Blocking Antibody (blocking + antibody)

Distribution by Scientific Domains
Distribution within Medical Sciences

Selected Abstracts

Endotoxin-stimulated macrophages decrease bile acid uptake in WIF-B cells, a rat hepatoma hybrid cell line

HEPATOLOGY, Issue 1 2000
Ekkehard Sturm
Endotoxemia leads to cytokine-mediated alterations of the hepatocellular sodium-taurocholate-cotransporting polypeptide (ntcp). We hypothesized that stimulated macrophages are essential transducers for down-regulating hepatocellular bile salt uptake in response to endotoxin (lipopolysaccharide [LPS]) exposure. Using an in vitro model, we exposed mouse macrophages (IC-21 cell line) to LPS for 24 hours. Concentrations of cytokines tumor necrosis factor-, (TNF-,), interleukin (IL)-1,, and IL-6 increased 10.6-fold, 12.5-fold, and 444-fold, respectively, in LPS-conditioned IC-21 medium (CM) versus unconditioned IC-21 medium (UM). WIF-B rat hepatoma hybrid cells were incubated with either CM or UM or treated directly with medium containing recombinant TNF-,, IL-1,, and IL-6. [3H]Taurocholate ([3H]TC) uptake decreased in WIF-B cells exposed to either TNF-, (54% of control), IL-1, (78%), IL-6 (55%) as single additives, or in triple combination (TCC) (43%). A virtually identical decrease was observed after exposing WIF-B cells to CM (52%, P < .001). LPS had no direct effect on [3H]TC uptake. CM treatment did not decrease L-alanine transport in WIF-B cells. Blocking antibodies against TNF-,, IL-1,, and IL-6 restored the diminished [3H]TC uptake in cells exposed to TCC and CM to 87% and 107% of controls, respectively. Northern blotting revealed that ntcp messenger RNA (mRNA) expression was significantly reduced in WIF-B cells after exposure to CM, and in primary rat hepatocytes exposed to CM or TNF-, (68%, 14%, and 29% of control, respectively). We conclude that macrophages and their ability to secrete the cytokines TNF-,, IL-1,, and IL-6 may be essential in mediating the endotoxin-induced cholestatic effect of decreased hepatocellular bile salt uptake. [source]

Transmembrane BAFF from rheumatoid synoviocytes requires interleukin-6 to induce the expression of recombination-activating gene in B lymphocytes

Caroline Rochas
Objective B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. Methods B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription,polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin , and , light chain expression and by ligation-mediated,PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. Results RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the ,-to-, ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. Conclusion Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA. [source]

Blocking antibodies in allergen immunotherapy: the Yin and Yang

C. L. Hardy
No abstract is available for this article. [source]

Cooperative expression of junctional adhesion molecule-C and -B supports growth and invasion of glioma

GLIA, Issue 5 2010
Mirna Tenan
Abstract Brain invasion is a biological hallmark of glioma that contributes to its aggressiveness and limits the potential of surgery and irradiation. Deregulated expression of adhesion molecules on glioma cells is thought to contribute to this process. Junctional adhesion molecules (JAMs) include several IgSF members involved in leukocyte trafficking, angiogenesis, and cell polarity. They are expressed mainly by endothelial cells, white blood cells, and platelets. Here, we report JAM-C expression by human gliomas, but not by their normal cellular counterpart. This expression correlates with the expression of genes involved in cytoskeleton remodeling and cell migration. These genes, identified by a transcriptomic approach, include poliovirus receptor and cystein-rich 61, both known to promote glioma invasion, as well as actin filament associated protein, a c-Src binding partner. Gliomas also aberrantly express JAM-B, a high affinity JAM-C ligand. Their interaction activates the c-Src proto-oncogene, a central upstream molecule in the pathways regulating cell migration and invasion. In the tumor microenvironment, this co-expression may thus promote glioma invasion through paracrine stimuli from both tumor cells and endothelial cells. Accordingly, JAM-C/B blocking antibodies impair in vivo glioma growth and invasion, highlighting the potential of JAM-C and JAM-B as new targets for the treatment of human gliomas. 2009 Wiley-Liss, Inc. [source]

Blockade of IL-15 activity inhibits microglial activation through the NF,B, p38, and ERK1/2 pathways, reducing cytokine and chemokine release

GLIA, Issue 3 2010
Diego Gomez-Nicola
Abstract Reactive glia formation is one of the hallmarks of damage to the CNS, but little information exists on the signals that direct its activation. Microglial cells are the main regulators of both innate and adaptative immune responses in the CNS. The proinflammatory cytokine IL-15 is involved in regulating the response of T and B cells, playing a key role in regulating nervous system inflammatory events. We have used a microglial culture model of inflammation induced by LPS and IFN, to evaluate the role of IL-15 in the proinflammatory response. Our results indicate that IL-15 is necessary for the reactive response, its deficiency (IL-15-/-) leading to the development of a defective proinflammatory response. Blockade of IL-15, both with blocking antibodies or with the ganglioside Neurostatin, inhibited the activation of the NF,B pathway, decreasing iNOS expression and NO production. Inhibiting IL-15 signaling also blocked the activation of the mitogen-activated protein kinase (MAPK) pathways ERK1/2 and p38. The major consequence of these inhibitory effects, analyzed using cytokine antibody arrays, was a severe decrease in the production of chemokines, cytokines and growth factors, like CCL17, CCL19, IL-12, or TIMP-1, that are essential for the development of the phenotypic changes of glial activation. In conclusion, activation of the IL-15 system seems a necessarystep for the development of glial reactivity and the regulation of the physiology of glial cells. Modulating IL-15 activity opens the possibility of developing new strategies to control gliotic events upon inflammatory stimulation. 2009 Wiley-Liss, Inc. [source]

MT1-MMP, but not secreted MMPs, influences the migration of human microvascular endothelial cells in 3-dimensional collagen gels

Teruhiko Koike
Abstract Matrix metalloproteinases (MMPs) and their specific inhibitors the TIMPs play significant roles in angiogenesis. We investigated how the expression of specific MMPs and TIMPs by human microvascular endothelial cells (hmECs) was modulated by culture of the cells in 3-dimensional (3D) type I collagen gels versus 2-dimensional (2D) collagen-coated surfaces. By reverse-transcription polymerase chain reaction (RT-PCR), levels of mRNA for MMPs-1, -2, and -13, MT1-MMP, and TIMPs-1 and -2 were similar in 2D versus 3D cultures. By Western blot assay, TIMP-1 and proMMP-1 were present and were expressed similarly in media from 2D versus 3D cultures, whereas active MMPs-1, -9, and -13 were not detected. Active MMP-13 was present in cell lysates (CL) and was increased in lysates from 3D cultures relative to 2D cultures. Relative to 2D cultures, CL and media from 3D cultures exhibited a decrease in expression of TIMP-2 and an increased conversion of proMMP-2 and proMT1-MMP to active or processed forms. The MMP inhibitor GM6001 interfered with the migration of hmECs in 3D cultures, but not in 2D cultures. Addition of active MMP-1 or blocking antibodies to TIMP-1 did not affect the migration of hmECs in 3D collagen. Migration in 3D collagen was decreased by TIMP-2 (an inhibitor of MT1-MMP), but not by TIMP-1 (a poor inhibitor of MT1-MMP, but an efficient inhibitor of MMP-2). Collectively, our data indicate that MT1-MMP contributes significantly to the movement of hmECs through 3D collagen, in contrast to secretory-type MMPs-1, -2, -9, and -13, which are not critical for this movement. J. Cell. Biochem. 86: 748,758, 2002. 2002 Wiley-Liss, Inc. [source]

The angiopoietin pathway is modulated by PAR-1 activation on human endothelial progenitor cells

Summary.,Objectives:,The importance of protease-activated receptor-1 (PAR-1) in blood vessel development has been shown in knock-out mice. As endothelial progenitor cells (EPCs) express functional PAR-1, we examined whether PAR-1 stimulation by the peptide SFLLRN interfered with the angiopoietin pathway, that is EPC commitment, proliferation and migration. Methods and results:,Given the strong PAR-1 expression on CD34+ cells, we tested the effect of SFLLRN 75 ,mol L,1 on the emergence of EPCs from cord blood. PAR-1 activation did not modify the number of colonies or the day of emergence, in keeping with the lack of induction of angiopoietin 1 gene expression. Conversely, SFLLRN treatment of EPCs induced angiopoietin 2 gene expression and protein synthesis. Experiments with polyclonal blocking antibodies showed that angiopoietin 2 was involved in the proliferative effect of PAR-1 activation. PAR-1 activation also enhanced migration toward angiopoietin 1 in a Boyden chamber assay. Conclusions:,Our study demonstrates that PAR-1-induced proliferation of EPCs involves angiopoietin 2. PAR-1 also enhances EPC migration toward angiopoietin 1. These findings might explain the role of thrombin in neovascularization via the angiopoietin pathway. [source]

Role of Platelets in Hypercholesterolemia-Induced Leukocyte Recruitment and Arteriolar Dysfunction

ABSTRACT Objective: To define the contribution of platelets, specifically platelet-associated P-selectin, to the altered venular and arteriolar responses induced by hypercholesterolemia. Methods: Leukocyte and platelet recruitment in cremasteric venules, and endothelium-dependent relaxation (EDR) in arterioles were determined using intravital videomicroscopy. Wild-type (WT) mice were placed on a normal or high cholesterol diet. Hypercholesterolemic mice were treated with blocking antibodies against either P-selectin or PSGL-1, or were depleted of neutrophils (ANS) or platelets (APS). Bone marrow chimeras (P-selectin deficiency in platelets, but not in endothelial cells) were produced by transplanting bone marrow from P-selectin,/, into WT mice (P-sel,/,, WT). Results: Hypercholesterolemia (HC) elicited the recruitment of adherent platelets and leukocytes in venules and an impaired EDR in arterioles. The exaggerated cell adhesion responses were absent in hypercholesterolemic mice treated with ANS, anti-P-selectin or anti-PSGL-1 antibodies and in P-sel,/,, WT chimeras. The hypercholesterolemia-induced impairment of arteriolar EDR was significantly blunted in mice rendered either neutropenic or thrombocytopenic, and in P-sel,/,, WT chimeras. Conclusions: The findings indicate that platelet-associated P-selectin contributes to the recruitment of leukocytes and platelets in venules of hypercholesterolemic mice and that the P-selectin-mediated adhesive interactions also contribute to the impaired arteriolar function induced by hypercholesterolemia. [source]

REVIEW ARTICLE: Anti-phospholipid Antibodies and Other Immunological Causes of Recurrent Foetal Loss , A Review of Literature of Various Therapeutic Protocols

Shrimati Shetty
Problem, An immune-based aetiology is one of the several accepted causes for recurrent foetal loss (RFL). However, most of the immunological theories have not fulfilled the criteria for causality. This is a review of the various immunological causes of RFL and the outcome of different treatment protocols. Method of study, Both auto- and alloimmune maternal immunological abnormalities have been proposed to account for foetal loss. Among the autoimmune factors, anti-phospholipid antibodies (APAs) have been demonstrated to be the strongest risk factors for foetal loss, the prevalence of which is as high as 40% in women with RFL. Other autoimmune antibodies implicated in RFL are anti-nuclear antibodies (ANAs), anti-thyroid antibodies and anti-endothelial cell antibodies. The alloimmune factors implicated in pregnancy loss of unknown aetiology include abnormal natural killer (NK) cell activity, alteration in T helper 1 (Th1) and T helper 2 (Th2) ratios, presence of alloimmune antibodies like anti-paternal cytotoxic antibodies, anti-idiotypic antibodies, mixed lymphocyte reaction blocking antibodies and abnormal expression of HLA-G molecules. Management of patients with RFL is mainly based on immunomodulatory (prednisolone, intravenous immunoglobulins, plasma exchange, paternal lymphocyte therapy), anti-aggregation (aspirin) or anti-coagulation (unfractionated or low molecular weight heparin) agents. Results, Low-molecular-weight heparin with low-dose aspirin has been found to be the most effective treatment for women with APAs and RFL. Differences in dosage, timing of treatment, inclusion criteria, outcome assessment parameters etc. are some of the factors which have resulted in discrepancies in various reports. Conclusion, Identification of the immunological mechanisms involved in pregnancy loss and the action of different therapeutic reagents is important so that effective therapies can be designed and investigated. [source]

Presentation of arthritogenic peptide to antigen-specific T cells by fibroblast-like synoviocytes

Chinh N. Tran
Objective To assess the ability of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) to function as antigen-presenting cells (APCs) for arthritogenic autoantigens found within inflamed joint tissues. Methods Human class II major histocompatibility complex (MHC),typed FLS were used as APCs for murine class II MHC,restricted CD4 T cell hybridomas. Interferon-, (IFN,),treated, antigen-loaded FLS were cocultured with T cell hybridomas specific for immunodominant portions of human cartilage gp-39 (HC gp-39) or human type II collagen (CII). T cell hybridoma activation was measured by enzyme-linked immunosorbent assay of culture supernatants for interleukin-2. Both synthetic peptide and synovial fluid (SF) were used as sources of antigen. APC function in cocultures was inhibited by using blocking antibodies to human class II MHC, CD54, or CD58, or to murine CD4, CD11a, or CD2. Results Human FLS could present peptides from the autoantigens HC gp-39 and human CII to antigen-specific MHC-restricted T cell hybridomas. This response required pretreatment of FLS with IFN,, showed MHC restriction, and was dependent on human class II MHC and murine CD4 for effective antigen presentation. Furthermore, FLS were able to extract and present antigens found within human SF to both the HC gp-39 and human CII T cell hybridomas in an IFN,-dependent and MHC-restricted manner. Conclusion RA FLS can function as APCs and are able to present peptides derived from autoantigens found within joint tissues to activated T cells in vitro. In the context of inflamed synovial tissues, FLS may be an important and hitherto overlooked subset of APCs that could contribute to autoreactive immune responses. [source]

Overcoming immunological tolerance to melanoma: Targeting CTLA-4

F Stephen HODI
Abstract The use of immunotherapeutics in melanoma has received much attention, and recent advances to further characterize the regulatory components of the immune system and the importance of co-stimulatory molecules have opened a new area for clinical investigation. Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) serves as a negative regulator of immunity. Recent trials administering fully human anti-CTLA-4 monoclonal antibodies to melanoma patients have demonstrated clinically meaningful responses. Treatment with CTLA-4 blocking antibodies, however, is not without potential toxicities. Autoimmune side-effects, the most common being colitis-associated diarrhea, are frequently associated with clinical responses. In efforts to build upon prior vaccination efforts as well as attempt to offer patients clinically meaningful immune responses with a CTLA-4 blockade but without significant toxicities, we conducted a clinical trial in patients who previously received autologous tumor cells engineered to secrete granulocyte-macrophage colony stimulating factor (GVAX; Cell Genesys, South San Francisco, CA, USA) with periodic infusions of CTLA-4 blocking antibodies. This sequential treatment resulted in clinically significant anti-tumor immunity without grade 3 or 4 toxicity in most patients. Pathological analyses following treatment of pre-existing tumors revealed a linear correlation between tumor necrosis and the ratio of intra-tumoral CD8+ effector cells to FoxP3+ regulatory cells (Tregs). Effective anti-tumor immunity and serious autoimmunity can be disassociated. Further targeting of anti-tumor Tregsin combinatorial therapy approaches may be a rich avenue of future investigation. [source]

Anaplasma phagocytophilum specifically induces tyrosine phosphorylation of ROCK1 during infection

Venetta Thomas
Summary Anaplasma phagocytophilum, an obligate intracellular pathogen that persists within polymorphonuclear leucocytes, is the second most common tick-borne agent in North America. We now show that infection of a promyelocytic cell line and neutrophils with A. phagocytophilum results in pathogen-specific tyrosine phosphorylation of ROCK1. Phosphorylation is associated with PSGL-1 and Syk, because PSGL-1 blocking antibodies and siRNA targeting Syk interfere with ROCK1 phosphorylation in A. phagocytophilum -infected cells. Knockdown of either Syk or ROCK1 also markedly impaired A. phagocytophilum infection. These data demonstrate a role for A. phagocytophilum -mediated ROCK1 phosphorylation in infection, and suggests that inhibiting this pathway may lead to new, non-antibiotic strategies to treat human granulocytic anaplasmosis. [source]

Hypoallergenic mutants of Ole e 1, the major olive pollen allergen, as candidates for allergy vaccines

E. G. Marazuela
Summary Background The C-terminal region of Ole e 1, a major allergen from olive pollen, is a dominant IgE-reactive site and offers a target for site-directed mutagenesis to produce variants with reduced IgE-binding capability. Objective To evaluate in vitro and in vivo the immunogenic properties of three engineered derivatives of Ole e 1. Methods One point (Y141A) and two deletion (135,10 and 140,5) mutants were generated by site-directed mutagenesis of Ole e 1-specific cDNA and produced in Pichia pastoris. Ole e 1 mutants were analysed for IgE reactivity by ELISA using sera from olive pollen-allergic patients. Their allergenicity was also investigated in both a mouse model of allergic sensitization and in basophil activation assays. IgG1 response was assayed by immunoblotting and competitive ELISA. T cell reactivity was evaluated by proliferation assays and cytokine production in splenocyte cultures. Results The 135,10 mutant showed the strongest reduction in the IgE-binding capability of sera from olive pollen-allergic patients. Rat basophil leukaemia assays identified the deletion mutant 135,10 as the variant with the lowest ,-hexosaminidase-releasing capacity. Furthermore, the same 135,10 mutant induced the lowest IgE levels in a BALB/c mouse model of sensitization. All Ole e 1 mutants retained their allergen-specific T cell reactivity. Immunization of mice with the mutants induced IgG1 antibodies, which cross-reacted with Ole e 1 and Ole e 1-like allergens from ash, lilac and privet pollens. The ability of the human IgE to block the binding of anti-Ole e 1 mutant-specific mouse IgG1 antibodies to natural Ole e 1 demonstrated that Ole e 1 mutants are able to induce in vivo antibodies reactive to the natural allergen. Conclusion The 135,10 mutant with reduced allergenicity, intact T cell reactivity and capacity to induce blocking antibodies could provide a suitable candidate vaccine for efficient and safer therapy of olive pollen allergy. [source]

Gonadotrophin receptor blocking antibodies measured by the use of cell lines stably expressing human gonadotrophin receptors are not detectable in women with 46,XX premature ovarian failure

Massimo Tonacchera
Summary background, Premature ovarian failure (POF) is defined by cessation of ovarian function after puberty and before the age of 40. The syndrome is characterized by amenorrhoea, oestrogen deficiency and elevated levels of gonadotrophins. Autoimmunity has been proposed as a mechanism for some cases of destruction or malfunction of ovarian follicles. POF is often associated with type I and type II polyglandular autoimmune syndromes. It has also been postulated that receptors such as the LH and FSH receptors might become targets for blocking antibodies and such antibodies could be a cause of ovarian failure. patients and methods, Sixty-nine patients with POF isolated or associated with other endocrine autoimmune diseases (autoimmune thyroid diseases, Addison's disease, type 1 diabetes mellitus, multiple sclerosis, myasthenia gravis) were studied. All the patients had secondary amenorrhoea. The patient group had a median age of 331 years (range 15,57). Ovarian failure had been diagnosed at a median age of 29 years (range 15,39). The median time since diagnosis was almost 1 year but in six patients gonadal insufficiency had appeared 10,30 years earlier. All had a normal chromosomal karyotype (46, XX). Patients with POF were characterized by duration of amenorrhoea > 1 year, with elevated FSH and LH levels and undetectable or low oestrogen levels. Cell lines stably expressing recombinant human LH (CHO-LHr) and FSH (CHO-FSHr) receptors were prepared and used to search for antibodies able to inhibit LH- or FSH-stimulated cAMP production. Immunoglobulins extracted from sera of patients with POF were incubated with CHO-LHr and CHO-FSHr in the presence of human recombinant CG and FSH, respectively. results and conclusions, None of the immunoglobulin G (IgG) preparations from patients with POF was able to inhibit the activity of the FSH- and CG-stimulated cAMP production. [source]

A luminescent bioassay for thyroid blocking antibodies

N. J. Jordan
Thyroid blocking antibodies (TBAb) have a role in the development of hypothyroidism and in the neonate are responsible for transient hypothyroidism. Specific measurement of TBAb requires a bioassay, but current methods are lengthy and cumbersome. We describe a rapid luciferase-based method for the detection of TBAb using the lulu* cell line which is suitable for the provision of a clinical service Chinese hamster ovary (CHO) cells were transfected with human TSH-R together with G418 resistance and a cAMP responsive luciferase construct. Stable pools of transfected cells were selected and clones identified by limiting dilution. Clone lulu* gave the best response to stimulation by TSH and was used to develop a bioassay for TBAb. The luminescent bioassay conditions have been optimized and validated using 12 serum samples from patients found to be TBAb positive in a bioassay using an established method quantifying cAMP by radioimmunoassay (RIA). The effect of thyroid stimulating antibodies (TSAb) on the calculation of Inhibition Index (InI) using two previously described formulae have been investigated and we have used serum containing both TSAb and TBAb to investigate detection of TBAb in samples containing more than one type of activity. Lulu* displays a dose dependent increase in luciferase expression in response to stimulation with bovine (b) TSH which is more effective in serum free medium than in salt free buffer. TSH stimulated luciferase expression can be inhibited by TBAb in either serum or an immunoglobulin preparation. Using optimized assay conditions, challenging 10% serum against 1 U/l bTSH in culture medium, we have tested 31 euthyroid sera to determine a reference range: InI values >23% were considered positive. Twelve samples previously shown to contain TBAb by an established method quantifying cAMP by RIA were positive by the luciferase-based assay. Of control sera, 20/20 systemic lupus erythematosus, 13/14 rheumatoid arthritis, 12/12 multinodular goitre were negative. We demonstrated that if more complex formulae are used to calculate InI, false positive TBAb results can be obtained in samples containing only TSAb. Finally, when sera contain both TSAb and TBAb, the net activity of stimulating and blocking antibodies is detected in the bioassay. Where TSAb are also present, analysis of serum may be required at several dilutions to detect TBAb. We describe the production of a new cell line, lulu*, and its use to develop a luminescent bioassay for TBAb suitable for clinical use. Comparing two established methods of calculating TBAb, we found that they do not give identical results. In light of this, the high prevalence reported for TBAb in some studies has to be considered with caution. [source]

Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLL

CYTOMETRY, Issue 4 2006
Mark Shenkin
Abstract Background: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity. Methods: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS. Results: Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling. Conclusions: Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes. 2006 International Society for Analytical Cytology [source]

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation,

CYTOSKELETON, Issue 4 2004
Mike Lorenz
Abstract Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-,-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured. Cell Motil. Cytoskeleton 57:207,217, 2004. 2004 Wiley-Liss, Inc. [source]

PD-1 signalling in CD4+ T cells restrains their clonal expansion to an immunogenic stimulus, but is not critically required for peptide-induced tolerance

IMMUNOLOGY, Issue 1 2010
Joanne E. Konkel
Summary The ultimate outcome of T-cell recognition of peptide,major histocompatibility complex (MHC) complexes is determined by the molecular context in which antigen presentation is provided. The paradigm is that, after exposure to peptides presented by steady-state dendritic cells (DCs), inhibitory signals dominate, leading to the deletion and/or functional inactivation of antigen-reactive T cells. This has been utilized in a variety of models providing peptide antigen in soluble form in the absence of adjuvant. A co-inhibitory molecule of considerable current interest is PD-1. Here we show that there is the opportunity for the PD-1/PD-L1 interaction to function in inhibiting the T-cell response during tolerance induction. Using traceable CD4+ T-cell receptor (TCR) transgenic cells, together with a blocking antibody to disrupt PD-1 signalling, we explored the roles of PD-1 in the induction of tolerance versus a productive immune response. Intact PD-1 signalling played a role in limiting the extent of CD4+ T-cell accumulation in response to an immunogenic stimulus. However, PD-1 signalling was not required for either the induction, or the maintenance, of peptide-induced tolerance; a conclusion underlined by successful tolerance induction in TCR transgenic cells genetically deficient for PD-1. These observations contrast with the reported requirement for PD-1 signals in CD8+ T-cell tolerance. [source]

Nmi (N-Myc interactor) inhibits Wnt/,-catenin signaling and retards tumor growth

Rebecca A. Fillmore
Abstract We found that the expression levels of N-Myc interactor (Nmi) were low in aggressive breast cancer cell lines when compared with less aggressive cell lines. However, the lower levels in the aggressive lines were inducible by interferon-, (IFN-,). Because Nmi has been reported to be a transcription cofactor that augments IFN-, induced transcription activity, we decided to test whether Nmi regulates expression of Dkk1, which is also inducible by IFN-,. We established stable clones constitutively expressing Nmi in MDA-MB-231 (breast) and MDA-MB-435 (melanoma) cell lines. Dkk1 was significantly up-regulated in the Nmi expressing clones concurrent with reduced levels of the critical transcription cofactor of Wnt pathway, ,-catenin. Treatment of the Nmi expressors with blocking antibody to Dkk1 restored ,-catenin protein levels. c-Myc is a known downstream target of activated ,-catenin signaling. Treatment of Nmi expressors with the proteosome inhibitor MG132, resulted in elevated ,-catenin levels with concomitant elevation of c-Myc levels. Our functional studies showed that constitutive expression of Nmi reduced the ability of tumor cells for the invasion, anchorage independent growth and tumor growth in vivo. Collectively, the data suggest that overexpression of Nmi inhibits the Wnt/,-catenin signaling via up-regulation of Dkk1 and retards tumor growth. 2009 UICC [source]

Mucous membrane pemphigoid, thymoma, and myasthenia gravis

Haideh Yazdani Sabet
In November 1997, approximately 1 year before being evaluated at the Mayo Clinic, Rochester, a 63-year-old woman presented with erosive tongue lesions that were diagnosed by her physician as oral lichen planus. The lesions responded well to 3 months of treatment with systemic and topical corticosteroids and topical antiyeast medication. She stopped taking the medications and had a relapse. A few months after the oral lesions developed, her left eyelid became ptotic. Results of magnetic resonance imaging of her brain were normal, and the ptosis resolved spontaneously after 2 weeks. One year later, her right eyelid began to droop, and the results of edrophonium testing were positive. She was prescribed prednisone, 30 mg daily, and pyridostigmine, as needed. The ptosis improved, but never fully resolved. Radiography revealed a left ,,thyroid nodule,'' but computed tomography did not show a mediastinal mass. She was advised to have the ,,nodule'' removed surgically and came to the Mayo Clinic, Rochester, for a second opinion. Her medical history was significant for the following: tinnitus, glaucoma, early bilateral cataracts, and long-standing hypertension, for which she took losartan, 50 mg twice daily. Other medications included: prednisone, 30 mg daily; pyridostigmine as needed; famotidine, 40 mg daily; and eyedrops for glaucoma. She denied any history of hyperthyroidism or hypothyroidism, head and neck irradiation, family history of thyroid disease, or diplopia. Hepatitis serologic studies revealed hepatitis B exposure and recovery, hepatitis C immunity, and a previous hepatitis A viral infection. On examination at the Mayo Clinic, Rochester, an erosive hypertrophic plaque was noted on the posterior dorsal half of the tongue, and vesicles and erythematous erosions on the hard and soft palates ( Fig. 1a). A lace-like white pattern was seen on the buccal mucosa bilaterally, and a small erosive patch on the left buccal mucosa ( Fig. 1b). Ocular and nasal mucous membranes were normal in appearance, and there were no pertinent skin findings. Dermatopathologic examination of an excisional biopsy specimen from the left dorsum of the tongue demonstrated an ulcer with epitheliomatous hyperplasia and a granulomatous reaction, presumably due to yeast infection. Silver staining showed hyphae and yeast at the base of the tongue ulcer. The results of the direct immunofluorescence study were negative and revealed no lichenoid changes on hematoxylin and eosin staining. Indirect immunofluorescence testing of the serum revealed a 1 : 80 titer of basement membrane zone antibodies, reflecting pemphigoid. This test was positive on repeat study. Salt-split skin on monkey esophagus revealed an epidermal pattern of basement membrane zone antibodies. Treatment included fluocinonide gel applied to the involved areas four times daily and oral antiyeast therapy (fluconazole, 200 mg once daily by mouth) while the rest of the evaluation was being completed. Figure 1(a). Erosive hypertrophic tongue plaque. Figure (b) ,. Erosive patch on the buccal mucosa. As part of the evaluation of the ptosis, a myasthenia gravis antibody panel was performed. It revealed the following abnormalities: striated muscle antibody at 1 : 480 (reference range, <1 : 60), acetylcholine receptor binding antibody at 6.33 nmol/L (reference range, ,,0.02 nmol/L), acetylcholine receptor blocking antibody at 31% (reference range, 0,25%), and acetylcholine receptor modulating antibody at 100% (reference range, 0,20%), suggesting thymoma. Treatment included pyridostigmine, 30,45 mg 3,4 times daily, to control the myasthenia symptoms, while the ill-defined neck mass was being evaluated. A mildly enlarged thyroid was noted on physical examination. Hematology panel revealed thyroid-stimulating hormone (TSH) levels in the low normal range; the thyroid microsomal antibody was normal. Chest radiography showed minor tracheal deviation, and a previous computed tomogram showed what appeared to be a 3-cm enlarged mass in the thyroid. Ultrasonographically guided thyroid biopsy did not show malignancy, but a benign mesenchymal-type tumor was found and surgical excision was planned. Intraoperatively, a thymoma of the left cervical thymic tongue was found. At 6 months' follow-up, the ptosis and oral mucosal lesions had improved significantly, although she continued topical corticosteroid therapy intermittently for minor erosive oral disease. [source]

Phosphatidylethanol Mediates its Effects on the Vascular Endothelial Growth Factor via HDL Receptor in Endothelial Cells

ALCOHOLISM, Issue 2 2009
Marja Katriina Liisanantti
Background:, Previous epidemiological studies have shown that light to moderate alcohol consumption has protective effects against coronary heart disease but the mechanisms of the beneficial effect of alcohol are not known. Ethanol may increase high density lipoprotein (HDL) cholesterol concentration, augment the reverse cholesterol transport, or regulate growth factors or adhesion molecules. To study whether qualitative changes in HDL phospholipids mediate part of the beneficial effects of alcohol on atherosclerosis by HDL receptor, we investigated whether phosphatidylethanol (PEth) in HDL particles affects the secretion of vascular endothelial growth factor (VEGF) by a human scavenger receptor CD36 and LIMPII analog-I (CLA-1)-mediated pathway. Methods:, Human EA.hy 926 endothelial cells were incubated in the presence of native HDL or PEth-HDL. VEGF concentration and CLA-1 protein expression were measured. Human CLA-1 receptor-mediated mechanisms in endothelial cells were studied using CLA-1 blocking antibody and protein kinase inhibitors. Results:, Phosphatidylethanol-containing HDL particles caused a 6-fold increase in the expression of CLA-1 in endothelial cells compared with the effect of native HDL. That emergent effect was mediated mainly through protein kinase C and p44/42 mitogen-activated protein kinase pathways. PEth increased the secretion of VEGF and that increase could be abolished by a CLA-1 blocking antibody. Conclusions:, High density lipoprotein particles containing PEth bind to CLA-1 receptor and thereby increase the secretion of VEGF from endothelial cells. Ethanol-induced protective effects against coronary heart disease may be explained, at least partly, by the effects of PEth-modified HDL particles on VEGF via CLA-1-mediated mechanisms in endothelial cells. [source]

Effect of the small molecule plasminogen activator inhibitor-1 (PAI-1) inhibitor, PAI-749, in clinical models of fibrinolysis

Summary.,Background:,The principal inhibitor of fibrinolysis in vivo is plasminogen activator inhibitor-1 (PAI-1). PAI-749 is a small molecule inhibitor of PAI-1 with proven antithrombotic efficacy in several preclinical models. Objective:,To assess the effect of PAI-749, by using an established ex vivo clinical model of thrombosis and a range of complementary in vitro human plasma-based and whole blood-based models of fibrinolysis. Methods:,In a double-blind, randomized, crossover study, ex vivo thrombus formation was assessed using the Badimon chamber in 12 healthy volunteers during extracorporeal administration of tissue-type plasminogen activator (t-PA) in the presence of PAI-749 or control. t-PA-mediated lysis of plasma clots and of whole blood model thrombi were assessed in vitro. The role of vitronectin was examined by assessing lysis of fibrin clots generated from purified plasma proteins. Results:,There was a dose-dependent reduction in ex vivo thrombus formation by t-PA (P < 0.0001). PAI-749 had no effect on in vitro or ex vivo thrombus formation or fibrinolysis in the presence or absence of t-PA. Inhibition of PAI-1 with a blocking antibody enhanced fibrinolysis in vitro (P < 0.05). Conclusions: Despite its efficacy in a purified human system and in preclinical models of thrombosis, the current study suggests that PAI-749 does not affect thrombus formation or fibrinolysis in a range of established human plasma and whole blood-based systems. [source]

Blockade of CCR4 in a humanized model of asthma reveals a critical role for DC-derived CCL17 and CCL22 in attracting Th2 cells and inducing airway inflammation

ALLERGY, Issue 7 2009
F. Perros
Background:, As Th2 type lymphocytes orchestrate the cardinal features of allergic asthma, inhibiting their recruitment to the lungs could be of therapeutic benefit. Although human Th2 cells express the CCR4 chemokine receptor and increased production of CCR4 ligands has been found in asthmatic airways, studies in animals have reached contradictory conclusions on whether blocking this pathway would be beneficial. Objective:, As a lack of efficacy might be due to differences between mouse and man, we readdressed this question using a humanized severe combined immunodeficiency model of asthma. Methods:, Mice received peripheral blood mononuclear cells from house dust mite (HDM) allergic asthmatic patients and then underwent bronchial challenge with HDM. Results:, This resulted in marked allergic inflammation and bronchial hyper-reactivity. Administration of CCR4 blocking antibody abolished the airway eosinophilia, goblet cell hyperplasia, IgE synthesis and bronchial hyperreactivity. In this chimeric system, human CD11c+ dendritic cells (DCs) were the predominant source of CCR4 ligands, suggesting that DC-derived chemokines attract Th2 cells. In separate experiments using human DCs, in vitro exposure to HDM of DCs from HDM allergic patients but not healthy controls caused CCL17 and CCL22 release that resulted in chemoattraction of polarized human Th2 cells in a CCR4-dependent way. Conclusions:, Taken together, our data provide proof of concept that CCR4 blockade inhibits the salient features of asthma and justify further clinical development of CCR4 antagonists for this disease. [source]

T-Lymphocytes Modulate the Microvascular and Inflammatory Responses to Intestinal Ischemia-Reperfusion

Takeharu Shigematsu
Objective: The overall objective of this study was to define the contribution of T-lymphocytes to the microvascular and inflammatory responses of the intestine to ischemia/reperfusion (I/R). Methods: The superior mesenteric artery of wild-type (WT) and SCID mice was occluded for 45 minutes, followed by 30 minutes or 6 hours of reperfusion. Intravital fluorescence microscopy was used to monitor the extravasation of FITC-labeled albumin or the adhesion of carboxy-fluorescein diacetate succinimidyl ester (CFSE)-labeled T-lymphocytes in mucosal venules of the postischemic intestine. Tissue myeloperoxidase (MPO) was used to monitor neutrophil accumulation in the intestine of WT and SCID mice. Results: Although the number of adherent T-cells was not increased above baseline at 1 hour after reperfusion, significant T-cell adhesion (both CD4+ and CD8+) was noted at 6 hours of reperfusion. The latter response was prevented by pretreatment with a blocking antibody directed against MAdCAM-1, but not ICAM-1 or VCAM-1. A significant increase in MAdCAM-1 expression was noted in both lymphoid (Peyer's patch) and nonlymphoid regions of the postischemic small bowel. The early (30 minutes after reperfusion) albumin extravasation elicited by gut I/R in WT mice was reduced in SCID mice. Reconstitution of SCID mice with T-lymphocytes restored the albumin leakage response to WT levels. The increased intestinal MPO caused by I/R (6 hours of reperfusion) in WT mice was attenuated in SCID mice; with reconstitution of SCID mice with T-cells the MPO response was restored. Conclusions: These findings indicate that intestinal I/R is associated with the recruitment of CD4+ and CD8+ T-cells, which is mediated by endothelial MAdCAM-1. T-cells seem to modulate the recruitment of neutrophils that occurs hours after reperfusion as well as the increased albumin extravasation that occurs within minutes after reperfusion. [source]

The clinical significance of food specific IgE/IgG4 in food specific atopic dermatitis

Geunwoong Noh
Food is closely associated with the pathogenesis of atopic dermatitis. Food allergy is usually mediated by IgE antibody to specific food proteins and determination of specific IgE antibody is the basis of the common diagnostic test for food allergy. IgG4 have been reported as blocking antibody and the protective effects of blocking antibody may be clear in inhalant allergy. However, the role of IgG4 in food allergy is still a matter of debate. In this study, the clinical significance of food allergen-specific IgE/IgG4 in atopic dermatitis was investigated and compared with that of IgE. A total of 97 patients who fulfilled the diagnostic criteria for atopic dermatitis participated in this study. Skin prick test and allergy patch test were performed. Specific IgE and IgG4 concentration were measured using allergy protein chip, ,AllergyChip'. Double blinded placebo controlled food challenge test (DBPCFC) was performed for the diagnosis of allergy to milk, egg white, wheat, and soybean. DBPCFCs for milk, egg white, soybean, and wheat were performed. The positive rates were 31.7% (19/60) in milk, 36.7% (18/49) in egg white, 30.4% (7/23) in soybean, and 34.8% (8/23) in wheat. Mean IgE/IgG4 levels in DBPCFC (+) subjects is higher than those in DBPCFC (,) subjects in all food items studied. Of them, there were significantly different between two groups in egg white and wheat (Egg white in DBPCFC (+) vs. (,): 0.4 0.3 vs. 0.2 0.2, wheat in DBPCFC (+) vs. (,): 1.2 1.2 vs. 0.3 0.3, p < 0.05). Allergen-specific IgE/IgG4 may provide one of the clues to understand the mechanism of food allergy in atopic dermatitis. The present study suggests that protein microarray can be one of the useful methods to assess ongoing status of allergic diseases. [source]

Nerve growth factor mediates steroid-resistant inflammation in respiratory syncytial virus infection,,

Lida Mohtasham MD
Abstract Neurotrophic factors and receptors are upregulated in the respiratory tract of humans and rodents infected by the respiratory syncytial virus, leading to airway inflammation and hyperreactivity. The contribution of neurotrophic pathways to the recruitment of immuno-inflammatory cells and their response to anti-inflammatory therapy remains unclear. We sought to determine whether selective nerve growth factor inhibition prevents the immuno-inflammatory response against infection, and explored the effect of inhaled corticosteroids on virus-induced neurotrophic upregulation and the consequent recruitment of immuno-inflammatory cells into the airways. We tried to inhibit the recruitment of lymphocytes and monocytes into the airways of infected weanling rats using immunologic inhibition of nerve growth factor with a specific blocking antibody, or chemical inhibition of receptor tyrosine kinase with K252a. The anti-inflammatory activity of inhaled corticosteroids was studied in infected rats treated with budesonide, fluticasone, or vehicle. Immunological or chemical inhibition of nerve growth factor or its high-affinity receptor tyrosine kinase pathway inhibited the recruitment of inflammatory cells triggered by nociceptive irritation of infected rat airways, thereby reducing local and systemic immuno-inflammatory responses against the virus. Neurotrophic upregulation in infected airways was not affected by inhaled corticosteroids. As a logical consequence, these commonly used drugs were also unable to stop the recruitment of immune and inflammatory effector cells into infected airways. Overexpression of neurotrophic factors and receptors in airways infected by respiratory syncytial virus is critical for the development of airway inflammation and hyperreactivity, which is resistant to the anti-inflammatory effect of inhaled corticosteroids. Pediatr Pulmonol. 2007; 42:496,504. 2007 Wiley-Liss, Inc. [source]

1141420984 Placental exosome suppression of T cell activation and differential target of T cell subsets

DD Taylor
Problem:, One immunoregulatory pathway that has received little attention is placental exosome release. In normal pregnancy, as factors linked with early immunomodulation decline, placental exosomes become key in modulating T-cell activation, maintaining the absence of effector T-cells by enhancing T cell apoptosis and loss of CD3-z. Method of Study:, Placental exosomes were isolated from the maternal peripheral circulation by a chromatographic/MACS procedure developed to specifically purify exosomes of placental origin. Exosomal FasL was identified by immunoelectron microscopy (IEM) and quantified by ELISA. Exosomal suppression of T cell signaling molecules and induction of apoptosis was determined by flow cytometry and DNA fragmentation, respectively. The role of FasL in these events was defined by use of FasL blocking antibody. The differential effect of exosomes on CD3-z on T subsets was analyzed by western immunoblot (WB). Results:, When T cells were co-incubated with placental exosomes, CD3-z was down-regulated, with placental exosomes treated cells expressing 18,900 7,000 MESF units of zeta, while control fraction-treated cells expressed 49,000 4,800 MESF units of zeta (P < 0.001). This down-regulation of CD3-z was partially reversed by pre-incubating T cells with ZB4 antibody, suggesting a role for FasL. IEM revealed FasL-antibody complexes located on the exterior of placental exosomes; however, only 20/27 preparations were FasL+. When placental exosomes were added to T cells, the level of CD3-z expression on CD8+ cells was 7.1%, (8.7% vs 59.7%) inhibited 1.43-fold more than in CD4+ cells (85.5 P < 0.01). On CD4+CD25+ cells, CD3-z was not significantly inhibited. (P < 0.05). Conclusions:, While all term placental exosome preparations tested induced apoptosis, exosomes obtained from 7/27 patients were negative for FasL but were able to induce DNA degradation. These results indicate that, while exosomal FasL is apoptogenic, additional exosome components are capable of inducing apoptosis, but these factors exhibit differential effect on T subsets. [source]

In Vivo Function of Immune Inhibitory Molecule B7-H4 in Alloimmune Responses

K. Yamaura
B7 ligands deliver both costimulatory and coinhibitory signals to the CD28 family of receptors on T lymphocytes, the balance between which determines the ultimate immune response. Although B7-H4, a recently discovered member of the B7 family, is known to negatively regulate T cell immunity in autoimmunity and cancer, its role in solid organ allograft rejection and tolerance has not been established. Targeting the B7-H4 molecule by a blocking antibody or use of B7-H4,/, mice as recipients of fully MHC-mismatched cardiac allografts did not affect graft survival. However, B7-H4 blockade resulted in accelerated allograft rejection in CD28-deficient recipients. B7-1/B7-2-double-deficient recipients are truly independent of CD28/CTLA-4:B7 signals and usually accept MHC-mismatched heart allografts. Blockade of B7-H4 in these mice also precipitated rejection, demonstrating regulatory function of this molecule independent of an intact CD28/CTLA-4:B7 costimulatory pathway. Accelerated allograft rejection was always accompanied by increased frequencies of alloreactive IFN-,-, IL-4- and Granzyme B-producing splenocytes. Finally, intact recipient, but not donor, B7-H4 is essential for prolongation of allograft survival by blocking CD28/CTLA4:B7 pathway using CTLA4-Ig. These data are the first to provide evidence of the regulatory effects of B7-H4 in alloimmune responses in a murine model of solid organ transplantation. [source]

Expression and role of E-cadherin and CD103,7 (,E,7 integrin) on cultured mucosal-type mast cells,

APMIS, Issue 2 2005
Mucosal-type mast cells (MMC) in the respiratory and/or gut epithelium play pivotal roles in the development of allergic inflammation and nematode clearance. To determine the role of E-cadherin and ,E,7 integrin in MMC localization to the epithelium, we analyzed the epithelial binding of two types of mouse bone marrow-derived mast cells: S3-BMMC, which developed in medium containing stem cell factor (SCF) plus IL-3, and S39T-BMMC, which developed with SCF, IL-3, IL-9 and TGF-,1. The latter cells were more similar to mature MMC than the former in terms of mouse mast cell protease (mMCP)-1 expression. FACS analyses revealed that S3-BMMC expressed E-cadherin and ,7 integrin but not ,E integrin, whereas S39T-BMMC expressed ,E,7 integrin as well as E-cadherin. Mn2+ promoted adhesion of S39T-BMMC to the monolayer of E-cadherin+F9 cells. The adhesion was suppressed significantly by the combined addition of blocking antibodies against integrin ,E and E-cadherin, whereas either blocking antibody alone failed to do so. S3-BMMC adhesion was suppressed by E-cadherin blocking antibody but not by ,E blocking antibody. These results suggested that E-cadherin and ,E,7 integrin, which are expressed on MMC-analog S39T-BMMC, play an important role in mast cell-epithelial cell interaction through homophilic as well as heterophilic binding to the epithelial E-cadherin molecule. [source]

Induction of bovine articular chondrocyte senescence with oxidized low-density lipoprotein through lectin-like oxidized low-density lipoprotein receptor 1

Satoshi Zushi
Objective Findings of recent in vivo and in vitro studies suggest that oxidized low-density lipoprotein (ox-LDL) plays a role in the degeneration of cartilage. The purpose of this study was to determine whether ox-LDL induces chondrocyte senescence through binding to lectin-like ox-LDL receptor 1 (LOX-1). Methods The effects of ox-LDL on senescence of cultured bovine articular chondrocytes (BACs) were investigated by observing senescence-associated (SA) ,-galactosidase (,-gal) activity, cell proliferation activity, and telomerase activity. Telomerase activity was measured after adding LY294002 (a specific inhibitor of phosphatidylinositol 3-kinase [PI3K]) or after adding insulin-like growth factor 1 (IGF-1; an activator of PI3K) plus ox-LDL to the culture medium to elucidate the involvement of the PI3K/Akt pathway. Immunoblot analysis was used to investigate whether ox-LDL affects the phosphorylation of Akt. To ascertain whether these effects were attributable to ox-LDL binding to LOX-1, BACs were preincubated with TS-20, an anti-bovine LOX-1 blocking antibody. Results The activity of SA ,-gal was increased and the incorporation of bromodeoxyuridine into BACs was decreased by ox-LDL in a dose-dependent manner. The telomerase activity of BACs was suppressed by the addition of ox-LDL in a time- and dose-dependent manner. LY294002 suppressed the telomerase activity of BACs, and IGF-1 reversed the ox-LDL,induced suppression of telomerase activity. In addition, ox-LDL rapidly decreased the amount of phosphorylated Akt in BACs. Pretreatment of cultured BACs with TS-20 recovered these effects. Conclusion These data show that ox-LDL binding to LOX-1 induces stress-induced premature senescence of chondrocytes and results in suppression of telomerase activity by inactivating the PI3K/Akt pathway. Oxidized LDL may play an important role in the pathogenesis of osteoarthritis by inducing chondrocyte senescence. [source]