagonists in AR rat models through comparison with desloratadine. In total, 20 adult Sprague-Dawley rats were used in this study. An ovalbumin-induced allergic rhinitis model was formed in three study groups except for the control group. Bosentan (100 mg/kg/day) was given to the bosentan-treated group for 7 days and desloratadine (10 mg/kg/day) was administered to the antihistaminic-treated group for 7 days. Nasal symptom scorings and histopathological examinations of the nasal tissues were carried out. Serum IgE levels and ET-1 and TNF-alpha mRNA expression levels were analysed. Between group comparisons for nasal symptoms, histopathological analysis, and molecular analyses were performed with a one-way ANOVA and Duncans multiple comparison tests. Significance was accepted at p smaller than 0.05. Bosentan inhibited nasal symptom more significantly than desloratadine. The IgE level, ET-1 and TNF-alpha mRNA expression levels statistically increased in the allergic rhinitis group when compared to other groups. Conversely, the bosentan-treatment group showed a significant recovery from the same parameters. The deterioration in histopathological parameters reached the highest levels in the allergic rhinitis group. The histopathological findings were close to those of the control group in the bosentan and antihistaminic-treated group. ET-1 is one of the mediators that impact AR development and ET-1 antagonists can be useful for symptom control and for decreasing allergic inflammation in AR patients.

Abstract 513: Evaluation of Proinflammatory Effects of Low Dose Angiotensin II Infusion in Intact Female Mice and Adiponectin Deficient Mice

Circulating vasoactive peptide Angiotensin II (ANGII) has a well-known role in the development of hypertension and other cardiovascular diseases. In addition it has significant proinflammatory actions in the vascular wall inducing the production of reactive oxygen species, inflammatory cytokines and adhesion molecules. It has been previously shown that estrogen in female mice protects against ANGII mediated hypertension and against proinflammatory effects. It is not clear if the protective effects of estrogen extend to adiponectin deficient mice. Adiponectin is one of the few peptides secreted by fat to have anti-inflammatory properties. The present study evaluates the effect of chronic ANGII infusion on expression of cytokines in female C57BL/6J and adiponectin deficient mice (adipo-/-). Female mice (24- 28wks), were implanted with osmotic pump containing either ANG II (800 ng/Kg /min) or saline. Blood samples and tissue were collected at the end of 14 days. Plasma levels of TNFalpha and IL6 were measured using enzyme linked immunoabsorbent assay. Renal tissue expression of the cytokines were quantified using real-time PCR (Eppendorf Realplex 4 mastercycler) and SYBR Green ROX mastermix. Plasma TNFα levels were similar in saline infused C57Bl/6J(11±1 pg/ml) and adiponectin deficient mice(10± 1 pg/ml). ANGII did not significantly increase TNFα in the control(14±3 pg/ml) or adipo-/- mice(13+1 pg/ml). Plasma IL6 levels were also not significantly different in the two groups. A microarray for mRNA expression of markers of endothelial activation and adhesion molecules was also performed in the renal tissue. Preliminary data show that TNFalpha mRNA expression levels were not increased by ANG II infusion and IL6 expression was undetectable. ANGII also did not alter E-Selectin,VCAM1, Collagen1a1 and eNOS expression. In conclusion, ANGII infusion did not result in a proinflammatory milieu in both female C57BL/6J and Adipo -/- mice.

Moxibustion Inhibits Apoptosis and Tumor Necrosis Factor-Alpha/Tumor Necrosis Factor Receptor 1 in the Colonic Epithelium of Crohn’s Disease Model Rats

Previous studies have shown that moxibustion on Tianshu (ST25) and Qihai (CV6) is effective for treating Crohn's disease. However, the mechanism of moxibustion has not been clearly elucidated. The purpose of this study was to investigate the effect of moxibustion on the inhibition of colonic epithelial cell apoptosis and on tumor necrosis factor alpha (TNF-alpha) and tumor necrosis factor receptor TNF receptor-1 (TNFR1) and TNFR2 and to determine the mechanism of its protective effect using Crohn's disease (CD) model rats. The experimental CD rat models were established by the administration of trinitrobenzene sulfonic acid. In the herbs-partitioned moxibustion (HPM) and mild-warm moxibustion (MWM) groups, moxibustion was administered to Tianshu (ST25) and Qihai (CV6) acupoints once daily for 14 days. In the salicylazosulfapyridine (SASP) group, SASP was administered twice daily for 14 days. A normal control (NC) group and a model control (MC) group were also studied. The levels of TNF-alpha and its mRNA, TNFR1 as well as the rate of colonic epithelial cell apoptosis were significantly decreased in the HPM, MWM and SASP groups compared with the MC group. The HPM and MWM groups had lower mRNA expression and lower protein levels of TNF-alpha compared to the SASP group. The HPM and MWM groups exhibited less apoptosis than the SASP group. Moxibustion may inhibit colonic epithelial cell apoptosis by reducing the high expression of TNF-alpha and TNFR1 to protect the defective colonic epithelial barrier in CD model rats.

Tu1174 Attenuated Progression of Gastric Dysplasia by Administration of Green Tea Catechins in INS-GAS Mice

Backgrounds: Green tea catechins (GTCs) have been shown to provide anti-carcinogenic effects in several laboratory studies. Although the effect of green tea consumption on the risk of gastric cancer has been studied in epidemiological studies, the results remain controversial. The anti-carcinogenic mechanisms of GTCs have not been fully evaluated in rodent models of gastric cancer. Previous reports indicate that transgenic INS-GASmice are hypergastrinemic and induced corpus gastritis and eventually developed gastric cancer at over 20 months old. Aim: The aim of our study was to investigate the molecular effects of GTCs on gastritis and premalignant lesions in INS-GAS mice. Methods: Thirty eight, male INSGAS mice on a FVB background were used in this study. Mice were divided into two groups: A GTCs group and control. Mice in the GTCs group were allowed free access to drinking water containing 2000ppm of GTCs from the age of 9 weeks. Mice were euthanized at the age of 13 or 37 weeks. Histological scores (inflammation and dysplasia) were graded on a blinded basis by pathologist on a scale from 0 to 4. The mRNA expressions of IFN-gamma and TNF-alpha in the corpus of stomachs were evaluated using quantitative PCR. Statistical analysis was performed using a Mann-Whitney U test. Results: Body weights in mice supplemented with GTCs were significantly lower than that of controls at both 13 and 37 weeks of age (p < 0.05). Treated and control mice developed minimal to mild corpus gastritis, although dysplasia was evident. The histological score of dysplasia in mice supplemented with GTCs was significantly lower than that in controls at 37 weeks of age (p < 0.05). GTCs supplementation did not affect the inflammation score. The mRNA level of IFN-gamma in mice administrated GTCs was significantly lower than that of controls at 13 weeks of age (p < 0.05), although it did not reach statistical significance at 34 weeks of age (p = 0.056). GTCs supplementation did not alter TNF-alpha mRNA expression levels between both groups. Conclusions: Administration of GTCs attenuated progression of gastric dysplasia in INS-GAS male mice. These results demonstrated that IFN-gamma dependent mechanisms may be involved in the protective role of GTCs in development of gastritis and premalignant lesions in this rodent model.

Tu1175 The Protective Influence of Vitamin D in Hepatocellular Carcinoma

Backgrounds: Green tea catechins (GTCs) have been shown to provide anti-carcinogenic effects in several laboratory studies. Although the effect of green tea consumption on the risk of gastric cancer has been studied in epidemiological studies, the results remain controversial. The anti-carcinogenic mechanisms of GTCs have not been fully evaluated in rodent models of gastric cancer. Previous reports indicate that transgenic INS-GASmice are hypergastrinemic and induced corpus gastritis and eventually developed gastric cancer at over 20 months old. Aim: The aim of our study was to investigate the molecular effects of GTCs on gastritis and premalignant lesions in INS-GAS mice. Methods: Thirty eight, male INSGAS mice on a FVB background were used in this study. Mice were divided into two groups: A GTCs group and control. Mice in the GTCs group were allowed free access to drinking water containing 2000ppm of GTCs from the age of 9 weeks. Mice were euthanized at the age of 13 or 37 weeks. Histological scores (inflammation and dysplasia) were graded on a blinded basis by pathologist on a scale from 0 to 4. The mRNA expressions of IFN-gamma and TNF-alpha in the corpus of stomachs were evaluated using quantitative PCR. Statistical analysis was performed using a Mann-Whitney U test. Results: Body weights in mice supplemented with GTCs were significantly lower than that of controls at both 13 and 37 weeks of age (p < 0.05). Treated and control mice developed minimal to mild corpus gastritis, although dysplasia was evident. The histological score of dysplasia in mice supplemented with GTCs was significantly lower than that in controls at 37 weeks of age (p < 0.05). GTCs supplementation did not affect the inflammation score. The mRNA level of IFN-gamma in mice administrated GTCs was significantly lower than that of controls at 13 weeks of age (p < 0.05), although it did not reach statistical significance at 34 weeks of age (p = 0.056). GTCs supplementation did not alter TNF-alpha mRNA expression levels between both groups. Conclusions: Administration of GTCs attenuated progression of gastric dysplasia in INS-GAS male mice. These results demonstrated that IFN-gamma dependent mechanisms may be involved in the protective role of GTCs in development of gastritis and premalignant lesions in this rodent model.

Effects of the angiotensin II receptor blocker losartan on the monocyte expression of biglycan in hypertensive patients

1. Recently, we demonstrated that biglycan (BGN) is increased in circulating monocyte cells from hypertensive patients and that angiotensin (Ang) II is able to increase BGN expression. The present study was designed to investigate the effects of treatment with the angiotensin AT(1) receptor antagonist losartan on monocyte BGN mRNA and protein expression in essential hypertension. 2. One hundred and twenty-six newly diagnosed hypertensive patients without additional risk factors for atherosclerosis and cardiovascular disease were treated with 100 mg losartan once daily for 6 months. Biglycan mRNA and protein expression was determined in monocytes isolated from peripheral blood before (T(0)) and after (T(1)) therapy. Plasma levels of interleukin (IL)-6, tumour necrosis factor (TNF)-alpha and high sensitivity C-reactive protein (hs-CRP) were also determined. In addition, BGN mRNA and protein expression was determined after the ex vivo addition of 1 micromol/L AngII to monocytes isolated from 20 randomly selected hypertensive patients. 3. Biglycan mRNA and protein expression, blood pressure and plasma levels of fibrinogen, IL-6, TNF-alpha and CRP were significantly lower at T(1) than at T(0). Variations in BGN expression were associated with inflammatory markers, but not directly with blood pressure. In AngII-stimulated monocytes, BGN mRNA and protein expression was significantly lower at T(1) that at T(0). Moreover, mean BGN mRNA expression in AngII-stimulated monocytes isolated from losartan-treated patients was similar to baseline expression in unstimulated monocytes from untreated patients. 4. The results of the present study show that losartan can reduce BGN expression in monocytes from hypertensive patients, without any linear association with blood pressure, suggesting that the effects of AngII on BGN expression in monocytes may be modulated, in part, by an AT(1) receptor blocker.

Insight into the Molecular Pathophysiology of Delayed Bone Healing in a Sheep Model

Delayed and nonunions are still challenging problems. In this study, we examined the endogenous mRNA expression of genes regulating cartilage formation, bone formation, endochondral ossification, and bone remodeling during mechanically induced delayed bone healing in a large animal model. A tibial osteotomy was performed in two groups of sheep and stabilized with either a rigid external fixator leading to standard healing or with a rotationally unstable fixator leading to delayed healing. At days 4, 7, 9, 11, 14, 21, and 42 after surgery, total RNA was extracted from the callus. Gene expressions of several molecules functionally important for bone healing were studied by quantitative reverse transcriptase-polymerase chain reaction. The expression profiles were related to callus tissue composition, analyzed by histomorphometry. Histomorphometry demonstrated a delayed, prolonged chondral phase and a reduction in bone formation in the experimental group. There was no differential expression of Runx2 between both groups until day 42, but mRNA expression levels of BMP2, BMP4, BMP7, noggin, Col1a1, IGF1, TGFbeta1, OPN, MMP9, MMP13, TIMP3, TNFalpha, MCSF, RANKL, and OPG were lower in the delayed healing group at several time points. This study provides insight into the temporal periods during which various factors may be deficient during a compromised bone-healing situation.

Leptin potentiates Prevotella intermedia lipopolysaccharide-induced production of TNF-α in monocyte-derived macrophages

PurposeIn addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α production in differentiated THP-1 cells, a human monocytic cell line.MethodsLPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-α and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-α and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis.ResultsLeptin enhanced P. intermedia LPS-induced TNF-α production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-α expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-α production was not mediated by the leptin receptor.ConclusionsThe ability of leptin to enhance P. intermedia LPS-induced TNF-α production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

Adhesion and immunomodulatory effects ofBifidobacterium lactisHN019 on intestinal epithelial cells INT-407

To elucidate the adherence and immunomodulatory properties of a probiotic strain Bifidobacterium lactis (B. lactis) HN019. Adhesion assays of B. lactis HN019 and Salmonella typhimurium (S. typhimurium) ATCC 14028 to INT-407 cells were carried out by detecting copies of species-specific genes with real-time polymerase chain reaction. Morphological study was further conducted by transmission electron microscopy. Interleukin-1beta (IL-1beta), interleukin-8, and tumor necrosis factor-alpha (TNF-alpha) gene expression were assessed while enzyme linked immunosorbent assay was used to detect IL-8 protein secretion. The attachment of S. typhimurium ATCC 14028 to INT407 intestinal epithelial cells was inhibited significantly by B. lactis HN019. B. lactis HN019 could be internalized into the INT-407 cells and attenuated IL-8 mRNA level at both baseline and S. typhimurium-induced pro-inflammatory responses. IL-8 secretion was reduced while IL-1beta and TNF-alpha mRNA expression level remained unchanged at baseline after treated with B. lactis HN019. B. lactis HN019 does not up-regulate the intestinal epithelium expressed pro-inflammatory cytokine, it showed the potential to protect enterocytes from an acute inflammatory response induced by enteropathogen.

Influence of baicalin on TNF-α mRNA, caspase-3 and P-selectin expression in pancreatic tissue of rats with severe acute pancreatitis

Baicalin reduces the severity of severe acute pancreatitis (SAP) in a rat model. This study was carried out to examine the effect of baicalin on TNF-alpha mRNA, caspase-3 and P-selectin protein expression in the pancreas of rats with SAP. Rats with SAP were randomly assigned to untreated, baicalin-treated, octreotide-treated and sham-operated group. The TNF-alpha mRNA expression level, caspase-3 and P-selectin protein expression levels of pancreatic tissue were measured 3, 6 and 12 hours after operation. The caspase-3 protein expression level was significantly higher in the treated groups than that in the untreated group at 3 hours (p <0.01). P-selectin protein expression level in pancreas of the treated groups was significantly lower than that of the untreated group (p <0.05 or <0.01). TNF-alpha mRNA expression level in pancreas decreased in baicalintreated group while untreated group increased at different time points (p <0.05). Baicalin increases caspase-3 expression and inhibits TNF-alpha mRNA and P-selectin protein expression in pancreatic tissue of SAP rats.

Eicosapentaenoic acid is more effective than docosahexaenoic acid in inhibiting proinflammatory mediator production and transcription from LPS-induced human asthmatic alveolar macrophage cells

The purpose of the study was to determine which of the active constituents of fish oil, eicosapentaenoic acid (EPA) or docosahexaenoic acid (DHA), is most effective in suppressing proinflammatory mediator generation and cytokine expression from LPS-stimulated human asthmatic alveolar macrophages (AMphi). The AMphi were obtained from twenty-one asthmatic adults using fiberoptic bronchoscopy. Cells were pretreated with DMEM, pure EPA, an EPA-rich media (45% EPA/10% DHA), pure DHA, a DHA-rich media (10% EPA/50% DHA) or Lipovenos (n-6 PUFA), and then exposed to Dulbecco's Modified Eagle's Medium (DMEM) (-) or LPS (+). Supernatants were analyzed for leukotriene (LT)B(4), prostaglandin (PG)D(2), tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production. Detection of TNF-alpha and IL-1beta mRNA expression levels was quantified by reverse transcriptase polymerase chain reaction. 120 microM pure EPA and EPA-rich media significantly (p<0.05) suppressed TNF-alpha and IL-1beta mRNA expression and the production of LTB(4), PGD(2) and TNF-alpha and IL-1beta in LPS-stimulated primary AMphi cells obtained from asthmatic patients to a much greater extent than 120 microM pure DHA and DHA-rich media respectively. This study has shown for the first time that EPA is a more potent inhibitor than DHA of inflammatory responses in human asthmatic AMphi cells.

Role of Kupffer Cells in Vascular Stress Genes During Trauma and Sepsis

Sepsis remains a serious complication after trauma. Although hepatic microvascular dysfunction occurs during trauma and sepsis, the mechanism responsible is unclear. Since Kupffer cells can provide the signals that regulate the hepatic response in inflammation and contribute to multiple organ failure, this study investigated the role of Kupffer cells in the imbalance of the expression of vasoactive and inflammatory mediators during trauma and sepsis. The Kupffer cells were inactivated by gadolinium chloride (GdCl(3), 7.5 mg/kg body weight, i.v.) 1 and 2 d before surgery. The animals then underwent femur fracture (FFx) followed 48 h later by cecal ligation and puncture (CLP). After 24 h, blood was obtained to determine the alanine aminotransferase (ALT) activity. Liver samples were also taken for RT-PCR analysis of the mRNA for genes of interest. Serum ALT levels increased in FFx and CLP. This increase was potentiated by FFx + CLP and was attenuated by GdCl(3). The mRNA expression levels in the FFx showed no change in the ET(A), ET(B), iNOS, and HO-1, and showed a slight increase of 2.6-fold, 2.2-fold, and 2.8-fold for ET-1, eNOS, and TNF-alpha, respectively. The ET-1 mRNA expression level increased after CLP and FFx + CLP. The ET(A) mRNA level showed no change, whereas the ET(B) transcripts increased after CLP. This increase was potentiated after FFx + CLP, and was attenuated by GdCl(3). After CLP alone, iNOS, eNOS, and TNF-alpha mRNA expression levels were increased 20.3-fold, 5.8-fold, and 11.9-fold, respectively. This increase was potentiated after FFx + CLP, and was attenuated by GdCl(3). HO-1 mRNA expression significantly increased after FFx + CLP, and this increase was attenuated by GdCl(3). Mild trauma alone causes little change in the expression of vasoactive mediators while sequential injury potentiates the imbalanced gene expression induced by sepsis. Kupffer cells might be essential for hepatic microvascular dysfunction after sequential stress.

The effects of ketorolac tromethamine and baicalein on the levels of inflammatory factors in human synoviocytes

This study examined the effects of ketorolac tromethamine (KT) and baicalein (BE) on the levels of inflammatory factors in human synoviocytes. The fibroblast-like synoviocytes (FLS) cells were used to determine the possible regulatory effects of KT and BE (KTBE) on the levels of inflammatory factors in FLS cells. In addition, the levels of TNF-alpha, IL-6, and IL-1beta mRNA expression in FLS cells induced by a TNF-alpha and IL-1beta co-treatment were largely inhibited by a KTBE treatment. The level of FLS cells proliferation was increased by IL-1beta and TNF-alpha, and strongly inhibited by KTBE treatment. The production of oxygen species (ROS) was inhibited by KTBE in FLS cells. KTBE appears to regulate the levels of mRNA that are important for regulating RA progression.

Analyses of Vaccination Protocols for Leptospira interrogans Serovar Autumnalis in Hamsters

Leptospirosis, caused by Leptospira spp., is a zoonotic disease found worldwide. Killed whole cell leptospiral vaccines have been used as effective vaccines to elicit specific antibodies for protection. However, the involvement of cytokine responses after vaccination is not well characterized. Hamsters were immunized with killed L. interrogans serovar Autumnalis before challenge to study cytokine mRNA expression levels (interferon [IFN]-gamma, tumor necrosis factor [TNF]-alpha, interleukin [IL]-10, and IL-4). Vaccinated groups showed 92-100% survival rates, whereas control hamsters died within 6-10 days. However, live organisms were detected in vaccinated groups, and mild to moderate pathology was observed early in infection. IFN-gamma and TNF-alpha mRNA expression levels correlated with the severity of infection and lung pathology, whereas IL-4 and IL-10 expression levels were significantly higher in vaccinated groups. In summary, commonly used vaccines changed the cytokine profiles and protected hamsters from death but failed to stimulate sterile immunity and were unable to prevent the occurrence of pathology.

Macrophages regulate tumor necrosis factor-α expression in adipocytes through the secretion of matrix metalloproteinase-3

Adipocytes accumulated in the visceral area change their function to induce tumor necrosis factor-alpha (TNF-alpha) secretion with concomitant matrix metalloproteinase (MMP)-3 induction in mice. This study was performed to clarify the role of macrophages (Mphi)-secreted MMP on the functional changes in adipocytes using a culture system. Cultures of 3T3-L1 adipocytes with THP-1 Mphi or the Mphi-conditioned medium were used to investigate the role of Mphi-MMP on the TNF-alpha gene in 3T3-L1 adipocytes by the addition of MMP inhibitors. For animal experiments, male C57BL/6J mice were rendered insulin resistant by feeding a high-fat diet, and the expression of an Mphi marker F4/80, and MMP-3 genes in mesenteric and subcutaneous fat tissue specimens were examined. Mphi-conditioned media (Mphi-CM) increased the levels of TNF-alpha mRNA expression in 3T3-L1 adipocytes, and these adipocyte responses were abolished by treatment with GM6001, a broad-spectrum MMP inhibitor, or NNGH (N-isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid), an MMP-3 inhibitor. The activated form of MMP-3 enhanced glycerol release as well as TNF-alpha protein secretion from 3T3-L1 adipocytes. The incubation of adipocytes with MMP-3 inhibited insulin-induced glucose uptake in adipocytes. Furthermore, a high-fat intake increased the expression of MMP-3, decreased the insulin-induced glucose uptake of adipocytes and induced expression of F4/80 in mesenteric fat tissue of C57BL/6 mice. Mphi may cause a pathological link with surrounding adipocytes through the secretion of MMP-3 followed by TNF-alpha expression in adipocytes in visceral fat tissue.

Immunomodulatory effects of an enzymatic extract from Ecklonia cava on murine splenocytes.

We investigated whether the brown seaweed Alariaceae Ecklonia cava (E. cava) has immunological effects on splenocytes in vitro. For that purpose, we prepared an enzymatic extract from E. cava (ECK) by using the protease, Kojizyme. Here, ECK administered to ICR mice dramatically enhanced the proliferation of their splenocytes and increased the number of their lymphocytes, monocytes and granulocytes. In flow cytometry assays performed to identify in detail the specific phenotypes of these proliferating cells after ECK treatment, the numbers of CD4(+) T cells, CD8(+) T cells and CD45R/B220(+) B cells increased significantly compared to those in untreated controls. In addition, the mRNA expression and production level of Th1-type cytokines, i.e., TNF-alpha and IFN-gamma, were down-regulated, whereas those of Th2-type cytokines, i.e., IL-4 and IL-10, were up-regulated by ECK. Overall, this dramatic increase in numbers of splenocytes indicated that ECK could induce these cells to proliferate and could regulate the production of Th1- as well as Th2-type cytokines in immune cells. These results suggest that ECK has the immunomodulatory ability to activate the anti-inflammatory response and/or suppress the proinflammatory response, thereby endorsing its usefulness as therapy for diseases of the immune system.

Influence of renal involvement on peripheral blood mononuclear cell expression behaviour of tumour necrosis factor- and interleukin-6 in type 2 diabetic patients

Type 2 diabetes is associated with a high cardiovascular risk, which is even increased if renal damage is superimposed. Peripheral blood mononuclear cells (PBMCs) and pro-inflammatory cytokines are key factors linking type 2 diabetes and atherosclerosis. We investigated the influence of renal damage on serum, urinary and PBMCs expression behavior of TNF-alpha and IL-6 in these patients. PBMCs were isolated by density gradient centrifugation (Ficoll-Paque method) from fasting blood samples of 22 non-diabetic control subjects and 78 diabetic patients with normal renal function and different stages of diabetic nephropathy (18 with normoalbuminuria, 29 with microalbuminuria and 31 with macroalbuminuria). Expression levels of TNF-alpha and IL-6 were analyzed by real-time quantitative RT-PCR. Serum and urinary TNF-alpha and IL-6 concentrations were measured by a solid-phase, chemiluminescent immunometric assay. The mean percent increases in the serum and urinary levels of TNF-alpha and IL-6 in diabetic patients with respect to control subjects were 176% (P < 0.0001), 250% (P < 0.0001), 114% (P < 0.0001) and 39.6% (P = 0.01), respectively. The mRNA expression level of TNF-alpha was higher by 68.8% (P < 0.001) and IL-6 mRNA levels were higher by 64.1% (P < 0.001) with respect to non-diabetic controls. TNF-alpha mRNA expression in patients with macroalbuminuria was higher by 84.8% with respect to subjects with normalbuminuria (P < 0.001) and by 29% with respect to individuals with microalbuminuria (P < 0.05). Likewise, microalbuminuric patients showed a 44.5% increase in TNF-alpha mRNA expression compared to subjects with normoalbuminuria (P < 0.05). Concerning IL-6, the mRNA expression levels of this cytokine was higher by 63.1% with respect to normoalbuminuric subjects (P < 0.01), and by 23.1% with respect to patients with microalbuminuria (P < 0.05). However, with respect to controls, diabetic patients with normoalbuminuria had similar serum TNF-alpha and urinary excretion of IL-6, without any differences in the mRNA expression levels of these cytokines in PBMCs. Partial correlation and multiple regression analysis using TNF-alpha and IL-6 mRNA levels as the dependent variables showed that urinary albumin excretion (UAE) was direct and independently associated with the expression profile of these pro-inflammatory cytokines in PBMCs. These data show for the first time the relationship between inflammatory activation of PBMCs (reflected by enhanced mRNA expression of TNF-alpha and IL-6) and renal involvement (reflected by increased UAE) in type 2 diabetic patients. These results provide potential insights for the increased inflammation, accelerated atherosclerosis and cardiovascular risk associated with nephropathy in type 2 diabetes.