Tissue Necrosis Factor Research Articles

Effect of maternal seaweed extract supplementation on suckling piglet growth, humoral immunity, selected microflora, and immune response after an ex vivo lipopolysaccharide challenge1

The present study was conducted to investigate the effect of maternal dietary supplementation (n = 10 sows/treatment) with seaweed extract (SWE: 0 vs. 10.0 g/d) from d 107 of gestation until weaning (d 26) on neonatal piglet growth, humoral immunity, intestinal morphology, selected intestinal microflora, and VFA concentrations. Furthermore, this study examined the effect of dietary treatment on the immune response after an ex vivo Escherichia coli lipopolysaccharide (LPS) tissue challenge at weaning in a 2 × 2 factorial arrangement. The main factors consisted of sow dietary treatment (SWE or control) and immunological challenge (yes or no). The SWE supplement (10.0 g/d) contained laminarin (1.0 g), fucoidan (0.8 g), and ash (8.2 g) and was extracted from a Laminaria spp. The SWE-supplemented sows had greater colostrum IgA (P < 0.01) and had a trend for greater IgG (P = 0.062) concentrations compared with non-SWE-supplemented sows. Piglets suckling SWE-supplemented sows had greater serum IgG (P < 0.05) concentrations on d 14 of lactation compared with those suckling non-SWE-supplemented sows. Dietary SWE supplementation decreased fecal Enterobacteriaceae populations in sows at parturition (P < 0.05), and piglets suckling SWE-supplemented sows had a decreased colonic E. coli population at weaning (P < 0.01) compared with non-SWE-supplemented sows. Lipopolysaccharide challenge increased the mRNA abundances of the pro-inflammatory cytokines IL-1α and IL-6 (P < 0.01) in ileal tissue and tumor necrosis factor (TNF)-α in colonic (P < 0.01) tissue. There was a treatment × LPS challenge interaction for ileal TNF-α mRNA expression (P < 0.05). Piglets suckling SWE-supplemented sows had greater TNF-α mRNA expression after ex vivo LPS challenge compared with non-SWE-supplemented sows (P < 0.05). However, there was no effect of sow dietary treatment on TNF-α mRNA expression in the unchallenged ileal tissue. Piglet BW at birth and weaning, and small intestinal morphology were unaffected by sow dietary treatment under current experimental conditions. In summary, these results demonstrate an important immunomodulatory role of SWE supplementation characterized by enhanced colostral IgA and IgG concentrations, greater piglet circulatory IgG concentrations on d 14 of lactation, and enhanced TNF-α mRNA expression in the ileum after an ex vivo LPS challenge. These results indicate that SWE supplementation enhanced piglet immune function and colonic microflora at weaning.

Proinflammatory cytokines in the prefrontal cortex of teenage suicide victims

Teenage suicide is a major public health concern, but its neurobiology is not well understood. Proinflammatory cytokines play an important role in stress and in the pathophysiology of depression—two major risk factors for suicide. Cytokines are increased in the serum of patients with depression and suicidal behavior; however, it is not clear if similar abnormality in cytokines occurs in brains of suicide victims. We therefore measured the gene and protein expression levels of proinflammatory cytokines interleukin (IL)-1β, IL-6, and tissue necrosis factor (TNF)-α in the prefrontal cortex (PFC) of 24 teenage suicide victims and 24 matched normal control subjects. Our results show that the mRNA and protein expression levels of IL-1β, IL-6, and TNF-α were significantly increased in Brodmann area 10 (BA-10) of suicide victims compared with normal control subjects. These results suggest an important role for IL-1β, IL-6, and TNF-α in the pathophysiology of suicidal behavior and that proinflammatory cytokines may be an appropriate target for developing therapeutic agents.

Bacteria, good and bad: Host–microbiota interactions in inflammatory bowel disease

Inflammatory bowel disease (IBD), a term encompassing the conditions ulcerative colitis (UC) and Crohn's disease, affects around 1 in 300 people in the UK and causes significant morbidity, although, thankfully, little mortality1. Although UC is limited to the colon and causes mucosal ulceration, Crohn's disease can affect any part of the intestine and causes ulceration, transmural inflammation, stricturing, fistula and abscess formation. Both are relapsing and remitting diseases and, despite advances in medical treatments, including immunosuppressants and drugs which specifically block pro-inflammatory molecules (tissue necrosis factor; TNF), about 25% with UC and 50–80% with Crohn's disease will require major surgery at least once in their lives.

Evaluation of SV40-transformed synovial fibroblasts in the study of rheumatoid arthritis pathogenesis

The SV40 T antigen has been used to generate immortalized cells from rheumatoid arthritis (RA) synovial fibroblasts (RASFs) that are commonly used in lieu of primary RASFs. In the current study, we investigated the effect of stimulation by tissue necrosis factor alpha (TNF-α) and interleukin 17 (IL-17) on primary and immortalized RASFs in order to gauge the appropriateness of the use of immortalized RASFs, the MH7A cell line, in the study of RA pathogenesis. Changes in the levels of secretion and expression of 8 proteins associated with RA upon stimulation were assessed by multiplex immunoassay. IL-17 stimulation had a minimal impact on protein secretion and expression for primary and immortalized cells. Basic fibroblast growth factor (FGF-2) was not detectable for the primary cells but was detectable for the immortalized cells. In contrast, monocyte chemoattractant protein 1 (MCP-1) was detectable for primary cells but was undetectable for immortalized cells. In general, protein expression and secretion by cells stimulated with TNF-α were significantly increased. For primary cells, several proteins were below the limit of detection for unstimulated cells and cells stimulated with IL-17, while levels for TNF-α-stimulated cells were within the detectable range. For the same proteins, expression was observed for immortalized cells, regardless of stimulation, suggestive of constitutive activation of the NF-κB signaling pathway. The current study therefore provides strong evidence that immortalized and primary RASFs differ in regard to protein expression and secretion and therefore may not be appropriate for use in the study of RA pathogenesis.

A Medicinal Plant Extract of Scutellaria Baicalensis and Acacia Catechu Reduced LPS-Stimulated Gene Expression in Immune Cells: A Comprehensive Genomic Study Using QPCR, ELISA, and Microarray

ABSTRACTA standardized, combined flavonoid extracts of Scutellaria baicalensis and Acacia catechu, UP446, demonstrates favorable anti-inflammatory properties. In this study, DNA microarray, quantitative polymerase chain reaction (QPCR), and enzyme-linked immunosorbent assay (ELISA) were used to study the effect of UP446 on the lipopolysaccharide (LPS)-induced pro-inflammatory gene regulation of both animal and human immortalized cell lines and also primary human cells. One consistent result from microarray was that the gene expression levels stimulated or suppressed by LPS were returned to normal levels by the UP446 co-treatment. This normalization effect from UP446 was also shown for pro-inflammatory genes cyclooxygenase (COX)-2, tissue necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6 using QPCR, and TNF-α using ELISA. The controlling transcriptional factor of these genes, NFκB, was also down-regulated by UP446 in the LPS-induced cell models. Microarray analysis for numerous genes, including cytokines, chemokines, receptors, transcriptional factors, caspase, growth factors, and phosphatases, suggests not only a genomic anti-inflammatory activity for UP446 but also signaling pathways of cell proliferation, cell death, and lipid metabolism demonstrated on different types of cells.

Activation of transglutaminase type 2 for aortic wall protection in a rat abdominal aortic aneurysm formation

The altered structure and composition of the vascular extracellular matrix (ECM) influences the formation of abdominal aortic aneurysms (AAA). Transglutaminase type 2 (TG2), which is a Ca(2+)-dependent cross-linking enzyme, has been proven the importance for ECM homeostasis, but there is no evidence of TG2 in AAA formation. The hypothesis was investigated that TG2 contributes to protect aortic walls during remodeling of the AAAs. In a rat abdominal aortic aneurysm model using a combination of intraluminal elastase infusion and extraluminal calcium chloride, TG2 expression and activity were evaluated at 1 and 8 weeks after the AAA preparation (n = 6 at each endpoint), compared with those of the non-prepared aorta (n = 6). Additionally, ex vivo experiments of isolated AAA tissue culture with recombinant human TG2, TG2 inhibitor cystamine, or tissue necrosis factor (TNF)-α were performed. TG2 mRNA expression in the AAAs was significantly upregulated at both 1 and 8 weeks (22.4-fold and 5.4-fold increases of the non-prepared aorta, P = .0022 and P = .0048, respectively). TG2 protein expression and activity were also enhanced by fluorescent staining of the AAAs. Similar mRNA upregulation of TNF-α, interleukin-1β, matrix metalloproteinases (MMP)-2, MMP-9, and tissue inhibitors of metalloproteinases (TIMP)-1 and TIMP-2 was observed in the AAAs, and TG2 and TNF-α were colocalized in the aortic walls at 1 week. Ex vivo experiments showed that mRNA expressions of TNF-α, MMP-2, and MMP-9 in the cultured AAA tissue were decreased by exogenous TG2, whereas were increased by cystamine. TNF-α exposure to the AAA tissues was significantly upregulated TG2 mRNA expression (P = .0333). TG2 expression and activity in AAA formation were enhanced, possibly due to compensatory reaction. TG2 has a potential role of ECM protector in aortic walls during remodeling of the AAAs.

On the Relevance of the Met-turn Methionine in Metzincins

The metzincins are a clan of metallopeptidases consisting of families that share a series of structural elements. Among them is the Met-turn, a tight 1,4-turn found directly below the zinc-binding site, which is structurally and spatially conserved and invariantly shows a methionine at position 3 in all metzincins identified. The reason for this conservation has been a matter of debate since its discovery. We have studied this structural element in Methanosarcina acetivorans ulilysin, the structural prototype of the pappalysin family, by generating 10 mutants that replaced methionine with proteogenic amino acids. We compared recombinant overexpression yields, autolytic and tryptic activation, proteolytic activity, thermal stability, and three-dimensional structure with those of the wild type. All forms were soluble and could be purified, although with varying yields, and three variants underwent autolysis, could be activated by trypsin, and displayed significant proteolytic activity. All variants were analyzed for the thermal stability of their zymogens. None of the mutants analyzed proved more stable or active than the wild type. Both bulky and small side chains, as well as hydrophilic ones, showed diminished thermal stability. Two mutants, leucine and cysteine, crystallized and showed three-dimensional structures that were indistinguishable from the wild type. These studies reveal that the Met-turn acts as a plug that snugly inserts laterally into a core structure created by the protein segment engaged in zinc binding and thus contributes to its structural integrity, which is indispensable for function. Replacement of the methionine with residues that deviate in size, side-chain conformation, and chemical properties impairs the plug-core interaction and prejudices molecular stability and activity.

Brain p44/42 mitogen‐activated protein kinase contributes to the sympathetic response to blood‐borne TNF‐α in rats

Blood‐borne pro‐inflammatory cytokines act upon the central nervous system to increase sympathetic drive in normal and pathophysiological conditions including heart failure. The underlying signaling pathways are still poor understood. We tested the hypothesis that brain p44/42 mitogen‐activated protein kinase (MAPK) mediates tissue necrosis factor ‐ alpha (TNF‐α) induced sympatho‐excitatory responses. In urethane‐anesthetized rats, a centrally directed intracarotid artery (ICA) injection of TNF‐α elicited significant (p&lt;0.05) increases in renal sympathetic nerve activity (RSNA), mean blood pressure (MBP) and heart rate (HR), and in hypothalamic phosphorylated p44/42 MAPK expression (Western blot and immunofluorescence). Intracerebroventricular (ICV) pretreatment (4 days before) with small interfering RNA (siRNA) using SMART pool siRNA targeting p44/42 MAPK significantly reduced the increases in RSNA, MBP and HR induced by ICA TNF‐α, but pretreatment with vehicle or control siRNA had no effect. TNF‐α induced increases in RSNA, MBP and HR were similarly diminished by a continuous ICV infusion of the p44/42 MAPK inhibitor PD98059. These data suggest that blood‐borne TNF‐α activates the brain p44/42 MAPK pathway to elicit sympatho‐excitatory responses. Supported by a VA Merit Review Award and NIH RO1 HL073986.

Bluetongue virus infection alters the impedance of monolayers of bovine endothelial cells as a result of cell death

Bluetongue virus (BTV) is the cause of bluetongue, an emerging, arthropod-transmitted disease of ungulates. Bluetongue is characterized by vascular injury with hemorrhage, tissue infarction and widespread edema, lesions that are consistent with those of the so-called viral hemorrhagic fevers. To further investigate the pathogenesis of vascular injury in bluetongue, we utilized an electrical impedance assay and immunofluorescence staining to compare the effects of BTV infection on cultured bovine endothelial cells (bPAEC) with those of inducers of cell death (Triton X-100) and interendothelial gap formation (tissue necrosis factor [TNF]). The data confirm that the adherens junctions of BTV-infected bPAECs remained intact until 24 h post-infection, and that loss of monolayer impedance precisely coincided with onset of virus-induced cell death. In contrast, recombinant bovine TNF-α caused rapid loss of bPAEC monolayer impedance that was associated with interendothelial gap formation and redistribution of VE-cadherin, but without early cell death. The data from these in vitro studies are consistent with a pathogenesis of bluetongue that involves virus-induced vascular injury leading to thrombosis, hemorrhage and tissue necrosis. However, the contribution of cytokine-induced interendothelial gap formation with subsequent edema and hypovolemic shock contributes to the pathogenesis of bluetongue remains to be fully characterized.

Mesalamine Restores Angiogenic Balance in Experimental Ulcerative Colitis by Reducing Expression of Endostatin and Angiostatin: Novel Molecular Mechanism for Therapeutic Action of Mesalamine

Mesalamine (5-aminosalicylate acid, 5-ASA) is an effective treatment for ulcerative colitis (UC). The mechanisms of its actions are not fully understood. Because angiogenesis is critical for healing UC, we examined whether 5-ASA alters the angiogenic balance between angiogenic factors [e.g., vascular endothelial growth factor (VEGF)] and antiangiogenic factors (e.g., endostatin and angiostatin) in the colon in experimental UC. Rats were treated with saline or 5-ASA (100 mg/kg) twice daily and euthanized 3 or 7 days after iodoacetamide-induced UC. Clinical signs (e.g., lethargy, diarrhea) and UC lesions were measured. Expression of VEGF, endostatin, angiostatin, tissue necrosis factor alpha (TNF-alpha), and matrix metalloproteinases (MMPs) 2 and 9 was determined by Western blots, enzyme-linked immunosorbent assay, and zymography in the distal colon. 5-ASA treatment reduced lethargy and diarrhea and significantly decreased colonic lesions (by approximately 50%) compared with saline treatment in UC (both, P < 0.05). 5-ASA did not reverse the increased levels of VEGF, but it significantly reduced expression of endostatin and angiostatin in UC compared with vehicle treatment (both, P < 0.05). Furthermore, 5-ASA treatment significantly diminished increased activity of TNF-alpha and MMP9 in UC. This is the first demonstration that 5-ASA treatment reverses an imbalance between the angiogenic factor VEGF and antiangiogenic factors endostatin and angiostatin in experimental UC. The effect of 5-ASA in UC may be caused by the down-regulation of expression of endostatin and angiostatin by modulation of MMP2 and MMP9 via inhibition of TNFalpha. The inhibition of antiangiogenic factors may represent a novel molecular mechanism of the therapeutic action of 5-ASA.

The 3'-untranslated region of ADAMTS1 regulates its mRNA stability

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1) is an inflammatory-induced gene. We have previously reported that ADAMTS1 was strongly but transiently expressed in the infarcted heart. In this study, we investigated whether a 3'-untranslated region (UTR) affects the mRNA stability of this gene. When stimulated with tissue necrosis factor (TNF)-alpha, the expression level of ADAMTS1 mRNA rapidly increased, but the induction of ADAMTS1 mRNA peaked at 6h after stimulation, after which the expression levels of ADAMTS1 mRNA decreased. The 3'-UTR ADAMTS1 mRNA contains multiple adenine and uridine-rich elements, suggesting that the 3'-UTR may regulate gene stability. The addition of actinomycin D, an RNA synthesis inhibitor, demonstrated the decay of induced ADAMTS1 mRNA by TNF-alpha. Furthermore, a region containing multiple AUUUA motifs within the ADAMTS1 3'-UTR destabilized transfected Enhanced Green Fluorescence Protein (EGFP) mRNA expression. These results demonstrated that the ADAMTS1 3'-UTR may regulate the expression of ADAMTS1 mRNA.

Suppression of nuclear factor kappa B ameliorates astrogliosis but not amyloid burden in APPswe/PS1dE9 mice

Neuroinflammation has been linked to the pathologies of Alzheimer's disease (AD), however, its effects on beta-amyloid (Aβ) burden are unclear. This study investigated the role of nuclear factor kappa B (NF-κB) in regulating neuroinflammation and Aβ deposition in a transgenic mouse model of AD. The APPswe/PS1dE9 mice and their wild-type controls received either the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC, i.p. 50 mg/kg daily) or saline starting at 7 months of age for 5 months. Expression of cyclooxygenase-2 (COX-2), tissue necrosis factor alpha (TNFα) precursor protein and microtubule-associated protein 2 was determined, and astrogliosis was assessed. Hippocampal and cortical levels of Aβ 1-40 and Aβ 1-42 were measured using ELISA. PDTC treatment effectively suppressed NF-κB signaling in APPswe/PS1dE9 mice as evidenced by the abolishment of COX-2 and TNFα induction. Inhibition of NF-κB further attenuated astrogliosis in the transgenic AD mice, yet markedly increased cerebral Aβ 1-42 burden. Our findings suggest that NF-κB can mediate induction of COX-2, TNFα and astrogliosis in APPswe/PS1dE9 mice. Additionally, these results support the idea that neuroinflammation contributes to the clearance of Aβ.

Role of perforin-mediated cell apoptosis in murine models of infusion-induced bone marrow failure

To investigate the role of perforin-mediated cell apoptosis in murine models of immune-mediated bone marrow (BM) failure. We compared C57BL/6J (B6) mice carrying a perforin gene deletion (Prf(-/-)) with wild-type (WT) controls for cellular composition in lymphohematopoietic tissues. Lymph node (LN) cells from Prf(-/-) mice were coincubated with BM cells from B10-H2(b)/LilMcdJ (C.B10) mice in an apoptosis assay in vitro. We then infused Prf(-/-) and WT B6 LN cells into sublethally irradiated C.B10 and CByB6F1 recipients with mismatches at the minor and major histocompatibility loci, respectively, in order to induce BM failure. Cellular composition was analyzed by flow cytometry. Prf(-/-) mice showed normal lymphoid cell composition, but Prf(-/-) LN cells had reduced ability to induce C.B10 BM cell apoptosis in vitro. Infusion of 5 to 10 x 10(6) Prf(-/-) LN cells produced obvious BM failure in C.B10 and CByB6F1 recipients; pancytopenia and BM hypocellularity were only slightly less severe than those caused by infusion of 5 x 10(6) WT B6 LN cells. Infused Prf(-/-) LN cells showed less T-cell expansion, normal T-cell activation, and higher proportions of T cells expressing gamma-interferon, tissue necrosis factor-alpha, and Fas ligand CD178, in comparison to infused WT B6 LN cells. Fas expression was equally high in residual BM cells in recipient of both Prf(-/-) and B6 LN cells. Perforin deficiency alters T-cell expansion but upregulates T-cell Fas ligand expression. Perforin-mediated cell death appears to play a minor role in mouse models of immune-mediated BM failure.

Fas- and Fas Ligand Rather Than Perforin-Mediated Apoptosis Has a Dominant Role in Target Cell Death in Murine Models of Immune- Mediated Bone Marrow Failure

Fas-Fas ligand and perforin-granzyme are two important cell death pathways associated with cytotoxic T cell induced target cell apoptosis. In patients with immune-mediated aplastic anemia, the development of bone marrow (BM) failure is associated with up-regulation in Fas ligand expression on effector cytotoxic T cells and elevated Fas expression on target BM cells. In some aplastic anemia patients, peripheral blood T lymphocytes also carry polymorphisms in the perforin gene which have been associated with familial hemophagocytosis. These findings suggested that Fas ligand/Fas might be the key signaling molecules mediating cell destruction while perforin might also play a role in the development of BM failure in patients with aplastic anemia. We have modeled immune-mediated BM failure in the mouse by infusing allogeneic lymph node (LN) cells from C57BL/6 (B6) donors into sublethally-irradiated CByB6F1 and C.B10 recipients that are mismatched at either major histocompatibility (MHC) or minor histocompatibility (minor-H) loci. Expansion and activation of allogeneic T cells results in increased production of the inflammatory cytokines gamma interferon and tissue necrosis factor alpha in recipient BM, massive BM cell destruction, severe marrow hypoplasia, and fatal pancytopenia. In the current study, we directly tested the roles of Fas, Fas ligand and perforin in the development of BM failure by using murine models with spontaneous mutations at the lymphoproliferation (lpr) and generalized lymphoproliferative disease (gld) loci, or with germline deletion of the gene perforin (prf−/−). Fas and Fas ligand-deficient lpr and gld mutant mice had no evidence of hematopoietic deficiency despite their autoimmune environment and marked lymphoproliferation. LN cells from lpr and gld mice caused significantly less apoptosis to minor-H mismatched C.B10 BM cells when co-cultured in a cytotoxicity assay in vitro, in comparison to LN cells from wild-type B6 mice. Infusion of lpr, gld, and B6 donor LN cells into sub-lethally irradiated CB10 recipients all caused massive T cell expansion in recipient BM with high level expression of CD11a, indicative of T cell activation, but only B6 LN cells caused severe BM destruction. In contrast, recipients of lpr and gld LN cells had only mild to moderate pancytopenia and marrow hypocellularity. We inferred from these results that disruption of the Fas ligand/Fas signaling pathway effectively abrogated immune mediated marrow destruction. To test the role of perforin in BM failure, we first analyzed prf−/−- mice and found no obvious change in cellular composition in lymphohematopoietic tissues in comparison to wild-type B6 controls. LN cells from prf−/− mice showed reduced ability to induce C.B10 BM cell apoptosis in an in vitro cytotoxicity assay when compared to wild-type B6 LN cells. Infusion of 5–10 million prf−/− LN cells into CByB6F1 and C.B10 recipients produced obvious BM failure in both recipient types with pancytopenia and marrow hypoplasia about 80–90% as severe as in control recipients of 5 million B6 LN cells. In both CByB6F1 and C.B10 recipients, infused prf−/− LN cells resulted in less T cell expansion, a similar level of T cell activation, higher proportions of T cells containing gamma-interferon and tissue necrosis factor-alpha, and a higher proportion of T cells expressing the Fas ligand CD178, in comparison to the infused B6 LN cells. We conclude that Fas-Fas ligand-mediated transmembrane signaling provides the major cell death pathway, while perforin-granzyme-mediated exocytosis plays a minor role, in BM cell destruction in animal models of immune-mediated BM failure.

Methylcellulose∕Agarose Hydrogel System to Release Therapeutic Agents

Our laboratory has previously developed an injectable hydrogel blend consisting of agarose and methylcellulose that solidifies at physiological temperatures. This study examined the feasibility of loading a chemical species into the hydrogel blend for controlled release. Free radical formation and excessive inflammation following initial central nervous system (CNS) trauma contribute to secondary injury. Therefore, the anti-oxidant glutathione and the anti-inflammatory cytokine interleukin-10 (IL-10) were loaded into the hydrogel blend for the purposes of neutralizing free radicals generated and inhibiting excessive inflammation following CNS injury, respectively. Using Ellman’s reagent, glutathione release from the hydrogel was monitored, and data from these experiments reveal that the agarose-methylcellulose hydrogel blend delivers glutathione for up to five days in vitro. Similar experiments were performed on IL-10 release, which was released for up to four days. Recent experiments have focused on implementing the glutathione-containing hydrogel in a neuronal culture model that contains elevated levels of free radical. In addition, other experiments have focused on implementing the IL-10-containing hydrogel in a monocyte culture model and observing the effects of tissue necrosis factor alpha (TNF‐α) production. Based on these preliminary findings, hydrogel blends consisting of agarose and methylcellulose loaded with glutathione and∕or IL-10 could potentially spare uninjured neurons from secondary injury. Future experiments will utilize a rat spinal cord injury model to further evaluate the efficacy of the hydrogel system. In addition to its use as an injury intervention, the hydrogel system also has the potential for use as a coating in implantable devices, especially in the CNS.

Modulating an oxidative-inflammatory cascade: potential new treatment strategy for improving glucose metabolism, insulin resistance, and vascular function.

Type 2 diabetes is a result of derangement of homeostatic systems of metabolic control and immune defense. Increases in visceral fat and organ adipose, environmental factors and genetic predisposition create imbalances of these homeostatic mechanisms, ultimately leading to a condition in which the oxidative environment cannot be held in check. A significant imbalance between the production of reactive oxygen species and antioxidant defenses, a condition called to oxidative stress, ensues, leading to alterations in stress-signalling pathways and potentially end-organ damage. Oxidative stress and metabolic inflammation upregulate the expression pro-inflammatory cytokines, including tissue necrosis factor alpha, monocyte chemoattractant protein-1 and interleukin-6, as well as activating stress-sensitive kinases, such as c-Jun N-terminal kinase (JNK), phosphokinase C isoforms, mitogen-activated protein kinase and inhibitor of kappa B kinase. The JNK pathway (specifically JNK-1) appears to be a regulator that triggers the oxidative-inflammation cascade that, if left unchecked, can become chronic and cause abnormal glucose metabolism. This can lead to insulin resistance and dysfunction of the vasculature and pancreatic β-cell. The series of events set in motion by the interaction between metabolic inflammation and oxidative stress constitutes an ‘oxidative-inflammatory cascade’, a delicate balance driven by mediators of the immune and metabolic systems, maintained through a positive feedback loop. Modulating an oxidative-inflammation cascade may improve glucose metabolism, insulin resistance and vascular function, thereby slowing the development and progression to cardiovascular diseases and type 2 diabetes.

Highly Crosslinked vs Conventional Polyethylene Particles—An In Vitro Comparison of Biologic Activities

Highly cross-linked polyethylenes (HXPEs) have been introduced to reduce wear after hip arthroplasty. The improved wear characteristics of HXPEs are well documented, but the relative biologic activity of HXPE and conventional polyethylene (CPE) particles remains unclear. Longevity (Zimmer, Warsaw, Ind; HXPE) and GUR 1050 (Zimmer; CPE) particles were isolated and characterized from a hip simulator and their in vitro inflammatory responses (tissue necrosis factor α, interleukin 1 α, and vascular endothelial growth factor levels) were compared using macrophages. The average diameter of Longevity particles (0.111 μm) was smaller than CPE particles (0.196 μm), and both were predominantly round (granular appearance). The inflammatory response to HXPE and CPE was concentration-dependent. No statistically significant differences were noted at low (0.1 surface area ratio [SAR]) and intermediate (0.75 SAR) doses. At the highest dose tested (2.5 SAR), HXPE was significantly more inflammatory than CPE based on relative tissue necrosis factor α and vascular endothelial growth factor secretion levels. Further study is needed to determine if similar findings would be noted in vivo over a broad concentration range.

Role of P-Glycoprotein and the Intestine in the Excretion of DPC 333 [(2R)-2-{(3R)-3-Amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide] in Rodents

The role of the intestine in the elimination of (2R)-2-{(3R)-3-amino-3-[4-(2-methylquinolin-4-ylmethoxy)phenyl]-2-oxopyrrolidin-1-yl}-N-hydroxy-4-methylpentanamide (DPC 333), a potent inhibitor of tissue necrosis factor alpha-converting enzyme, was investigated in mice and rats in vivo and in vitro. In Madine-Darby canine kidney cells stably transfected with P-glycoprotein (P-gp) and DPC 333, the transport from B-->A reservoirs exceeded the transport from A-->B by approximately 7-fold. In Caco-2 monolayers and isolated rat ileal mucosa, DPC 333 was transported from basolateral to apical reservoirs in a concentration-dependent, saturable manner, and transport was blocked by N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), confirming the contribution of P-gp/breast cancer resistance protein in B-->A efflux of DPC 333. In quantitative whole body autoradiography studies with [(14)C]DPC 333 in mice and rats, radioactivity was distributed throughout the small intestine in both species. In GF120918-pretreated bile duct-cannulated rats, radioactivity in feces was reduced 60%. Using the in situ perfused rat intestine model, approximately 20% of an i.v. dose of [(14)C]DPC 333 was measured in the intestinal lumen within 3 h postdose, 12% as parent. Kinetic analysis of data suggested that excreted DPC 333 may be further metabolized in the gut. Intestinal clearance was 0.2 to 0.35 l/h/kg. The above data suggest that in the rodent the intestine serves as an organ of DPC 333 excretion, mediated in part by the transporter P-gp.

A medicinal plant extract of Scutellaria baicalensis and Acacia catechu reduced LPS stimulated gene expression in immune cells: A comprehensive genomic study using QPCR, ELISA, and microarray

A standardized flavonoid extracts from Scutellaria baicalensis and Acacia catechu, UP446 (Univestin™), was profiled favorably in its anti‐inflammatory properties. In this study, quantitative polymerase chain reaction (QPCR), enzyme‐linked immunosorbent assay (ELISA), and cDNA microarray were used to study the effect of UP446 on lipopolysaccharide (LPS)‐induced pro‐inflammatory reactions. One consistent result was that the gene expression levels stimulated or suppressed by LPS were normalized by UP446 co‐treatment. This normalization effect was observed for the well‐known pro‐inflammatory genes cyclooxygenase (COX)‐2, tissue necrosis factor (TNF)‐ 1 □, interleukin (IL)‐1β, NFκB, and IL‐6 using QPCR and TNF‐α using ELISA. This was also observed by microarray. In addition to the above genes, microarray results also included cytokines, chemokines, chemokine receptor, immunoglobulin receptors, transcriptional factors and coactivator, caspase, growth factors, and phosphatases. The microarray results supported not only the anti‐inflammatory property of UP446, but also suggested that UP446 may participate in additional signaling pathways of cell proliferation, cell death and lipid metabolism. This research work was supported by Unigen.

Expression of cytokines and cytokine receptors in human Schwann cells

The expression of cytokines and cytokine receptors was investigated in enriched populations of human fetal Schwann cells by reverse transcribed-PCR and enzyme-linked immunosorbent assay. Human fetal Schwann cells constitutively expressed mRNA of IL-1beta, IL-6, IL-8, IL-11, IL-12, IL-15 and TGF-beta, and also cytokine receptors for IL-1, IL-4, IL-6, IL-8, IL-13, tissue necrosis factor and gp130. The expression of IL-1beta, IL-6 and IL-15 was upregulated following treatment with IL-1beta or TGF-beta. The protein levels of IL-6 were increased with IL-1beta treatment, but were decreased with IFN-gamma treatment. Human Schwann cells may respond to cytokine signals in the nerve injury sites and modify the pathological conditions by secreting cytokines. The secreted cytokines may play a role in leukocyte recruitment and exacerbation of axonal injury process.