Mouse Tumor Necrosis Factor Alpha Research Articles

INHIBITION OF NITRIC OXIDE SYNTHASE ENHANCES THE PRODUCTION OF TUMOR NECROSIS FACTOR ‐ALPHA IN MACROPHAGE CELLS

We demonstrated earlier that systemic infusion of nitric oxide (NO) synthase inhibitor, nitro-L-arginine methyl ester (L-NAME) caused an increase in the plasma level of tumor necrosis factor-alpha (TNF-α) in mice. However, the source of TNF-α release during systemic NO blockade is not known. To determine the source, cultured macrophage cells were treated with L-NAME (100 μM; Sigma) or vehicle, and were incubated for 1, 2, 4 and 6 hours (n=6 in each treatment). TNF-α concentration in the condition media, harvested after each incubation period, was measured using a quantitative TNF-α ELISA kit. In another set, L-NAME treated cells were co-treated with a NO donor compound, Diethylenetriaamine NONOate, (DETA NONOate; 1 mM). The mRNA levels of T TNF-α was measured in cell extract by real time RT-PCR. TNF-α level in the condition media was increased in a time dependent manner in cells treated with L-NAME compared to vehicle (316 ± 68 vs 143 ± 32 pg/mL in 1st hour and 1459 ± 348 vs 414 ± 82 pg/mL in 6th hour). Compared to vehicle, there were also increases in TNF-α mRNA (0.62 ± 0.02 vs 1.68 ± 0.05 relative folds changes at 6thhour period) in cells treated with L-NAME. Co-treatment with L-NAME and DETA NONOate prevented the increased TNF-α production/release as well as mRNA expression of TNF-α. These data demonstrate that NO production exerts a tonic inhibition on TNF-α production and release in macrophage cells.

Antinociceptive and gastroprotective actions of ethanolic extract from Pluchea sagittalis (Lam.) Cabrera

Pluchea sagittalis, an herbaceous plant widely distributed in South America, is used in folk medicine for the treatment of digestive diseases and inflammation. This study was designed to investigate the antinociceptive and gastroprotective effects of the ethanolic extract (EE) of aerial parts from Pluchea sagittalis in rodents. The antinociceptive effects of EE was evaluated in mice after oral administration in chemical tests (acetic-acid, glutamate and formalin) or by biting behavior following intrathecal administration of cytokines such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in mice. Furthermore, rats were treated with EE and subsequently exposed to acute gastric lesions induced by 80% ethanol. Afterwards the gastric lesion extension and the mucus levels of gastric mucosa were measured. The oral administration of EE showed a dose-dependent inhibition of acetic acid-induced abdominal constrictions and glutamate-induced pain in mice, with ID(50) values of 624.0 (523.0-746.0) mg/kg and 368.0 (216.0-628.0) mg/kg, respectively. In the formalin test, the EE also produced significant inhibition of the inflammatory phase, with an ID(50) value of 411.0 (183.0-721.0) mg/kg; however, it was ineffective in the neurogenic phase caused by formalin. In addition, oral treatment with EE caused a significant inhibition of biting behavior induced by i.t. injection of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). The antinociception caused by the EE (300 mg/kg, p.o.) was not reversed by naloxone (1 mg/kg, i.p.) when assessed in the acetic acid writhing test. The EE (300-1000 mg/kg, p.o.) did not affect the motor coordination of animals in an open-field model. Oral treatment with the EE protected rats against gastric lesions induced by ethanol, with an ID(50) value of 55.0 (46.6-64.9) mg/kg, and increased the mucus levels of gastric mucosa to levels found in the non-lesioned group. The mechanism by which the extract produced antinociception still remains unclear, but this effect seems to be primarily related to the modulation or inhibition of the action of pro-inflammatory mediators. Furthermore, these data support, at least in part, the ethnomedical use of Pluchea sagittalis.

Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations

Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor. In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered. Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities. The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled, e.g., by appropriate medium formulation and substrate dosing. Overflow metabolism as well as high biomass growth rates must be avoided because they reduce protein yields. Further investigation of the intracellular metabolic fluxes should be conducted to fully unravel and identify ways to relieve the metabolic burden of plasmid maintenance and heterologous protein production and to prevent overflow.

Effects of Qingyi Huaji decoction on serum levels of interleukin-6, interleukin-8 and tumor necrosis factor-α in nude mice bearing pancreatic tumors

To investigate the effects of Qingyi Huaji (QYHJ) decoction, a compound traditional Chinese herbal medicine, on tumor inhibition rate and serum levels of interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor-alpha (TNF-alpha) in nude mice with transplanted tumors of human pancreatic cancer. The tumor-bearing mice model was established by subcutaneously inoculating with xenografts of pancreatic cancer into the right armpit of 40 BALB/c nude mice. After successful modeling, the mice were randomly divided into untreated group (Arabic gum), capecitabine group, low-dose QYHJ decoction group (36 g/kg) and high-dose QYHJ decoction group (72 g/kg), with 10 mice in each group. Citrate buffer solution (containing 5% Arabic gum), capecitabine suspension and QYHJ decoction were administered to four groups by gavage respectively. After 5-week treatment, the concentrations of serum IL-6, IL-8 and TNF-alpha were examined by enzyme-linked immunosorbent assay (ELISA) using blood sample from eye socket. Then the mice were euthanized by cervical dislocation. Tumor weight and the tumor inhibition rate were calculated. Tumor weight in the low-dose QYHJ decoction group decreased significantly as compared with the untreated group (P<0.05). Serum levels of IL-6 and TNF-alpha in low- and high-dose QYHJ groups were extremely significantly lower than those in the untreated group (P<0.01). Serum level of IL-8 in the low-dose QYHJ group was significantly lower than that in the untreated group (P<0.05). Correlation analysis showed that transplanted tumor weight of the mice was linearly positively correlated with serum levels of IL-6, IL-8 or TNF-alpha (P<0.01). Conventional dose of QYHJ decoction is effective in suppressing pancreatic carcinoma in nude mice. The mechanism may be related to down-regulation of serum cytokines such as IL-6, IL-8 and TNF-alpha.

Hepatocyte nuclear factor-kappa β (NF-κB) activation is protective but is decreased in the cholestatic liver with endotoxemia

Obstructive jaundice (OJ) is an important clinical consideration associated with a high risk of bacteremia. Hepatocyte nuclear factor-kappa B (NF-kappaB) activation confers an antiapoptotic function. Although the occurrence of hepatocyte apoptosis has been shown in OJ, the activation and role of NF-kappaB over the time course of OJ in conjunction with endotoxemia have not yet been well defined. We hypothesized that NF-kappaB activation may be decreased over the time course of OJ and endotoxemia, which leads to severe liver injury. The aim of the current study was to examine whether NF-kappaB activation can decrease hepatocyte apoptosis and liver injury over the time course of OJ in response to lipopolysaccharide (LPS) administration. Male C57BL/6 mice were subjected to bile duct ligation and were administered LPS intravenously at 3 days (OJ3) or 14 days (OJ14) after bile duct ligation. NF-kappaB activation; protein expressions of NF-kappaB p65, IkappaB-alpha, Ikappabeta-b, and Pin1; immunohistochemistry of poly adenosine diphosphate (ADP)-ribose polymerase p85 fragment (PARP); and serum alanine transaminase (ALT) levels were examined. Hepatocyte NF-kappaB activation was observed during OJ. After LPS administration, the hepatic NF-kappaB activation defined by electrophoretic mobility shift assay was decreased in the OJ14 group compared with the OJ3 group, which is consistent with a decrease in NF-kappaB p65 protein expression. Changes in phosphorylated Ikappa-B-beta but not phosphorylated IkappaB-alpha mirrored these results. Significant hepatocyte apoptosis defined by PARP immunohistochemistry was observed in the LPS-treated OJ14 relative to the LPS-treated OJ3. Hepatic expressions of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the LPS OJ14 mice were upregulated relative to those in the LPS OJ3. Serum ALT levels increased significantly in the LPS OJ14 relative to other mice. The survival rate was significantly less in the LPS OJ14 relative to other mice. After prolonged OJ, exposure to endotoxemia was associated with a decrease in hepatocyte NF-kappaB activation and an increase in hepatocyte apoptosis and secondary necrosis, thus resulting in liver dysfunction.

Ca2+/calmodulin-dependent protein kinase II inhibitors potentiate superoxide production in polymorphonuclear leukocytes.

The possible role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in superoxide anion (O2-) production induced by formyl-methionyl-leucyl-phenylalanine (FMLP) was investigated in mouse polymorphonuclear leukocytes (PMNs). KN-93 and KN-62, specific CaMK II inhibitors, augmented FMLP-induced O2- production. KN-92, an analogue which did not inhibit CaMK II, did not affect O2- production. W-7, a calmodulin inhibitor, augmented O2- production when administered at 30 mM for 5 min. KN-93 and recombinant mouse tumour necrosis factor-alpha (rmTNF-alpha) each augmented the maximal production of O2- induced by FMLP, and an additive effect of a combination of KN-93 and rmTNF-alpha was observed. CaMK II activity in the PMNs was increased by FMLP, and the increase was inhibited by KN-93 but not by rmTNF-alpha. These results suggest that the inhibition of CaMK II resulted in the augmentation of FMLP-induced O2- production in PMNs by a mechanism different from that of the augmentation shown by TNF-alpha.

Assessment of adaptive focused acoustics versus manual vortex/freeze-thaw for intracellular metabolite extraction from Streptomyces lividans producing recombinant proteins using GC-MS and multi-block principal component analysis

We compared the gas chromatography-mass spectrometry (GC-MS) metabolite profiles of mouse tumour necrosis factor alpha (mTNF-alpha) secreting Streptomyces lividans TK24 to the non-secreting wild type and the wild type harbouring the empty pIJ486 plasmid by multi-block principal component analysis (PCA). The multi-block PCA model successfully identified peaks that were statistically different between the protein secreting and non-secreting strains, and at the same time also uncovered the efficiency of intracellular metabolite extraction by an ultrasonic adaptive focused acoustics (AFA) technique compared to a manual vortex/freeze-thaw method. Fifty-one metabolites were significantly different between the three biological strains and 17 of these were abundant in the mTNF-alpha secreting strain compared to the non-secreting strains. No significant differences in the number of detected metabolite peaks were observed between the two extraction techniques. However, from the loadings of the multi-block PCA model, as well as univariate statistical analysis, we observed that the relative peak response ratios to the internal standard of 10 metabolites were higher for the AFA extraction, suggesting a more efficient recovery of these metabolites than achieved with the manual vortex/freeze thaw method.

Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis

Wolbachia endosymbiotic bacteria have been implicated in the inflammatory pathogenesis of filariasis. Inflammation induced by Brugia malayi female worm extract (BMFE) is dependent on Toll-like receptors 2 and 6 (TLR2/6) with only a partial requirement for TLR1. Removal of Wolbachia, lipids, or proteins eliminates all inflammatory activity. Wolbachia bacteria contain the lipoprotein biosynthesis genes Ltg and LspA but not Lnt, suggesting Wolbachia proteins cannot be triacylated, accounting for recognition by TLR2/6. Lipoprotein databases revealed 3–11 potential lipoproteins from Wolbachia. Peptidoglycan-associated lipoprotein (PAL) and Type IV secretion system-VirB6 were consistently predicted, and B. malayi Wolbachia PAL (wBmPAL) was selected for functional characterization. Diacylated 20-mer peptides of wBmPAL (Diacyl Wolbachia lipopeptide (Diacyl WoLP)) showed a near identical TLR2/6 and TLR2/1 usage compared with BMFE and bound directly to TLR2. Diacyl WoLP induced systemic tumor necrosis factor-α and neutrophil-mediated keratitis in mice. Diacyl WoLP activated monocytes induce up-regulation of gp38 on human lymphatic endothelial cells and induced dendritic cell maturation and activation. Dendritic cells primed with BMFE generated a non-polarized Th1/Th2 CD4+ T cell profile, whereas priming with Wolbachia depleted extracts (following tetracycline treatment; BMFEtet) polarized to a Th2 profile that could be reversed by reconstitution with Diacyl WoLP. BMFE generated IgG1 and IgG2c antibody responses, whereas BMFEtet or inoculation of TLR2 or MyD88−/− mice produced defective IgG2c responses. Thus, in addition to innate inflammatory activation, Wolbachia lipoproteins drive interferon-γ-dependent CD4+ T cell polarization and antibody switching.

Chronic restraint stress impairs T-cell immunity and promotes tumor progression in mice

Long-term exposure to stressful situations can affect the immune system. The T-cell response is an important component of anti-tumoral immunity. Hence, impairment of the immune function induced by a chronic stressor has been postulated to alter the immunosurveillance of tumors, thus leading to a worse neoplastic prognosis. Here, we show that chronic restraint stress affects T-cell mediated immunity in mice. This was evidenced by a decrease of mitogen-induced T-cell proliferation, a reduction in CD4+T lymphocyte number and a decrease of tumor necrosis factor-alpha (TNF-α) and Interferon-gamma (IFN-γ) production in stressed mice. Additionally, mice subjected to chronic restraint stress displayed an enhancement of tumor growth in a syngeneic lymphoma model, i.e. an increase of tumor proliferation and a reduction of animal survival. Finally, stressed mice had a reduced specific cytotoxic response against these tumor cells. These results suggest that chronic exposure to stress promotes cancer establishment and subsequent progression, probably by depressing T-cell mediated immunity. The T-cell immunity impairment as well as the tumor progression enhancement emphasize the importance of the therapeutic management of stress to improve the prognosis of cancer patients.

Effects of Administration of a Monoclonal Antibody against Mouse Tumor Necrosis Factor Alpha during Pregnancy and Lactation on the Pre- and Postnatal Development of the Mouse Immune System

Monoclonal antibodies directed against tumor necrosis factor alpha (TNFalpha) are currently employed in the treatment of various immune-mediated diseases. These studies were designed to evaluate potential effects of anti-TNFalpha treatment in mice during pregnancy and lactation on the development of the immune system in the F1 generation. Pregnant CD-1 mice were treated with vehicle or with 10 or 40 mg/kg of an anti-mouse TNFalpha monoclonal antibody (mAb) (cV1q) on days 6, 12, and 18 of gestation and on days 3, 9, and 15 of lactation. Evaluation of immune system functionality was conducted in F1 generation mice at 11 weeks of age. Immune function was evaluated by splenocyte phenotyping, immunoglobulin M (IgM) antibody response to sheep red blood cells (SRBCs), spleen cell proliferative response to anti-CD3, and natural killer cell activity. Treatment of pregnant mice with cV1q produced no adverse effects in the dams and no adverse effects in the F1 generation. In general, the functioning of the immune system of the F1 generation did not appear to be adversely affected following exposure to cV1q in utero and during lactation. The only statistically significant change was a slight (approximately 20%) reduction in the spleen cell expansion in response to SRBC immunization in the female F1 mice from the 40 mg/kg cV1q treatment group. In conclusion, administration of a monoclonal antibody against mouse TNFalpha during pregnancy and lactation had little or no effect on selected immune parameters in mice, with only a possible minor attenuation of spleen cell response to immunization noted in the female F1 generation at 11 weeks of age.

Co-expression of IL-18 binding protein and IL-4 regulates Th1/Th2 cytokine response in murine collagen-induced arthritis

We constructed a recombinant adenoviral vector containing a murine interleukin (IL)-18 binding protein (mIL-18BP) and murine IL-4 (mIL-4) fusion gene (AdmIL-18BP/mIL-4) and used a gene therapy approach to investigate the role of IL-18BP and IL-4 in modulating the T-helper1 and T-helper2 (Th1/Th2) balance in mice with collagen-induced arthritis (CIA). Mice with CIA were intra-articularly injected with 107 pfu/6 microl of either AdmIL-18BP/mIL-4, or a control adenovirus, or with the control vehicle (phosphate-buffered saline). After intra-articular gene therapy with AdmIL-18BP/mIL-4, the serum levels of tumor necrosis factor-alpha (TNF-alpha), gamma-interferon (IFN-gamma), IL-4, IL-10, and IL-18 in mice with CIA were assessed by ELISA. IFN-gamma-expressing and IL-4-expressing CD4+ T cells from mice splenocytes were monitored by flow cytometry. Mice with CIA at weeks 1, 2, and 4 after intra-articular injection of AdmIL-18BP/mIL-4 showed significantly increased serum concentrations of IL-4 and IL-10 (P<0.01 at all time points) but greatly decreased serum concentrations of IFN-gamma, TNF-alpha, and IL-18 (P<0.01 at all time points) compared to both the control adenovirus and phosphate-buffered saline control groups. The percentage of IFN-gamma-producing CD4+ T cells was significantly decreased in response to local AdmIL-18BP/mIL-4 treatment. The percentage of IL-4-producing CD4+ T cells increased significantly at 1 week after local injection of AdmIL-18BP/mIL-4 then returned to normal by week 4. These data indicated the significant modifying effects on the Th1/Th2 imbalance in murine CIA produced by local overexpression of IL-18BP and IL-4. Combination treatment with IL-18BP and IL-4 is a promising potential therapy for rheumatoid arthritis.

Protective Effect of Lignans against Sepsis from the Roots of &lt;i&gt;Saururus chinensis&lt;/i&gt;

In the course of isolating preventive agents from sepsis based on the in vivo assay model from the EtOAc extract of the roots of Saururus chinensis, twelve lignans, sarisan (1), erythro-austrobailignan-6 (2), meso-dihydroguaiaretic acid (3), saucerneol B (4), manassantin B (5), manassantin A (6), rel-(8R,8'R)-dimethyl-(7S,7'R)-bis(3,4-methylenedioxyphenyl)tetrahydro-furan (7), (+)-saururinone (8), sauchinone (9), sauchinone B (10), nectandrin B (11) and machilin D (12), were isolated. Compounds 9 and 10, at a dose of 10 mg/kg, increased survival rates to 80% from 20% for the control experiment, and decreased the plasma levels of tumor necrosis factor-alpha (TNF-alpha) and alanine aminotransferase (ALT) activity in mice administered LPS/D-GalN.

Immunosensors Based on Functional Nanoparticle Labels

The electrochemical immunosensors based on functional nanoparticle (NP) labels were described. We have developed functionalized silica NPs conjugates and functionalized gold nanoparticles for sensitive immunoassay of proteins such as mouse IgG and tumor necrosis factor-alpha (TNF-alpha). These functionalized NPs were characterized by AFM, TEM, X-ray photoelectron spectroscopy, or electrochemistry. And the analytical performance of these immunosensors was examined. It is found that these methods are very sensitive and the limit of detection was found to be down to 1.0 pM level. The work demonstrates the feasibility of developing a cheap, sensitive, and portable device for multiplexed diagnosis of different proteins. This method is simple, selective and reproducible for trace protein analysis and can be extended to study protein/protein, peptide/protein, and DNA/ protein interactions.

Adriamycin‐mediated nitration of manganese superoxide dismutase in the central nervous system: insight into the mechanism of chemobrain

Adriamycin (ADR), a potent anti-tumor agent, produces reactive oxygen species (ROS) in cardiac tissue. Treatment with ADR is dose-limited by cardiotoxicity. However, the effect of ADR in the other tissues, including the brain, is unclear because ADR does not pass the blood-brain barrier. Some cancer patients receiving ADR treatment develop a transient memory loss, inability to handle complex tasks etc., often referred to by patients as chemobrain. We previously demonstrated that ADR causes CNS toxicity, in part, via systemic release of cytokines and subsequent generation of reactive oxygen and nitrogen species (RONS) in the brain. Here, we demonstrate that treatment with ADR led to an increased circulating level of tumor necrosis factor-alpha in wild-type mice and in mice deficient in the inducible form of nitric oxide (iNOSKO). However, the decline in mitochondrial respiration and mitochondrial protein nitration after ADR treatment was observed only in wild-type mice, not in the iNOSKO mice. Importantly, the activity of a major mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), was reduced and the protein was nitrated. Together, these results suggest that NO is an important mediator, coupling the effect of ADR with cytokine production and subsequent activation of iNOS expression. We also identified the mitochondrion as an important target of ADR-induced NO-mediated CNS injury.

A new chapter opens in anti-inflammatory treatments:The antidepressant bupropion lowers production of tumor necrosis factor-alpha and interferon-gamma in mice

In a wide range of human diseases of inflammatory nature like Crohn's disease, pathology is mediated in part by pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF) or interferon-gamma. We show here that a commonly used generic antidepressant bupropion, in wide use worldwide to treat depression in humans for a decade now, profoundly lowers levels of TNF, interferon-gamma, and interleukin-1 beta in vivo, in a mouse lipopolysaccharide (LPS) induced inflammation model. Mice challenged with an otherwise lethal dose of LPS were protected by bupropion and levels of the anti-inflammatory cytokine interleukin-10 were increased. Previous data in rodents and humans indicate antidepressant effects of bupropion are mediated by its weak reuptake inhibition of norepinephrine and dopamine. Concordant with this, TNF suppression by bupropion in our mouse LPS model was largely abrogated by beta-adrenergic or dopamine D1 receptor antagonists but not by a D2 antagonist. TNF synthesis is controlled by an inverse relationship with intracellular cyclic adenosine monophosphate (cAMP) and stimulation of either beta-adrenoreceptors or D1 dopaminergic receptors result in increased cAMP but stimulation of D2 receptors lowers cAMP. We conclude that bupropion may suppress TNF synthesis by mediating increased signaling at beta-adrenoreceptors and D1 receptors, resulting in increased cAMP that inhibits TNF synthesis. Bupropion is well tolerated also in non-psychiatric populations and has less risk with long term use than current anti-inflammatory, immunosuppressive or TNF suppressive treatments such as prednisone, azathioprine, infliximab, or methotrexate. New anti-inflammatory treatments are needed. We believe a new chapter in antiinflammatory, TNF lowering treatment of disease has been opened. Bupropion's use for this in humans should be explored.

Contribution of Transmembrane Tumor Necrosis Factor to Host Defense against Mycobacterium bovis Bacillus Calmette-Guerin and Mycobacterium tuberculosis Infections

To study the specific role of transmembrane tumor necrosis factor (TmTNF) in host defense mechanisms against bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis infections, we compared the immune responses of TNF/lymphotoxin (LT)-alpha(-/-) mice expressing a noncleavable transgenic TmTNF (TmTNF tg) to those of TNF/LT-alpha(-/-) and wild-type mice. Susceptibility of TNF/LT-alpha(-/-) mice to BCG infection was associated with impaired induction of systemic RANTES but not of monocyte chemoattractant protein 1 (MCP-1), the development of excessive local and systemic Th1-type immune responses, and a substantially reduced inducible nitric oxide synthase (iNOS) activity. Resistance of TmTNF tg mice to BCG infection was associated with efficient activation of iNOS in granulomas and with the regulated release of local and systemic chemokines and Th1-type cytokines. However, M. tuberculosis infection of TmTNF tg mice resulted in longer survival and enhanced resistance compared to TNF/LT-alpha(-/-) mice but higher sensitivity than wild-type mice. TmTNF tg mice exhibited reduced pulmonary iNOS expression and showed an exacerbated cellular infiltration in the lungs despite a modest bacillary content. Our data thus indicate a role for TmTNF in host defense against mycobacteria by contributing to induction and regulation of Th1-type cytokine and chemokine expression leading to development of bactericidal granulomas expressing iNOS, which critically determines susceptibility versus resistance of the host to mycobacterial infections.

Agents Protecting against Sepsis from the Roots of Angelica dahurica

In the course of isolating agents preventing sepsis from the EtOAc extract of the roots of Angelica dahurica, four known furanocoumarins, isoimperatorin (1), oxypeucedanin (2), (+/-)-byakangelicin (3), and (+)-oxypeucedanin hydrate (4), were isolated as active compounds based on the in vivo assay model of sepsis induced by lipopolysaccharide (LPS) and D-galactosamine (D-GalN). Among them, 3 showed the highest survival rate (100% with a dose of 30 mg/kg versus 20% for the control experiment) and decreased the plasma levels of tumor necrosis factor-alpha and alanine aminotransferase in mice adminstered LPS/D-GalN.

Five Cysteine-Containing Compounds Delay Diabetic Deterioration in Balb/cA Mice

The effects of n-acetyl cysteine (NAC), s-allyl cysteine (SAC), s-ethyl cysteine, s-methyl cysteine and s-propyl cysteine (SPC) activity on Balb/cA mice against diabetic complications were examined. These complications included hyperglycemia, hyperlipidemia, oxidation stress, blood coagulation, and cytokine imbalance. To induce diabetes, mice were treated with streptozotocin i.p. for 5 consecutive days. Five cysteine-containing compounds at 1 g/L were added to the drinking water. After intake of the 5 cysteine-containing agents for 4 wk, body weight loss, plasma concentrations of glucose and insulin, and fibronectin levels were improved (P < 0.05) in diabetic mice. The administration of these agents restored the glutathione level (P < 0.05), reduced the loss of catalase and glutathione peroxidase activities in kidney and liver (P < 0.05), and decreased glucose-induced lipid oxidation, as assessed by malondialdehyde formation (P < 0.05). In all diabetic mice, the intake of these agents reduced triglyceride levels in plasma and liver (P < 0.05); however, only NAC, SAC and SPC treatments reduced cholesterol level in liver (P < 0.05). These cysteine-containing agents elevated the activity of 2 fibrinolytic factors, protein C and antithrombin III (P < 0.05). The overexpression of interleukin-6 and tumor necrosis factor-alpha in diabetic mice was suppressed by the intake of the 5 cysteine-containing agents (P < 0.05). Via their antioxidant activities, the 5 compounds effectively improved glycemic control, delayed oxidation damage, downregulated inflammatory cytokines, and enhanced anticoagulant activity in diabetic mice. These data support the multiple roles of these agents as potential protective agents for delaying diabetic deterioration.

Azithromycin blocks neutrophil recruitment in Pseudomonas endobronchial infection.

Macrolides exert their effects on the host by modulation of immune responses. In this study, we assessed the therapeutic efficacy of azithromycin in a murine model of mucoid Pseudomonas aeruginosa endobronchial infection. The clearance of Pseudomonas from the airway of mice treated with the macrolide azithromycin was not different than untreated mice challenged with Pseudomonas beads. However, the azithromycin-treated mice showed a remarkable reduction in lung cellular infiltrate in response to Pseudomonas beads, as compared with untreated mice. This effect was associated with significant decreases in lung levels of tumor necrosis factor-alpha and keratinocyte-derived chemokine in azithromycin-treated mice compared with untreated mice. Furthermore, there was a significant reduction in the response of both mouse and human neutrophils to chemokine-dependent and -independent chemoattractants when studied in vitro. Inhibition of chemotaxis correlated with azithromycin-mediated inhibition of extracellular signal-regulated kinase-1 and -2 activation. This study indicates that the azithromycin treatment in vivo results in significant reduction in airway-specific inflammation, which occurs in part by inhibition of neutrophil recruitment to the lung through reduction in proinflammatory cytokine expression and inhibition of neutrophil migration via the extracellular signal-regulated kinase-1 and -2 signal transduction pathway.

Protective effect of exogenous recombinant mouse interferon-gamma and tumour necrosis factor-alpha on ectromelia virus infection in susceptible BALB/c mice.

The resistance to mousepox is correlated with the production of type I cytokines: interleukin (IL)-2, IL-12, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha. We intend to describe the modulation of generalized ectromelia virus (EV) infection with exogenous administration of mrIFN-gamma and mrTNF-alpha separately and in combination using susceptible BALB/c mice. The treatment schemes presented resulted in the localization of the generalized EV infection and its development into non-fatal sloughing of the infected limb. This was accompanied by low virus titres in the treated mice due to control of systemic virus replication and virus clearance. The balance of type I versus type II cytokines was dominated by a type I response in the treated groups. The group treated with the combination of IFN-gamma and TNF-alpha exhibited the best survival with Th1-dominant (IFN-gamma and IL-12) cytokine profiles, whereas the TNF-alpha-treated group of mice was less successful in clearance of virus and demonstrated the lowest survival rate. The successful cytokine treatment schemes in this orthopoxvirus model system may have important implications in the treatment of viral diseases in humans and, in particular, of variola virus infection.