sepharose and Inflammation

This review presents a summary of various types of scaffold biomaterials used alone or together with therapeutic drugs and cells to regenerate spinal cord injury (SCI). The inhibitory environment and loss of axonal connections after SCI give rise to critical obstacles to regeneration of lost tissues and neuronal functions. Biomaterial scaffolds can provide a bridge to connect lost tissues, an adhesion site for implanted or host cells, and sustained release of therapeutic drugs in the injured spinal cord. In addition, they not only provide a structural platform, but can play active roles by inhibiting apoptosis of cells, inflammation and scar formation, and inducing neurogenesis, axonal growth and angiogenesis. Many synthetic and natural biomaterial scaffolds have been extensively investigated and tested in vitro and in animal SCI models for these purposes. We summarized the literature on the biomaterials commonly used for spinal cord regeneration in terms of historical backgrounds and current approaches.

Topics: Alginates; Animals; Apoptosis; Axons; Biocompatible Materials; Chitosan; Collagen; Disease Models, Animal; Drug Delivery Systems; Fibrin; Humans; Hyaluronic Acid; Inflammation; Lactic Acid; Materials Testing; Peptides; Polyesters; Polymers; Sepharose; Spinal Cord; Spinal Cord Injuries; Spinal Cord Regeneration; Stem Cells; Tissue Engineering; Tissue Scaffolds

Topics: Cell Adhesion; Cell Communication; Cell Count; Cell Movement; Chemotaxis, Leukocyte; Cineradiography; Colchicine; Dose-Response Relationship, Immunologic; Fibroblasts; Humans; Inflammation; Micropore Filters; Neutrophils; Sepharose; Stimulation, Chemical; Videotape Recording

A combination of polysaccharides and tea polyphenols can enhance immune activity synergistically, depending on the type and structure of polysaccharides, but the mechanism remains unknown. This study is aimed to investigate the regulating effects of different seaweed polysaccharide (ι-carrageenan, agarose) and tea polyphenol blends on intestinal flora and intestinal inflammation using an

Topics: Carrageenan; Fermentation; Gastrointestinal Microbiome; Humans; Inflammation; Polyphenols; Polysaccharides; Seaweed; Sepharose; Tea

A long T. To fabricate and assess a phantom dedicated to the quality assurance of T. A T. The phantom embodies 9 internal agarose-containing T. The T

Topics: Humans; Inflammation; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Myocardium; Phantoms, Imaging; Polyethylene; Predictive Value of Tests; Reproducibility of Results; Sepharose

Articular cartilage exhibits reduced self-healing following degeneration. This research evaluated the effects of hydrogels derived from various polysaccharides-gellan gum (GG), alginate, and agarose-on cartilage regeneration compared with that of hyaluronic acid (HA), which is commonly used in cartilage tissue engineering. Chondrocytes were isolated from the articular cartilage of New Zealand White (NZW) rabbits and stimulated with IL-1β followed by incubation with polysaccharides. The expressions of NF-κB and Cox-2 were decreased and those of IκBα, Sox-9, aggrecan, and type II collagen were increased in HA, GG, and Alginate groups. Osteochondral defects in NZW rabbits were treated with intra-articular polysaccharide injections; all except alginate resulted in tissue regeneration. Significant improvements were observed in cartilage regeneration in the GG and agarose groups. These results show that GG and agarose improve cartilage regeneration by suppressing inflammatory mediators and inducing cartilage formation and autophagy-related gene expression, indicating their potential for cartilage tissue engineering.

Topics: Alginates; Animals; Autophagy; Biomarkers; Cartilage, Articular; Cell Survival; Cells, Cultured; Chondrocytes; Chondrogenesis; Cross-Linking Reagents; Disease Models, Animal; Gene Expression Regulation; Hyaluronic Acid; Hydrogels; Inflammation; Male; Osteoarthritis; Polysaccharides; Polysaccharides, Bacterial; Rabbits; Regeneration; Rheology; RNA, Messenger; Sepharose; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Blood loss remains a major concern during surgery and can increase the morbidity of the intervention. The use of topical haemostatic agents to overcome this issue therefore becomes necessary. Fibrin sealants are promising haemostatic agents due to their capacity to promote coagulation, but their effectiveness and applicability need to be improved. We have compared the haemostatic efficacy of a novel nanostructured fibrin-agarose hydrogel patch, with (c-NFAH) or without cells (a-NFAH), against two commercially available haemostatic agents in a rat model of hepatic resection. Hepatic resections were performed by making short or long incisions (mild or severe model, respectively), and haemostatic agents were applied to evaluate time to haemostasis, presence of haematoma, post-operative adhesions to adjacent tissues, and inflammation factors. We found a significantly higher haemostatic success rate (time to haemostasis) with a-NFAH than with other commercial haemostatic agents. Furthermore, other relevant outcomes investigated were also improved in the a-NFAH group, including no presence of haematoma, lower adhesions, and lower grades of haemorrhage, inflammation, and necrosis in histological analysis. Overall, these findings identify a-NFAH as a promising haemostatic agent in liver resection and likely in a range of surgical procedures.

Topics: Animals; Fibrin; Hemorrhage; Hemostatics; Hydrogels; Inflammation; Liver; Male; Nanostructures; Necrosis; Rats, Wistar; Sepharose

In this work, we evaluate the tissue response and tolerance to a designed 3D porous scaffold composed of nanocrystalline carbonate-hydroxyapatite and agarose as a preliminary step in bone repair and regeneration. These scaffolds were subcutaneously implanted into rats, which were sacrificed at different times. CD4+, CD8+ and ED1+ cells were evaluated as measurements of inflammatory reaction and tolerance. We observed some inflammatory response early after subcutaneous implantation. The 3D interconnected porosity increased scaffold integration via the formation of granulation tissue and the generation of a fibrous capsule around the scaffold. The capsule is initially formed by collagen which progressively invades the scaffold, creating a network that supports the settlement of connective tissue and generating a compact structure. The timing of the appearance of CD4+ and CD8+ cell populations is in agreement with the resolved inflammatory response. The appearance of macrophage activity evidences a slow and gradual degradation activity. Degradation started with the agarose component of the scaffold, but the nano-apatite was kept intact for up to 30 days. Therefore, this apatite/agarose scaffold showed a high capacity for integration by a connective network that stabilizes the scaffold and results in slow nano-apatite degradation. The fundamental properties of the scaffold would provide mechanical support and facilitate bone mobilization, which is of great importance in the masticatory system or large bones.

Topics: Absorbable Implants; Animals; Bone Regeneration; Carbonates; Durapatite; Female; Inflammation; Porosity; Rats; Rats, Wistar; Sepharose; Tissue Scaffolds

IgG is an important defence protein. To exhibit optimum function the molecule must maintain its native structure. Peroxynitrite is a potent oxidizing and nitrating agent produced in vivo under pathophysiological conditions. It can oxidize and/or nitrate various amino acids causing changes in the structure and function of proteins. Such proteins may be involved in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis. In the present work, peroxynitrite-induced structural changes in IgG have been studied by UV-visible, fluorescence, CD, FT-IR, DLS spectroscopy and DSC as well as by SDS-PAGE. Peroxynitrite-modified IgG exhibited hyperchromicity at 280 nm, quenching of tryptophan fluorescence, increase in ANS fluorescence, loss of β-sheet, shift in the positions of amide I and amide II bands, appearance of new peak in FT-IR, attachment of nitro residues and increase in melting temperature, compared to native IgG. Furthermore, peroxynitrite-modified IgG exhibited an additional peak at 420 nm, quenching in tyrosine fluorescence and enhancement in dityrosine fluorescence compared to native IgG. Generation of nitrotyrosine, dityrosine and nitrotryptophan was also observed in peroxynitrite-modified IgG. Gross structural changes in IgG caused by peroxynitrite and observed in vitro may favour autoantibodies induction in vivo under similar conditions.

Topics: Arthritis, Rheumatoid; Calorimetry, Differential Scanning; Circular Dichroism; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Immunoglobulin G; Inflammation; Light; Microscopy, Fluorescence; Oxygen; Peroxynitrous Acid; Protein Structure, Secondary; Scattering, Radiation; Sepharose; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Temperature; Tryptophan; Tyrosine

Quiescent fibroblasts possess unique genetic program and exhibit high metabolic activity distinct from proliferative fibroblasts. In response to inflammatory stimulation, quiescent fibroblasts are more active in expressing cyclooxygenase-2 and other proinflammatory genes than proliferative fibroblasts. The underlying transcriptional mechanism is unclear. Here we show that phorbol 12-myristate 13-acetate (PMA) and cytokines increased p300 histone acetyltransferase activity to a higher magnitude (> 2 fold) in quiescent fibroblasts than in proliferative fibroblasts. Binding of p300 to cyclooxygenase-2 promoter was reduced in proliferative fibroblasts. By ultrahigh-performance liquid chromatography coupled with a quadrupole time of flight mass spectrometer and enzyme-immunoassay, we found that production of 5-methoxytryptophan was 2-3 folds higher in proliferative fibroblasts than that in quiescent fibroblasts. Addition of 5-methoxytryptophan and its metabolic precursor, 5-hydroxytryptophan, to quiescent fibroblasts suppressed PMA-induced p300 histone acetyltransferase activity and cyclooxygenase-2 expression to the level of proliferative fibroblasts. Silencing of tryptophan hydroxylase-1 or hydroxyindole O-methyltransferase in proliferative fibroblasts with siRNA resulted in elevation of PMA-induced p300 histone acetyltransferase activity to the level of that in quiescent fibroblasts, which was rescued by addition of 5-hydroxytryptophan or 5-methoxytryptophan. Our findings indicate that robust inflammatory gene expression in quiescent fibroblasts vs. proliferative fibroblasts is attributed to uncontrolled p300 histone acetyltransferase activation due to deficiency of 5-methoxytryptophan production. 5-methoxytryptophan thus is a potential valuable lead compound for new anti-inflammatory drug development.

Topics: Anti-Inflammatory Agents; Cell Differentiation; Cell Proliferation; Chromatography, High Pressure Liquid; Cyclooxygenase 2; Fibroblasts; Gene Silencing; Humans; Immunoenzyme Techniques; Inflammation; Mass Spectrometry; p300-CBP Transcription Factors; Promoter Regions, Genetic; RNA, Small Interfering; Sepharose; Streptavidin; Tryptophan

Pulmonary surfactant protein-D (SP-D) is a soluble collagenous C-type lectin with important anti-microbial and anti-inflammatory properties. Although it is subject to functionally relevant modification by common polymorphisms and unregulated inflammation, the functional status of SP-D in cystic fibrosis (CF) remains unclear. Given the importance of infection and inflammation in CF lung pathology we have undertaken the first systematic analysis of SP-D lectin activity in this population. By ELISA, we found that airway lavage fluid SP-D expression was greater in CF compared to control patients but was reduced in CF patients with infection and correlated negatively with markers of neutrophilic inflammation. In a functional assay, the percentage of SP-D capable of binding zymosan rarely exceeded 60% in CF or control patients and similarly restricted binding activity was observed towards maltose-agarose. SP-D lectin activity also correlated negatively with infection and neutrophilic inflammation but there was little evidence of major proteolytic degradation amongst the non-bound material. SP-D which failed to bind zymosan exhibited features of lower oligomeric form compared to bound material when tested by native gel electrophoresis. Furthermore, when separated by gel chromatography, high and low oligomeric populations of SP-D were observed in CF lavage fluid but only high oligomeric forms exhibited substantial lectin activity towards yeast derived mannan. Our data demonstrate that oligomeric heterogeneity underlies functional diversity amongst SP-D in health and disease and that dynamic regulation of oligomerisation is an important feature of SP-D biology.

Topics: Adolescent; Bacterial Infections; Biomarkers; Blotting, Western; Bronchoalveolar Lavage Fluid; Case-Control Studies; Child; Child, Preschool; Chromatography, Gel; Cohort Studies; Cystic Fibrosis; Enzyme-Linked Immunosorbent Assay; Female; Humans; Infant; Inflammation; Male; Maltose; Protein Multimerization; Pulmonary Surfactant-Associated Protein D; Sepharose; Zymosan

Agarose is hydrolyzed easily to yield oligosaccharides, designated as agaro-oligosaccharides (AGOs). Recently, it has been demonstrated that AGOs induce heme oxygenase-1 (HO-1) expression in macrophages and that they might lead to anti-inflammatory property. Nevertheless, the molecular mechanism of AGO-mediated HO-1 induction remains unknown, as does AGOs' ability to elicit anti-inflammatory activity in vivo. This study was undertaken to uncover the mechanism of AGO-mediated HO-1 induction and to investigate the therapeutic effect of AGOs on intestinal inflammation.. Mice were treated with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to induce colitis. The respective degrees of mucosal injury of mice that had received AGO and control mice were compared. We investigated HO-1 expression using Western blotting, quantitative real-time PCR (qRT-PCR), and immunohistochemistry. The expression of tumor necrosis factor-α (TNF-α) was measured using qRT-PCR and enzyme-linked immunosorbent assay.. AGO administration induced HO-1 expression in colonic mucosa. The induction was observed mainly in F4/80 positive macrophages. Increased colonic damage and myeloperoxidase activity after TNBS treatment were inhibited by AGO administration. TNBS treatment induced TNF-α expression, and AGO administration suppressed induction. However, HO inhibitor canceled AGO-mediated amelioration of colitis. In RAW264 cells, AGOs enhanced HO-1 expression time-dependently and concentration-dependently and suppressed lipopolysaccharide-induced TNF-α expression. Furthermore, agarotetraose-mediated HO-1 induction required NF-E2-related factor 2 function and phosphorylation of c-jun N-terminal kinase.. We infer that AGO administration inhibits TNBS-induced colitis in mice through HO-1 induction in macrophages. Consequently, oral administration of AGOs might be an important therapeutic strategy for inflammatory bowel disease.

Topics: Animals; Anti-Inflammatory Agents; Blotting, Western; Cell Line; Colitis; Disease Models, Animal; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Heme Oxygenase-1; Inflammation; Macrophages; Male; Mice; Mice, Inbred C57BL; Oligosaccharides; Real-Time Polymerase Chain Reaction; Sepharose; Time Factors; Trinitrobenzenesulfonic Acid

Anchorage-dependent cells including hepatocytes, the main functional cellular constituent comprising liver tissue, require a substrate for cell adhesion when cultured outside their native tissue. The challenge with hepatocyte culture is that material substrates and designs supporting hepatocyte attachment, phenotype, and function are not readily available. Our laboratory previously published that type I collagen found in the liver extracellular matrix supports hepatocyte culture. We hypothesized that micropatterned agarose with a coating of collagen covalently bound to the surface would facilitate hepatocyte adhesion and phenotype. To test this hypothesis, primary canine hepatocytes and neoplastic human HepG2 hepatocellular carcinoma cells were cultured on these substrates. Hepatocyte adhesion was dependent on the cell type and also the micropattern design. Viable normal and neoplastic hepatocytes attached to the microchannel troughs rather than on the ridges. In contrast, hepatocyte adhesion on the microcircular patterns was similar to control agarose as cells did not sense differences in surface topology on these substrates. Neoplastic cells exhibited a distinct difference in growth behavior following 7 days in culture on the microchannel patterns, exhibiting aberrant proliferation relative to normal hepatocytes which did not proliferate. Our results suggest that patterned microchannel agarose may be useful to evaluate hepatoprotective and noxious agents.

Topics: Animals; Cell Adhesion; Cell Shape; Cells, Cultured; Collagen; Dogs; Hep G2 Cells; Hepatocytes; Humans; Inflammation; Liver Neoplasms; Microtechnology; Oxidative Stress; Sepharose

In the present study, an acidic PLA(2), designated Bl-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000Da and pI was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-α, IL-1β and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA(2) induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism.

Topics: Amino Acid Sequence; Animals; Bothrops; Cells, Cultured; Chromatography, Ion Exchange; Crotalid Venoms; Cytokines; Edema; Electrophoresis, Polyacrylamide Gel; Humans; Inflammation; Interleukin-10; Interleukin-12 Subunit p40; Interleukin-6; Interleukin-8; Leukocytes, Mononuclear; Male; Mice; Molecular Weight; Phospholipases A2; Sepharose; Sequence Homology, Amino Acid; Tumor Necrosis Factor-alpha

Extracellular histones released from cells during acute inflammation contribute to organ failure and death in a mouse model of sepsis, and histones are known to exert in vitro cytotoxicity in the absence of serum. Since addition of histones to serum and plasma is known to induce protein aggregation, we reasoned that plasma proteins may afford protection from cytotoxicity. We found that MODE-K mouse small intestinal epithelial cells were protected from histone-induced toxicity in the presence of 10% FCS. Therefore, the main aim of this study was to identify histone-interacting plasma proteins that might be involved in cytoprotection. The precipitate formed following addition of calf thymus histones to human EDTA plasma was characterised by shotgun proteomics, identifying a total of 36 protein subunits, including complement components, coagulation factors, protease inhibitors and apolipoproteins. The highly sulphated glycosaminoglycan heparin inhibited histone-induced plasma protein aggregation. Moreover, histones bound to heparin agarose were capable of pulling down plasma proteins from solution, indicating their effective cross-linking properties. It was particularly notable that inter-alpha-trypsin inhibitor was prominent among the histone-precipitated proteins, since it contains a chondroitin sulphate glycan chain, and suggests a potential role for this protein in histone sequestration during acute inflammation in vivo.

Topics: Animals; Blood Proteins; Cattle; Chemical Precipitation; Cytoprotection; Glycosaminoglycans; Heparin; Histones; Humans; Immobilized Proteins; Inflammation; Mice; Protein Interaction Mapping; Proteomics; Sepharose; Thymus Gland

Nuclear histones have previously been shown to aggregate LDL in vitro, suggestive of a possible pro-atherogenic role. Recent studies indicate that histones are released during acute inflammation, and therefore might interact with circulating lipoproteins in vivo. In view of the associative link between inflammation and cardiovascular disease, the behaviour of histones was investigated using in vitro models of LDL retention and foam cell formation.. Heparin agarose beads were used as a model of a matrix rich in sulphated glycosaminoglycans, to which histones bind strongly. Histone-modified beads were observed to pull down more LDL from solution than untreated beads, indicating that histones can function as bridging molecules, enhancing LDL retention. Furthermore, addition of heparin inhibited histone-induced aggregation of LDL. To model foam cell formation, murine RAW 264.7 macrophages were incubated for 24 h in the presence of LDL, histones, LDL plus histones or vehicle control. Cells incubated with LDL in the presence of histones accumulated significantly more intracellular lipid than with LDL or histone alone.. These results are consistent with a potential pro-atherogenic role for extracellular histones, which should be investigated further.

Topics: Animals; Atherosclerosis; Cell Nucleus; Foam Cells; Heparin; Histones; In Vitro Techniques; Inflammation; Lipoproteins, HDL; Lipoproteins, LDL; Macrophages; Mice; Sepharose

Intracellular heat shock protein 72 (Hsp72) is known to serve a broad cytoprotective role. Recent data indicate that stressed cells can release Hsp72 into the extracellular compartment, although the biological function of extracellular Hsp72 remains to be fully elucidated. Because extracellular Hsp72 has been demonstrated to interact with Toll-like receptor 4, we hypothesized that endogenously produced and released Hsp72 would reprogram the mononuclear cell responses to LPS. THP-1 cells treated with LPS were used as a model for nuclear factor (NF)-kappaB activation. Heat shock conditions consisted of incubation at 43 degrees C for 1 h. Control cells were incubated at 37 degrees C. Twenty four hours after incubation, heat shock conditioned media (HSCM) and control media (CM) were centrifuged, and the respective cells were discarded. A separate group of naive THP-1 cells were then incubated with either HSCM or CM for 18 h and then stimulated with LPS (1 mug/mL). Heat shock significantly increased Hsp72 in HSCM compared with CM. In THP-1 cells transfected with an NF-kappaB luciferase reporter plasmid, the addition of HSCM attenuated subsequent LPS-mediated luciferase activity compared with cells incubated in CM. The addition of HSCM also attenuated LPS-mediated NF-kappaB-DNA binding and IkappaBalpha degradation. Heat shock protein 72-mediated inhibition of NF-kappaB activation was further corroborated by a significant decrease in TNF-alpha production. When HSCM and CM were subjected to Hsp72 depletion via adenosine triphosphate-agarose binding, LPS-mediated activation of NF-kappaB was partially restored, suggesting that Hsp72 is partially responsible for cellular reprogramming in response to HSCM. These data demonstrate that endogenously produced and released extracellular Hsp72 has the ability to reprogram the in vitro response to endotoxin in cultured human mononuclear cells.

Topics: Cell Line, Tumor; Endotoxins; HSP72 Heat-Shock Proteins; Humans; Hypoxia; Immune System; Inflammation; Ischemic Preconditioning; Leukocytes, Mononuclear; Lipopolysaccharides; Models, Biological; Monocytes; NF-kappa B; Sepharose; Signal Transduction

Patients with cystic fibrosis (CF) develop chronic Pseudomonas aeruginosa lung infection with mucoid strains of P. aeruginosa; these infections cause significant morbidity. The immunological response in these infections is characterized by an influx of neutrophils to the lung and subsequent lung damage over time; however, the underlying mediators to this response are not well understood. We recently reported that IL-23 and IL-17 were elevated in the sputum of patients with CF who were actively infected with P. aeruginosa; however, the importance of IL-23 and IL-17 in mediating this inflammation was unclear. To understand the role that IL-23 plays in initiating airway inflammation in response to P. aeruginosa, IL-23p19(-/-) (IL-23 deficient) and wild-type (WT) mice were challenged with agarose beads containing a clinical, mucoid isolate of P. aeruginosa. Levels of proinflammatory cytokines, chemokines, bacterial dissemination, and inflammatory infiltrates were measured. IL-23-deficient mice had significantly lower induction of IL-17, keratinocyte-derived chemokine (KC), and IL-6, decreased bronchoalveolar lavage (BAL) neutrophils, metalloproteinase-9 (MMP-9), and reduced airway inflammation than WT mice. Despite the reduced level of inflammation in IL-23p19(-/-) mice, there were no differences in the induction of TNF and interferon-gamma or in bacterial dissemination between the two groups. This study demonstrates that IL-23 plays a critical role in generating airway inflammation observed in mucoid P. aeruginosa infection and suggests that IL-23 could be a potential target for immunotherapy to treat airway inflammation in CF.

Topics: Animals; Bronchoalveolar Lavage Fluid; Cell Count; Chemokines; Colony Count, Microbial; Gene Expression Regulation; Glycosaminoglycans; Inflammation; Interleukin-17; Interleukin-23; Lung; Matrix Metalloproteinase 9; Mice; Mice, Inbred C57BL; Microspheres; Pseudomonas aeruginosa; Pseudomonas Infections; RNA, Messenger; Sepharose

The present study demonstrates the interest of two slow-release systems as vaccination tools in cattle. Two experiments show that a first intradermal administration of one DNA vaccine dose combined with the slow-release of a second dose conduct to a priming of the bovine herpesvirus 1-specific immune response similar to the one generated by two discrete administrations 4 weeks apart. The first experiment demonstrates the efficacy of the slow-release system with well-characterized Alzet osmotic pumps, whereas the second experiment extends the same concept with innovative agarose hydrogel implants. These latter implants are cheaper and more convenient than the osmotic pumps or repeated intradermal administrations since they contribute to an efficient priming of the immune response in a single manipulation of the animals.

Topics: Animals; Cattle; Cattle Diseases; Delayed-Action Preparations; Diffusion; Drug Implants; Excipients; Feces; Herpesvirus 1, Bovine; Hydrogels; Immunization; Immunization Schedule; Inflammation; Neutralization Tests; Osmotic Pressure; Plasmids; Sepharose; Vaccination; Vaccines, DNA; Viral Vaccines

Rheumatoid arthritis (RA) is a chronic inflammatory disease that mainly destroys cartilages or bones at the joints. This inflammatory disorder is initiated by self-attack using own immune system, but the detail of pathological mechanism is unclear. Features of autoantigens leading to autoimmune disease are also under veil although several candidates including type II collagen have been suggested to play a role in pathogenesis. In this report, we tried to identify proteins responding to antibodies purified from RA patients and screen proteins up-regulated or down-regulated in RA using proteomic approach. Fibronectin, semaphorin 7A precursor, growth factor binding protein 7 (GRB7), and immunoglobulin mu chain were specifically associated with antibodies isolated from RA synovial fluids. In addition, some metabolic proteins such as adipocyte fatty acid binding protein, galectin-1 and apolipoprotein A1 precursor were overexpressed in RA synovium. Also, expression of peroxiredoxin 2 was up-regulated in RA. On the contrary, expression of vimentin was severely suppressed in RA synoviocytes. Such findings might give some insights into understanding of pathological mechanism in RA.

Topics: Adult; Aged; Arthritis, Rheumatoid; Autoantigens; Collagen Type II; Female; Gene Expression Regulation; Humans; Inflammation; Male; Middle Aged; Proteomics; Sepharose; Synovial Fluid

Transcription factor NF-kappaB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF). In the absence of TNF, NF-kappaB is sequestered in the cytoplasm by inhibitory IkappaB proteins. Phosphorylation of IkappaBby the beta-catalytic subunit of IKK, a multicomponent IkappaB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-kappaB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKbeta, which greatly stimulates IkappaB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IkappaB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKKgamma regulatory subunit. Third, deletion of the PP2A-binding site in IKKgamma attenuates T loop phosphorylation and catalytic activation of IKKbeta in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK.PP2A complexes is required for the proper induction of IkappaB kinase activity in vivo.

Topics: Active Transport, Cell Nucleus; Adenosine Triphosphate; Alkenes; Animals; B-Lymphocytes; Catalysis; Cell Line; Chromatography, Liquid; Cytoplasm; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Gene Deletion; Humans; I-kappa B Kinase; Immunoblotting; Immunoprecipitation; Inflammation; Jurkat Cells; Mice; Mice, Inbred C57BL; Mutation; Okadaic Acid; Phosphoprotein Phosphatases; Phosphorylation; Polyenes; Protein Phosphatase 2; Pyrones; Sepharose; Serine; Signal Transduction; Spleen; Threonine; Time Factors; Transfection; Tumor Necrosis Factor-alpha

Topics: Animals; Cystic Fibrosis; Disease Models, Animal; Inflammation; Lung; Mice; Pseudomonas aeruginosa; Sepharose; Spleen

We studied the effects of alpha1-acid glycoprotein on tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to alpha1-acid glycoprotein: first, stimulation of TNF-alpha and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-alpha/IL-10 ratio remained unchanged after addition of native alpha1-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of alpha1-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.

Topics: Cell Division; Cells, Cultured; Concanavalin A; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Humans; Inflammation; Interleukin-10; Leukocytes; Models, Biological; Orosomucoid; Phytohemagglutinins; Sepharose; Tumor Necrosis Factor-alpha

It has been suggested that elastase released from activated neutrophils degrades cortisol binding globulin. A novel assay for serum cortisol binding capacity was therefore devised and applied to assess whether such degradation was evident in patients showing a recent inflammatory response as indicated by a raised serum C-reactive protein. In 49 patients with evidence (C-reactive protein > 50 mg/l) of a recent inflammatory response, mean serum cortisol binding capacity (288 nmol/l, S.D. = 82.9) was significantly lower (P < 0.05, t test) than in 48 patients (320 +/- 75.8 nmol/l) whose response was quiescent (C-reactive protein < 6 mg/l) or in 49 healthy controls (335 +/- 72.4 nmol/l). Four patients with septic shock had markedly reduced values (167 +/- 49.9 nmol/l) but low values were not restricted to this condition. It is concluded that a population experiencing a recent inflammatory response exhibits reduced serum cortisol binding capacity but a role for elastase in this process remains to be defined.

Topics: Adult; C-Reactive Protein; Carrier Proteins; Female; Humans; Hydrocortisone; Inflammation; Kinetics; Male; Middle Aged; Pregnancy; Protein Binding; Reference Values; Sensitivity and Specificity; Sepharose

Cytokines incorporated into agarose blocks and implanted subcutaneously into mice establish an in vivo gradient which can be used to mimic a local inflammatory process. We have developed a model in which cellular influx into cytokine impregnated blocks parallels the normal cellular reaction to infections or wounds. Agarose blocks containing supernatants from ConA activated rat spleen cells attracted neutrophils within 4 h. These cells were followed by lymphocytes and macrophages in 24 h. Flow cytometry analysis of lymphoid cells on day 1 revealed that 38% were Ig+ (B cell marker), 60% MAC-2,3+ and 20% Thy 1.2+ of which only a small fraction were expressing CD4 on their surface. These numbers changed with time following implantation of the blocks. Initially, isolated adherent cells (macrophages) were resting, with low phagocytic activity. Cells isolated from blocks at later time points were activated, as evidenced by their increased ability to ingest fluoresceinated beads. The secretion patterns of cells trafficking to murine rIL-1 containing agarose blocks were examined. TNF, IL-6 and antibody secreting cells were found. No IL-2 was detected at any time. We believe that this model will be of value in studies of local actions of cytokines.

Topics: Animals; Cell Adhesion; Cells, Cultured; Cytokines; Disease Models, Animal; Female; Humans; Inflammation; Injections, Subcutaneous; Lymphocytes; Macrophages; Mice; Mice, Inbred BALB C; Models, Biological; Phagocytosis; Rats; Recombinant Proteins; Sepharose; Spleen; Wound Infection

Microencapsulation of adrenal cells is proposed for reducing the nonspecific inflammatory reaction observed around polymer implants. This hypothesis was tested by comparing both host cellular reaction and the surrounding graft cell populations which appeared either when agarose embedded cells or when empty agarose beads were implanted. Our results showed that the fibrotic material that surrounded the implanted empty agarose microbeads was not as severe and important when adrenal cells were present. Similarly, T lymphocyte population surrounding the graft was considerably reduced together with the percentage of CD4 and CD8 positive cell subpopulations. The activation macrophage marker IaD disappeared. Our results support the hypothesis that embedded adrenal cells may be a suitable solution for reducing early inflammatory events due to microcapsules implantation.

Topics: Adrenal Cortex; Animals; Capsules; Flow Cytometry; Graft Survival; Granuloma; Inflammation; Lymphocytes; Male; Mice; Mice, Inbred BALB C; Mice, Inbred Strains; Microscopy, Electron; Sepharose

Coincubation of neutrophils with TNF inhibited the chemoattractant-directed migration of neutrophils under agarose and enhanced their migration in the multiwell chemotaxis chamber. To assess the physiological significance of these differing in vitro TNF effects, ex vivo and in vivo investigations were performed using animal models. Neutrophils from the peripheral blood of rabbits preadministered systemic TNF showed impaired ability to migrate toward chemoattractants in vitro. In addition, systemic TNF administration suppressed zymosan-activated plasma-induced local accumulation of leukocytes in mouse skin. The results indicate that circulating TNF may act as a suppressor for local inflammatory reaction.

Topics: Animals; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Humans; In Vitro Techniques; Inflammation; Mice; Mice, Inbred BALB C; Neutrophils; Peroxidase; Rabbits; Recombinant Proteins; Sepharose; Skin; Time Factors; Tumor Necrosis Factor-alpha

Serotherapy and plasma therapy have proved to be effective in the treatment of diverse neoplasms. The mechanisms of the tumoricidal or growth-inhibitory effects are unknown. We previously reported that activation of the alternative pathway of complement in absorbed sera correlated with the presence of anti-tumor activity. Complement components generated during absorption may serve as the initial mediators of cytotoxicity; for example, C5a may function in its role as a chemo-attractant. To further investigate the anti-tumor mechanisms, we undertook a series of sequential histological studies of in vivo changes in tumors following i.v. serotherapy. We found diffuse inflammatory cellular infiltrates in the interstitial compartments of primary mammary carcinomas of rats within 3-4 hr of administration of protein A-Sepharose absorbed syngeneic serum. The number of inflammatory cells was significantly higher in tumors from treated rats: total infiltrating cells (p = 0.002), eosinophils (p = 0.001), neutrophils (p = 0.001), macrophages (p = 0.001), lymphocytes (p = 0.004) and plasma cells (p = 0.001). Also, the mitotic index of tumor cells was significantly lower 4 hr after serotherapy when compared with that of untreated rat tumor cells. C3 in tumor tissue was decreased at 4 hr following serotherapy. Fibrosis was present in tumor nodules with retarded growth 5 weeks after the start of serotherapy. Localization of the infiltrating cells to tumor interstitial compartments prevents direct contact between inflammatory cells and neoplastic cells, making it unlikely that direct cell-cell killing occurs. Indirect cell killing within the tumor bed apparently occurs through several mechanisms involving interactions between serotherapy-initiated humoral mediators and inflammatory cells. The resulting anti-tumor effects include microvascular injury leading to localized ischemia, tumor infarction, and fibroblastic reactions obstructing tumor invasion and growth.

Topics: Animals; Antibodies; Blood Cell Count; Chromatography, Affinity; Complement Activation; Complement C3; Complement C5; Complement C5a; Cytotoxicity, Immunologic; Female; Immunization, Passive; Immunoenzyme Techniques; Inflammation; Mammary Neoplasms, Experimental; Methylnitrosourea; Mitotic Index; Rats; Sepharose

Polymorphonuclear leukocytes harvested from a skin chamber were compared with peripheral blood leukocytes by examining their migration under agarose, their bactericidal capacity and their chemiluminescence. The chamber leukocytes were chemotactically de-activated and their bactericidal capacity reduced. The chemiluminescent response of chamber leukocytes was increased. In response to stimulation with formylmethionyl-leucyl-phenylalanine (first peak), chemiluminescence was far more marked in chamber leukocytes (10-30 times) than in blood leukocytes, whereas in response to stimulation with opsonized zymosan it was only moderately more marked (3-5 times). Exposed to zymosan activated serum, blood leukocytes showed a similar functional modification as leukocytes harvested from a skin chamber, and our findings suggest that the altered function of leukocytes at an inflammatory focus is largely the result of their exposure to chemotactic factors.

Topics: Adult; Blood Bactericidal Activity; Cell Survival; Chemotaxis, Leukocyte; Female; Humans; Inflammation; Luminescent Measurements; Male; Middle Aged; Neutrophils; Phagocytosis; Sepharose; Skin

An in vivo skin chamber method using lesions obtained by suction was evaluated. Neither dyspigmentation nor scarring were seen after 2 mth. The number of leucocytes accumulated in the collection chamber was correlated to the area of the lesion. Reproducibility remained essentially unchanged over an extended period and was 19% for one chamber and 13.6% for determinations with two chambers. No correlation was found between results obtained with the skin chamber technique and with chemotaxis or random migration as determined by an underagarose technique. When factors influencing in vivo and in vitro migration were studied, it was found that nonsteroidal anti-inflammatory drugs when given to arthritis patients or healthy volunteers inhibit leucocyte migration in vivo while in vitro migration was unchanged. Activated serum and LTB4 attracted leucocytes both in vivo and in vitro. The attraction by serum appeared at least partly to be caused by C5a. Polymorphonuclear leucocytes harvested from skin chambers were chemotactically deactivated and their bactericidal capacity reduced. The chemiluminescent response to formyl-methionyl-leucyl-phenylalanine and opsonized zymosan was increased. Exposed to zymosan activated serum, blood leucocytes showed a similar functional modification as leucocytes harvested from a skin chamber, and our findings suggest that the altered function of leucocytes in an inflammatory focus is largely the result of their exposure to chemotactic factors.

Topics: Anti-Inflammatory Agents; Arthritis; Bacteria; Cell Movement; Chemotaxis, Leukocyte; Humans; Inflammation; Kinetics; Leukocytes; Methods; Phagocytosis; Sepharose; Skin

A model of chronic pulmonary infection was used for studying cellular events in a sequential manner. In this model, agarose beads containing Pseudomonas aeruginosa were instilled endotracheally into cats. Nine cats were inoculated with agarose beads containing P. aeruginosa, and four others were inoculated with sterile beads. With a fiberoptic bronchoscope, bronchial washings were obtained biweekly for up to 30 weeks. The quantitative pulmonary inflammatory cell response and alveolar macrophage morphology of the animals exposed to P. aeruginosa were compared with those for the animals exposed to a chronic irritant (agarose beads). Bronchial washings of all animals before inoculation showed that 70 to 90% of the cells were macrophages. After inoculation with P. aeruginosa, a persistent inflammatory response was observed (60 to 70% granulocytes). In the sterile-bead-inoculated group, the response was less prominent (30 to 40% granulocytes). As early as 2 weeks after inoculation, alveolar macrophages from infected animals were larger and had cytoplasmic features that differed from those of controls. Electron microscope examination showed prominent surface alterations in alveolar macrophages from the infected cats. These alterations persisted from 2 to 12 weeks after infection. In animals inoculated with sterile beads, alveolar macrophages exhibited less extensive surface changes that had resolved by week 8. Histologically, chronic bronchiolitis and pneumonia were more severe in the infected animals than in controls. This model of chronic inflammation and macrophage stimulation, which is similar to the chronic pneumonia of cystic fibrosis, may be a useful approach to answer questions on the role of macrophage activation in chronic lung disease.

Topics: Animals; Cats; Female; Inflammation; Leukocyte Count; Macrophages; Male; Microscopy, Electron; Pneumonia; Pseudomonas aeruginosa; Pseudomonas Infections; Sepharose

The effect of locally applied calcium on the vascular permeability is studied under the conditions of an acute inflammatory reaction. Using the Sephadex-inflammation, local elevated calcium concentration neither inhibited oedema formation, nor were reduction of inflammatory signs at all observed on the microscopical level.

Topics: Animals; Calcium; Capillary Permeability; Edema; Inflammation; Rats; Sepharose

Horseradish peroxidase (HRP) or bovine serum albumin (BSA) were covalently linked to polyacrylamide or agarose beads and were injected into control Syrian hamsters and hamsters previously immunized with either HRP or BSA. Animals sensitized to soluble antigen and subsequently challenged intravenously with the same antigen immobilized on beads developed an acute focal inflammatory response within 2 to 6 hours after injection. The acute response involved local deposition of IgG and complement (beta1A/beta1C globulin), polymorphonuclear leukocyte exudation, and variable amounts of hemorrhage. A focal vasculitis was occasionally present. Within 72 hours the reaction had become largely mononuclear or granulomatous in nature, and giant cell formation was seen within 4 days after immobilized antigen injection. Severe reactions developed only upon recognition of specific antigenic determinants; thus hamsters immunized against soluble HRP developed characteristic lesions only upon intravenous challenge with HRP-coated beads but not with beads coated with unrelated antigen (BSA). The beads elicited only a mild foreign body reaction in the control hamsters at 5 to 7 days after injection which was temporally and histopathologically distinct from the lesions in immunized hamsters. Thus, the state of existing immunity can influence the character and severity of the local pulmonary inflammatory response.

Topics: Acrylamides; Animals; Complement System Proteins; Cricetinae; Fluorescent Antibody Technique; Foreign-Body Reaction; Hypersensitivity; Immunoglobulin G; Inflammation; Injections, Intramuscular; Injections, Intravenous; Leukocytes; Lung; Lung Diseases; Microspheres; Peroxidases; Pulmonary Artery; Sepharose; Serum Albumin, Bovine