Quercetin Alleviates Asthma-Induced Airway Inflammation and Remodeling through Downregulating Periostin via Blocking TGF-ß1/Smad Pathway.

INTRODUCTION: The aim of the study was to discuss whether the anti-asthmatic effect of quercetin is related to periostin and the downstream molecular pathway of quercetin's anti-asthmatic effect. METHODS: We constructed asthmatic mice, sensitized by ovalbumin, and administrated different treatments into mice according to the experimental design. In this study, we mainly observed the inflammatory response, airway fibrosis, and airway hyperresponsiveness in asthmatic mice. Pathological stains (H&E, PAS, and Masson) were performed. We also detected the inflammation factors and fibrosis-related cytokines by enzyme-linked immunosorbent serologic assay. In addition, we also explored the level of periostin by enzyme-linked immunosorbent serologic assay and Western blot. At the same time, TGF-ß1/Smad pathway was also determined by Western blot. RESULTS: A high expression of periostin was found in asthmatic mice, and quercetin decreases periostin content in bronchoalveolar lavage fluid. Quercetin and OC-20 inhibit airway inflammation response, airway fibrosis, and airway hyperreactivity. Quercetin downregulated TGF-ß1/Smad pathway in the lung tissues of asthmatic mice. Anti-asthma role of quercetin is related to periostin. Then deeper mechanical study revealed that inhibiting TGF-ß1 could improve asthmatic symptoms, and quercetin exerted the protective effect on asthmatic mice through inhibition of TGF-ß1/Smad pathway. CONCLUSION: Quercetin provided a protective role against asthma via periostin, manifested by mild inflammatory infiltration, reduced goblet cell proliferation, and reduced airway fibrosis. TGF-ß1/Smad pathway is an important transduction system, participating in the protective effect of quercetin on asthma.

Potential roles of recombinant acetylcholine receptor α subunit 1-211 in immunoadsorbent and DNA immunization.

The open reading frame of the α-subunit (amino acids 1-211) of human muscle nicotinic acetylcholine receptor (hAChR(α211)) was inserted into eukaryotic expression vector of pPIC9K to form a recombinant plasmid. After transformation and expression, the target protein rhAChR(α211) (recombinant hAChR(α211)) was secretively expressed in recombinant strain P. pastoris GS115/pPIC9K-hAChR(α211) with a yield of 25 mg/L. rhAChR(α211) was purified by Q Sepharose column and gel filtration chromatography. Furthermore, the purified protein was coupled with CNBr-actived Sepharose 4B to form a special immunoadsorbent. By this immunoadsorbent, the removal rate for AChRAb in two myasthenia gravis (MG) patient sera reached 84% and 94%, respectively. The DNA fragment of hAChR(α211) was cloned into shuffle vector of pcDNA3.0 to form the recombinant plasmid pcDNA-hAChR(α211). Then the gene vaccine was directly injected intramuscularly into C57BL/6 mice. After immunization, the corresponding antibody, AChRAb, was detected in mice sera by ELISA. The target gene could be re-amplified by PCR in muscle, liver, spleen and kidney of immunized mice. It provides rapid and efficient methods to remove specific acetylcholine receptor antibody from the patient's sera and establish an animal model of myasthenia gravis by recombinant hAChR(α211) immunization.

Allosensitization of islet allograft recipients.

BACKGROUND: The immune monitoring of islet transplant recipients includes the assessment of panel reactive antibodies (PRA). A negative association of PRA+ with allogeneic solid organ graft survival has been recognized, but scattered data is available for islet transplantation. METHODS: We performed a retrospective analysis of PRA status in 66 patients with type 1 diabetes mellitus recipient of islet allografts between 1985 and 2006. RESULTS: Pretransplant PRA+ was observed in 10 subjects in the old trials and associated with kidney transplantation and/or pregnancies. Thirteen subjects displayed PRA+ at follow-up, eight of whom were de novo. Overall, PRA+ did not correlate with islet graft outcome: long-term graft survival was observed in the presence of basal or persistent PRA+ and graft dysfunction occurred also in the absence of PRA+. Loss of graft function was associated with PRA+ after lowering of immunosuppression or after infection episodes. Loss of C-peptide did not affect kidney graft function even in simultaneous islet-kidney transplant recipients. Mostly, PRA remained negative under adequate immunosuppression. Patients whose immunosuppression was discontinued invariably developed PRA+. CONCLUSIONS: Monitoring of PRA under immunosuppression may have little clinical value under adequate immunosuppression in islet transplant recipients. The implications of allosensitization after discontinuation of immunosuppression need to be evaluated to define the real clinical impact in this patient population.

A quantitative assay for Epstein-Barr Virus-specific immunity shows interferon-gamma producing CD8+ T cells increase during immunosuppression reduction to treat posttransplant lymphoproliferative disease.

Reduction of immunosuppression (RIS) to allow development or recovery of Epstein-Barr virus (EBV) immunity can be used to treat EBV-associated posttransplant lymphoproliferative disease (PTLD). Quantification of EBV-specific immunity would help assessment of the efficacy of RIS therapy. Use of intracellular cytokine staining and analysis by flow cytometry to monitor functional EBV-specific T-cell immunity was evaluated in healthy volunteers. The technique was then used to monitor EBV immunity in nine renal transplant patients with PTLD during RIS. The number of interferon (IFN)-gamma producing CD8+ T cells specific for EBV increased distinctly before regression of EBV+ PTLD tumors occurred. The findings confirm the importance of IFN-gamma producing CD8+ T cells in controlling the malignant EBV-transformed B cells of PTLD. The assay effectively quantified EBV immunity during RIS in transplant patients with PTLD.

Extracellular domains of the beta, gamma and epsilon subunits of the human acetylcholine receptor as immunoadsorbents for myasthenic autoantibodies: a combination of immunoadsorbents results in increased efficiency.

Myasthenia gravis (MG) is usually caused by autoantibodies against the human muscle acetylcholine receptor (AChR). Plasmapheresis offers a therapeutic option, but, as well as removing the pathogenic anti-AChR autoantibodies, it non-specifically removes indispensable immunoglobulins. An attractive alternative to plasmapheresis would be the extracorporeal specific removal of the autoantibodies using AChR-based immunoadsorbents. Previously, we used the N-terminal extracellular domain (ECD) of the AChR alpha subunit to immunoadsorb anti-alpha subunit autoantibodies from MG sera. In this study, we immobilised the beta -, gamma- and epsilon-AChR ECDs on Sepharose and tested them as immunoadsorbents on 50 MG sera. A given ECD removed a different percentage of autoantibodies from different sera and different ECDs removed different percentages from the same serum; on average, the beta-, gamma- and epsilon-ECDs removed 22%, 20% and 15.5% of the autoantibodies, respectively. Immunoadsorption was completed in 3 min, 1 mug of ECD removed approximately 2 pmol of autoantibodies, and the immunoadsorbent could be recycled approximately 4 times. The combined use of two (alpha+gamma) or four (alpha+beta+gamma+epsilon) ECDs in a single immunoadsorbent resulted in much higher (often additive) immunoadsorption. These results show that MG sera have autoantibodies against several AChR subunits, and suggest that the combined use of all AChR ECDs could provide the basis for a novel, antigen-specific therapy for MG.

A serial copolymerization procedure for manufacturing immunoadsorption walls as a potential unit in conjunction with hemodialysis filters.

A functional immunoadsorption wall for removal of beta-2-microglobulin has been made by partially incomplete two-stage copolymerization of acrylamide with immunoadsorbent. However, a substantial amount of immunoadsorbent needs to be flushed away after the copolymerization process. Thus, to enhance the utilization efficiency of immunoadsorbent, the flushed-away immunoadsorbent was further recovered, and the copolymerization was conducted in series to produce three consecutive immunoadsorption walls in this study. Preliminary removal tests show that similar removal patterns were obtained for these immunoadsorption walls. Although it is not timely to conclude that a clinically applicable immunoadsorption wall has taken shape, the development of a partially incomplete two-stage polymerization method and its associated techniques indeed provide a good basis for large-scale manufacturing immunoadsorption walls.

Moderate effect of enamel matrix derivative (Emdogain Gel) on Porphyromonas gingivalis growth in vitro.

OBJECTIVE: The aim of this study was to analyse the antibacterial effects of Emdogain Gel or its constituents on the growth of the suspected periodontopathogen Porphyromonas gingivalis. STUDY DESIGN: The effects of the proteins of enamel matrix derivative (EMD), the commercial product Emdogain Gel or its vehicle propylene glycol alginate (PGA) (Straumann, Switzerland) on P. gingivalis growth were determined by two methods: broth dilution assay (BDA) and agar diffusion assay (ADA). RESULTS: BDA-Emdogain Gel inhibited moderately the growth of P. gingivalis, whereas EMD showed no effect. The PGA vehicle inhibited the growth completely. ADA-Emdogain Gel resulted in some inhibition in growth but was not significantly different from control. EMD revealed no zone of inhibition. PGA demonstrated statistically significant zones of inhibition. CONCLUSION: Emdogain Gel shows moderate antibacterial activities against P. gingivalis. These properties seem to be due to the PGA component of the gel preparation.

In vitro assessment of a new ABO immunosorbent with synthetic carbohydrates attached to sepharose.

Transplantation across the ABO barrier is sometimes done in cases of emergency, such as acute liver failure, but is also carried out in elective cases, e.g. kidneys from living donors. Reducing the recipient anti-A/B antibody titres is often necessary in ABO-incompatible kidney transplantation. This is usually done by the use of techniques such as plasmapheresis and protein A- or sepharose-linked anti-human Ig immunoadsorption. A new ABO immunosorbent with synthetic A- or B-trisaccharide carbohydrate epitopes linked to a sepharose matrix has been tested. Columns made of this material have been tested in vitro with plasma from A- and B-individuals, assessed for antibody reduction capacity, flow characteristics, biocompatibility, and unspecific protein adsorption. The columns have a high capacity for ABO antibody removal, reducing titres by three to seven steps in one passage. We noted a high biocompatibility, with no unspecific protein adsorption, no activation of coagulation factors, and a low activation of complement, no immune complex formation and no cytotoxicity towards cultured mammalian L929 cells.

Efficacy of immunoadsorbent devices for maintaining hepatic function in ex vivo direct xenogenic hemoperfusion.

We have developed a new system for direct xenogenic hemoperfusion of a bioartificial liver support system adopting two types of immunoadsorbent devices. In this study, we compared the efficacy of each immunoadsorbent device in maintaining porcine hepatocyte function during 3 h perfusion treatment in a canine liver failure model. Suppression of humoral immunity by the immunoglobulin adsorber prevented immunogenic hepatocyte injury more effectively, and the system showed higher hepatic function when compared with suppression of cell-mediated immunity by the leukocyte adsorber. However, single use of immunoglobulin adsorber was less effective in reducing patients' systemic ammmonia levels and modulating the Fischer's ratio compared with the case of combined use of both immunoadsorbent devices. These results suggest that suppression of humoral immunity was of primary importance in preventing immunogenic hepatocyte injury, however the adsorption of leukocytes may have a synergic effect on maintaining hepatocyte function in direct xenogenic hemoperfusion.

An effective and rapid method for functional characterization of immunoadsorbents using POROS beads and flow cytometry.

To facilitate the construction, functional characterization, and use of immunoadsorbents, we have developed a flow cytometry method that allows rapid assessment of large numbers of particle-bound antibodies. Protein G derivitized POROS beads were used to bind affinity-purified antibodies specific for synthetic peptides designed from human plasma proteins. The antibodies were covalently coupled to the beads and used to capture and release synthetic peptides that had been labeled at the C-terminus with the fluorochrome Alexa Fluor 488. Antibody coupling and specificity of antigen binding and release were measured by analysis of the POROS affinity beads by flow cytometry. The affinity-capture matrixes were also used through several antigen-binding and release cycles without loss of peptide binding efficiency. The ability to produce and characterize extremely small amounts of POROS affinity matrices will facilitate their use in protein microchemical procedures such as protein chip technology, monoclonal antibody screening and mass spectrometry, applications where analytes are limiting or present in low abundance in complex mixtures.

Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome.

Guillain-Barré syndrome is a postinfectious, autoimmune neuropathy resulting in neuromuscular paralysis. Auto-antibodies, often induced by bacterial infection, bind to human gangliosides possessing monosialoside and diasialoside epitopes and impair the function of nerve junctions, where these ganglioside structures are highly enriched. Truncated gangliosides representive of GD3, GQ1b and GM2 epitopes have been synthesized as methyl glycosides and as a glycosides of an eleven carbon tether. The synthetic oligosaccharide ligands are structural mimics of these highly complex ganglioside epitopes and via their ability to neutralize or remove auto-antibodies have the potential for therapy, either as soluble blocking ligands administered systemically, or as immuno-affinity ligands for use as extracorporeal immunoadsorbents.

Synthetic disialylgalactose immunoadsorbents deplete anti-GQ1b antibodies from autoimmune neuropathy sera.

Acute and chronic autoimmune neuropathies, including Guillain-Barré syndromes (GBS) are often characterized by the presence of autoantibodies that react with neural gangliosides. Evidence from human and animal studies indicates that anti-ganglioside antibodies play a primary neuropathogenic role, and their rapid elimination from the circulation through specific immunoadsorption therapy thus has the potential to ameliorate the course of the disease. Here we have tested this therapeutic principle in the Miller Fisher variant of GBS that is associated serologically with acute phase anti-GQ1b ganglioside immunoglobulin G (IgG) antibodies, and in chronic ataxic neuropathies associated with persistently elevated immunoglobulin M (IgM) antibodies that react with GQ1b, GD3 and other disialylated gangliosides. Human and mouse anti-GQ1b IgG and IgM antibodies may also react with GD3, suggesting the shared terminal disialoside epitope could be involved in antibody binding. We thus synthesized the terminal trisaccharide, NeuAc(alpha2-8)NeuAc(alpha2-3)Gal common to GQ1b and GD3, and conjugated it to bovine serum albumin (BSA). This disialylgalactose glycoconjugate (DSG-BSA) binds anti-GQ1b antibodies in 32/58 (55%) human sera containing IgG or IgM anti-GQ1b antibodies at titres up to 1/130 000; it also binds a wide range of mouse monoclonal anti-GQ1b and -GD3 antibodies. When conjugated to Sepharose as mock therapeutic immmunoaffinity columns, the immobilized trisaccharide (DSG-Sepharose) eliminates anti-GQ1b antibodies from positive sera in proportion to their level of binding to DSG-BSA. Oligosaccharide-specific immunoadsorption therapy thus provides a new therapeutic approach to anti-GQ1b antibody-associated syndromes that could be applied to clinical practice. Furthermore, modification of the immobilized oligosaccharide epitopes to incorporate other glycan structures may allow this approach to be adapted to other forms of autoimmune neuropathy associated with uniform anti-glycolipid antibody profiles.

Treatment of coagulation inhibitors with extracorporeal immunoadsorption (Ig-Therasorb).

Coagulation inhibitors may occur as alloantibodies in patients with congenital factor deficiencies or as autoantibodies in patients with a previously normal coagulation. We treated 10 patients with factor VIII inhibitors (three haemophiliacs and seven patients with acquired factor VIII inhibitors) and one patient with a factor V inhibitor using extracorporeal immunoadsorption to immobilized antibodies against human immunoglobulins (Ig-Therasorb). The initial inhibitor titre was between 18 BU/ml and 540 BU/ml. Nine patients had signs of bleeding. Eighty-nine immunoadsorption sessions were performed in the 11 patients (8.1 +/- 5.1 per patient), each processing 6980 +/- 880 ml of plasma in 3.8 +/- 0.5 h. The mean reduction of the inhibitor titre was 71.9 +/- 19.4% per session. Serum IgG, IgA and IgM levels decreased by 68.7 +/- 10.1%, 55.7 +/- 12.7% and 48.6 +/- 11.1% respectively. In two haemophiliac patients, an initial titre reduction prior to an immune tolerance protocol was performed. Another haemophiliac patient was treated because of acute cerebral bleeding. In six out of eight patients with acquired inhibitors, a durable elimination was achieved within a median of 18 d. Treatment was safe and well-tolerated and seems to be a promising method in the treatment of patients with coagulation inhibitors, especially when a fast inhibitor titre reduction is necessary.

Immunosorbent for removal of b2-microglobulin from human blood plasma.

Immunosorbent removing beta2-microglobulin from human blood plasma was synthesized on the basis of sheep monospecific polyclonal antibodies to beta2-microglobulin and conditions ensuring effective regeneration of the immunosorbent for its repeated use were selected. Relationships between adsorption capacity and the volume of immobilized ligand, antigen concentration in the solution, and duration of incubation were studied. The adsorbent can be used for effective and specific removal of beta2-microglobulin from the plasma of patients on chronic hemodialysis.

Immunoadsorption, current status and future developments.

The association of abnormalities in the cellular and humoral immune system with various autoimmune diseases provides the rationale for apheresis technologies. While plasmapheresis or plasma exchange is limited by its non-selective removal of all plasma components, modern apheresis techniques aim to provide more specific elimination according to clinical needs and avoid plasma product replacement. However, the commercialisation has not met the expectations in the early 80's and the number of patients treated by extracorporeal immunoadsorption remains small due to a lack of well-defined controlled trials and limited reimbursement. This review highlights the immunological and technical basis for extracorporeal immunoadsorption, as well as its current status in the treatment of immunologically-mediated diseases.

[Changes in the lymph nodes of rats under the influence of natural sorbents in damaging exposure to carbophos]. / Izmeneniia limfaticheskikh uzlov u krys pod vliianiem prirodnykh sorbentov pri povrezhdaiushchem vozdeistvii karbofosa.

The experiment was performed in male Wistar rats to study the influence of natural sorbents (Tyumen diatomites and Novosibirsk kudurites) administered with food at dose 6% of its dry weight on lymph nodes. The data obtained demonstrated that addition of natural sorbents to food does not exert pathological influence on lymph nodes of experimental animals. Sorbent diet affects follicular and paracortical lymph node structures, changing their sizes and cytological composition; natural sorbents increase immune potential which is realized after the impairing action.

Immune modulation associated with extracorporeal immunoadsorption treatments utilizing protein A/silica columns.

The Prosorba column is designed for the removal of IgG and IgG containing immune complexes from plasma. Clinical studies employing patients presenting with idiopathic thrombocytopenic purpura (ITP) indicate that this new form of therapy is effective in approximately 40% of treated patients. Responding patients exhibit a significant increase in platelet numbers associated with decreases in antiplatelet antibody and immune complexes suggesting the induction of immune modulation. Preliminary studies indicate that ITP patients presenting with antiplatelet IgG antibody are those most likely to respond. In addition, this subgroup of ITP patients also exhibit elevated levels of antiidiotypic IgG antibody, which may contribute to an exacerbation of the autoimmune process due to antigen mimicry of the platelet autoantigen. Interestingly, antiidiotypic IgG antibody levels appear to decrease in association with antiplatelet IgG autoantibody levels suggesting that removal of immune complexes composed of IgG autoantibody and platelet autoantigen and/or antiidiotypic IgG antibody may be related to the observed clinical responses. Additional studies with alloimmune patients refractory to platelet transfusion suggest that transfused platelet retention time may be increased as a consequence of immunoadsorption therapy. This clinical response appears to be related to decreases in IgG alloantibody, again suggesting the induction of immune modulation. Alloimmune thrombocytopenic patients also appear to present with elevated levels of antiidiotypic IgG antibody which may contribute to an exacerbation of the alloimmune process due to antigen mimicry of platelet alloantigen(s). Preliminary studies indicate that both IgG alloantibody and corresponding antiidiotypic IgG antibody levels appear to decrease during immunoadsorption therapy, which suggests that removal of these antibodies, possibly in the form of immune complexes, may be related to clinical responses. Finally, studies in rheumatoid arthritis patients suggest that immunoadsorption therapy may be of clinical benefit in this autoimmune disorder. Consistent with the results observed above, preliminary studies in patients responding to immunoadsorption treatments again suggest that there is a concomitant decrease in idiotypic IgG (rheumatoid factor) and antiidiotypic IgG antibodies levels during therapy.

In vitro evaluation of the efficacy and biocompatibility of new, synthetic ABO immunoabsorbents.

Synthetic ABO immunoabsorbents (known as Synsorbs) were in use for several years to specifically eliminate ABO antibodies from the patient's circulation before ABO-incompatible organ or bone marrow transplantation. Because Synsorbs are no longer available, we have developed new ABO immunoabsorbents. These substances, termed BioSorbents A and B, respectively, consist of synthetic A or B trisaccharides covalently coupled to macroporous glass beads via polyacrylamide. Here we report the evaluation of BioSorbents in regard to efficacy, specificity, and biocompatibility. Using a closed-circuit in vitro system, representing a 1:10-1:20 scale as compared with the immunoabsorption procedure with an adult patient, blood group O plasma was run through columns filled with ethylene oxide-sterilized BioSorbent. Hemagglutination was reduced by 4 titer steps after absorption, and anti-A and/or anti-B IgM/G/A, as measured by ABO ELISA, dropped by 85% or more, while no nonspecific absorption of immunoglobulins occurred. No significant changes could be observed for complement (C3, C4, and total hemolytic complement of the classical pathway) or for coagulation parameters (fibrinogen, prothrombin time, activated partial thromboplastin time). As monitored by immunoblotting, neither factor XII nor high molecular weight kininogen was cleaved. In addition, a monocyte phagocytosis inhibition test provided evidence that no significant aggregation of IgG had occurred during absorption. We conclude that BioSorbents A and B are efficient, specific, and biocompatible with human plasma.

Use of protein A immunoadsorption as a treatment for thrombocytopenia in HIV-infected homosexual men: a retrospective evaluation of 37 cases.

Thirty-seven HIV-infected homosexual men with thrombocytopenia (less than 100 x 10(9)/l) received protein A immunoadsorption treatments to remove platelet-sensitizing immunoglobulin (Ig) G and circulating immune complexes (CIC) from plasma. Patients received an average of six treatments each, consisting of 250 ml plasma over a 3-week period. Clinical improvement in hemorrhagic symptoms associated with substantial increase in platelet counts was achieved in 18 patients. These responses were maintained over a median follow-up period of more than 7 months in 14 evaluable patients who were not lost to follow-up (three patients relapsed in 2 weeks and one received another therapy). Generally, moderate transient treatment-related side-effects included fever, musculoskeletal pain, chills and nausea. A transient serum sickness-like reaction was observed in seven patients, leading to termination of treatment in two. Clinical responses were associated with significant decreases in levels of platelet-sensitizing Ig, including CIC. Stimulation of broadly cross-reactive anti-antigen-binding fragment [F(ab)2], antibodies contributed to these responses. Protein A immunoadsorption is an effective alternative treatment for HIV-associated thrombocytopenia.

Immunoadsorbents with synthetic oligosaccharide hapten representing blood group A substances.

Immunoadsorbents with a synthetic oligosaccharide hapten representing human blood group A specific substances are prepared. The synthetic hapten, known as A-trisaccharide, which carries a space arm, is chemically attached to various solid supports, either directly through a suitable functional group at the end of the spacer arm or indirectly via a protein conjugated to the hapten. The preparation involves simple and mild procedures for the activation and/or derivatization of the supports. The latter includes naturally occurring polyhydroxy materials such as agarose, cellulose, or cellulose derivatives, and other particulate materials such as inorganic diatomites and a synthetic organic copolymer. The methods used for the coupling concern specifically the preparation of controlled-capacity and high-efficiency immunoadsorbents, with limited incorporations, which may be prepared easily and used for the selective removal, or affinity chromatographic separation, of specific antibodies from plasma environment or blood. It has been found that while hapten incorporation to the support may be varied rather easily, the physical nature of the support as well as the form of the hapten is important in determining the efficiency of an immunoadsorbent.