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1.
Clin Exp Immunol ; 189(3): 372-382, 2017 09.
Article in English | MEDLINE | ID: mdl-28509333

ABSTRACT

The goal of this study was to investigate the glycosylation profile of native immunoglobulin (Ig)G present in serum immune complexes in patients with rheumatoid arthritis (RA). To accomplish this, lectin binding assays, detecting the accessibility of glycans present on IgG-containing immune complexes by biotinylated lectins, were employed. Lectins capturing fucosyl residues (AAL), fucosylated tri-mannose N-glycan core sites (LCA), terminal sialic acid residues (SNA) and O-glycosidically linked galactose/N-acetylgalactosamine (GalNac-L) were used. Patients with recent-onset RA at baseline and after 3-year follow-up were investigated. We found that native IgG was complexed significantly more often with IgM, C1q, C3c and C-reactive protein (CRP) in RA patients, suggesting alterations of the native structure of IgG. The total accessibility of fucose residues on captured immune complexes to the respective lectin was significantly higher in patients with RA. Moreover, fucose accessibility on IgG-containing immune complexes correlated positively with the levels of antibodies to cyclic citrullinated peptides (anti-CCP). We also observed a significantly higher accessibility to sialic acid residues and galactose/GalNAc glyco-epitopes in native complexed IgG of patients with RA at baseline. While sialic acid accessibility increased during treatment, the accessibility of galactose/GalNAc decreased. Hence, successful treatment of RA was associated with an increase in the SNA/GalNAc-L ratio. Interestingly, the SNA/GalNAc-L ratio in particular rises after glucocorticoid treatment. In summary, this study shows the exposure of glycans in native complexed IgG of patients with early RA, revealing particular glycosylation patterns and its changes following pharmaceutical treatment.


Subject(s)
Antigen-Antibody Complex/immunology , Arthritis, Rheumatoid/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Polysaccharides/chemistry , Polysaccharides/immunology , Adult , Aged , Antigen-Antibody Complex/chemistry , Arthritis, Rheumatoid/therapy , C-Reactive Protein/immunology , C-Reactive Protein/metabolism , Complement C1q/immunology , Complement C1q/metabolism , Complement C3c/immunology , Complement C3c/metabolism , Female , Fucose/metabolism , Galactose/metabolism , Glycosylation , Humans , Immunoglobulin A/immunology , Immunoglobulin A/metabolism , Immunoglobulin G/chemistry , Immunoglobulin G/metabolism , Immunoglobulin M/metabolism , Lectins/metabolism , Male , Middle Aged , Polysaccharides/metabolism , Sambucus nigra , Sialic Acids/metabolism
2.
Clin Exp Immunol ; 184(1): 110-7, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26618514

ABSTRACT

The Fc portion of immunoglobulin (Ig)G harbours a single glycosylation site. Glycan sialylation is critical for structure and for certain effector functions of IgG. Anti-histone IgG of patients with systemic lupus erythematosus is reportedly responsible for the recruitment of polymorphonuclear cells (PMN) to the clearance of apoptotic cells. Autoantibodies decorating secondary necrotic cells (SNEC) induce proinflammatory responses after activation of blood-borne phagocytes. Analysing the sialylation status of affinity-purified anti-histone IgG in patients with systemic lupus erythematosus (SLE), we demonstrated that the anti-histone IgG was contained preferentially in the non-sialylated fraction. In functional ex-vivo phagocytosis studies, non-sialylated anti-SNEC IgG directed SNEC preferentially into PMN but did not change their cytokine secretion profiles. In contrast, sialylated IgG reduced the phagocytosis by monocytes of SNEC. Moreover, the sialylated anti-SNEC IgG was not simply anti-inflammatory, but switched the cytokine secretion profiles from interleukin (IL)-6/IL-8 to tumour necrosis factor (TNF)-α/IL-1ß. Here we describe how different sialylation statuses of IgG autoantibodies contribute to the complex inflammatory network that regulates chronic inflammation.


Subject(s)
Autoantibodies/metabolism , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Lupus Erythematosus, Systemic/metabolism , Neutrophils/metabolism , Phagocytosis , Adult , Aged , Aged, 80 and over , Apoptosis/immunology , Autoantibodies/immunology , Case-Control Studies , Gene Expression Regulation , Glycosylation , Histones/immunology , Histones/metabolism , Humans , Interleukin-1beta/genetics , Interleukin-1beta/immunology , Interleukin-6/genetics , Interleukin-6/immunology , Interleukin-8/genetics , Interleukin-8/immunology , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/pathology , Middle Aged , Neutrophils/immunology , Neutrophils/pathology , Primary Cell Culture , Sialic Acids/metabolism , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology
3.
Lupus ; 24(6): 569-81, 2015 May.
Article in English | MEDLINE | ID: mdl-25389233

ABSTRACT

In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the highest activity of systemic lupus erythematosus. Our results show that native circulating IgG complexes from active systemic lupus erythematosus patients expose fucosyl residues and their glycan core is accessible to soluble lectins. Two putative mechanisms may contribute to the increased exposure of these glycans: (1) the canonical N-glycosylation site of the IgG-CH2 domain; (2) an IgG binding non-IgG molecule, like complement or C-reactive protein. In both cases the complexed IgG may be alternatively targeted to lectin receptors of effector cells, e.g. dendritic cells.


Subject(s)
Antigen-Antibody Complex/blood , Antigen-Antibody Complex/immunology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/immunology , Adolescent , Adult , C-Reactive Protein/metabolism , Cohort Studies , Complement C1q/immunology , Complement System Proteins/immunology , Enzyme-Linked Immunosorbent Assay/methods , Female , Glycosylation , Humans , Lectins , Lens Plant , Male , Middle Aged , Predictive Value of Tests , Young Adult
4.
Clin Exp Immunol ; 179(1): 39-49, 2015 Jan.
Article in English | MEDLINE | ID: mdl-24673456

ABSTRACT

Most cases of systemic lupus erythematosus (SLE) are characterized by an impaired clearance of apoptotic cells in various tissues. Non-cleared apoptotic waste is considered an immunogen driving the autoimmune response in patients with SLE. During the execution of apoptosis, membrane blebs are formed and filled with cellular components. Here, we evaluate the cytoskeletal pathway(s) responsible for the loading of SLE prototypic nuclear autoantigens into the apoptotic cell-derived membranous vesicles (ACMV) generated during late phases of apoptosis. HeLa cells expressing a fusion protein of histone H2B with green fluorescent protein (GFP) were irradiated with ultraviolet (UV)-B to induce apoptosis. The appearance and trafficking of chromatin-derived material was monitored by fluorescence microscopy. Specific inhibitors of cytoskeletal pathways were employed to identify the motile elements involved in translocation and trafficking of the nuclear components. We observed that immediately after their appearance the ACMV did not contain histone H2B(GFP) ; in this phase the fluorescence was contained in the nuclear remnants and the cytoplasm. Within consecutive minutes the ACMV were loaded with chromatin-derived material, whereas the loading of simultaneously created ACMV with histone H2B(GFP) was not uniform. Some ACMV were preferentially filled and, consequently, showed a remarkably higher histone H2B(GFP) accumulation. Inhibitors of the cytoskeleton revealed that functional microtubules and myosin light chain kinase are required for nuclear shrinkage and loading of nuclear material into the ACMV, respectively.


Subject(s)
Apoptosis/immunology , Autoantigens/immunology , Cell-Derived Microparticles/immunology , Cell-Derived Microparticles/metabolism , Lupus Erythematosus, Systemic/immunology , Lupus Erythematosus, Systemic/metabolism , Microtubules/metabolism , Myosin-Light-Chain Kinase/metabolism , Apoptosis/radiation effects , Cell Line , Cell Nucleus Size , Cytoskeleton/metabolism , Humans , Models, Biological
6.
J Vet Pharmacol Ther ; 36(3): 275-84, 2013 Jun.
Article in English | MEDLINE | ID: mdl-22834856

ABSTRACT

The anti-parasitic drugs ivermectin (IVM) and moxidectin (MOX) normally show limited brain penetration in vertebrates because of effective drug efflux at the blood-brain barrier by P-glycoprotein, encoded by the multi-drug resistance (MDR1) gene. However, dogs with homozygous nt230(del4) mutation in the MDR1 gene do not express a functionally active P-glycoprotein and show increased brain penetration of these drugs, resulting in neurological toxicity to different degrees. Thus, whereas IVM provokes neurological toxicity at 0.1 mg/kg, MOX is tolerated at this dosage. To investigate whether this difference is attributable to lower brain penetration of MOX in the absence of P-glycoprotein or to their neurotoxic potential, we applied IVM and MOX to P-glycoprotein-deficient CF-1 mice and comparatively analysed the absolute drug concentrations in the brain. Furthermore, we quantified drug-induced neurotoxicity by measuring the walking performance of the mice on a rotarod setup. We found that at a dosage of 0.2 mg/kg, representing 0.23 µmol/kg IVM and 0.31 µmol/kg MOX, the absolute drug concentrations in the brain were comparable with 100.8 pmol/g and 140.2 pmol/g, respectively. However, MOX induced the same degree of neurotoxicosis at the higher dosage of 1.09 µmol/kg (0.7 mg/kg) compared with IVM at 0.40 µmol/kg (0.35 mg/kg), demonstrating the 2.7-fold lower neurotoxic potential of MOX compared to IVM. This could be explained by a lower binding affinity or lower intrinsic activity of MOX at the relevant central nervous system receptors compared with IVM.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B/metabolism , Blood-Brain Barrier/metabolism , Central Nervous System Diseases/chemically induced , Ivermectin/toxicity , Macrolides/toxicity , ATP Binding Cassette Transporter, Subfamily B/genetics , Animals , Dose-Response Relationship, Drug , Genotype , Ivermectin/pharmacokinetics , Macrolides/administration & dosage , Macrolides/pharmacokinetics , Mice , Mutation , Tissue Distribution
7.
Lupus ; 21(7): 781-3, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22635232

ABSTRACT

Autoantibodies against opsonins of dying and dead cells mediate Fcγ receptor-dependent phagocytosis of autologous apoptotic and necrotic cells and hereby tend to elicit inflammation instead of silent clearance. We analysed sera of patients with chronic autoimmune diseases for the occurrence of IgG autoantibodies recognizing galectins. These pluripotent effectors can also bind to apoptotic or necrotic cells. Patients with antiphospholipid syndrome (APS; n = 104) and systemic lupus erythematosus (SLE; n = 62) were examined, healthy donors (n = 31) served as controls. Selected peptides of galectin (Gal)-2 were employed for peptide-based ELISAs. Levels of anti-Gal-2(PEP)-IgG were significantly increased in SLE and APS when compared with controls. In addition, patients with APS showed significantly higher levels of anti-Gal-2(PEP)-IgG compared with patients with SLE. Anti-Gal-2(PEP)-IgG may, therefore, be considered novel biomarkers for APS.


Subject(s)
Antiphospholipid Syndrome/immunology , Autoantibodies/blood , Galectin 2/immunology , Lupus Erythematosus, Systemic/immunology , Adult , Antiphospholipid Syndrome/blood , Biomarkers/blood , Case-Control Studies , Female , Humans , Immunoglobulin G/blood , Lupus Erythematosus, Systemic/blood , Male , Middle Aged
8.
J Immunotoxicol ; 7(3): 194-204, 2010.
Article in English | MEDLINE | ID: mdl-20205624

ABSTRACT

Most of the classical therapies for solid tumors have limitations in achieving long-lasting anti-tumor responses. Therefore, treatment of cancer requires additional and multimodal therapeutic strategies. One option is based on the vaccination of cancer patients with autologous inactivated intact tumor cells. The master requirements of cell-based therapeutic tumor vaccines are the: (a) complete inactivation of the tumor cells; (b) preservation of their immunogenicity; and (c) need to remain in accordance with statutory provisions. Physical treatments like freeze-thawing and chemotherapeutics are currently used to inactivate tumor cells for vaccination purposes, but these techniques have methodological, therapeutic, or legal restrictions. For this reason, we have proposed the use of a high hydrostatic pressure (HHP) treatment (p >or= 100 MPa) as an alternative method for the inactivation of tumor cells. HHP is a technique that has been known for more than 100 years to successfully inactivate micro-organisms and to alter biomolecules. In the studies here, we show that the treatment of MCF7, B16-F10, and CT26 tumor cells with HHP >or= 300 MPa results in mainly necrotic tumor cell death forms displaying degraded DNA. Only CT26 cells yielded a notable amount of apoptotic cells after the application of HHP. All tumor cells treated with >or= 200 MPa lost their ability to form colonies in vitro. Furthermore, the pressure-inactivated cells retained their immunogenicity, as tested in a xenogeneic as well as syngeneic mouse models. We conclude that the complete tumor cell inactivation, the degradation of the cell's nuclei, and the retention of the immunogeneic potential of these dead tumor cells induced by HHP favor the use of this technique as a powerful and low-cost technique for the inactivation of tumor cells to be used as a vaccine.


Subject(s)
Antigens, Neoplasm/immunology , Breast Neoplasms/immunology , Cancer Vaccines , Colorectal Neoplasms/immunology , Hydrostatic Pressure , Animals , Antibodies, Neoplasm/blood , Breast Neoplasms/chemistry , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Colorectal Neoplasms/chemistry , Colorectal Neoplasms/pathology , Colorectal Neoplasms/therapy , DNA Fragmentation , Female , Histocompatibility Antigens/immunology , Humans , Immunity, Humoral , Melanoma, Experimental , Mice , Neoplasm Transplantation , Neoplastic Stem Cells/chemistry , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology
9.
Autoimmun Rev ; 8(1): 9-12, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18703173

ABSTRACT

Deficiencies in the recognition and phagocytosis of dead and dying cells have been shown to be one of the main alterations in patients with systemic lupus erythematosus (SLE). Cellular as well as humoral elements play an important role in the clearance of apoptotic and necrotic cells. Non-ingested nuclear material may provide survival signals for autoreactive B-cells and consequently antibodies directed against nuclear structures will be produced. In healthy individuals, nuclear fragments are not phagocytosed in whole blood. Instead, they are mainly degraded by the action of plasma DNases and complement factors. In contrast, the uptake of nuclear fragments by blood-borne phagocytes is increased in most patients with SLE. The phagocytosis of this kind of prey, which might be opsonised by autoantibodies, may contribute to the maintenance of inflammatory responses in SLE.


Subject(s)
Antigens, Nuclear/metabolism , Apoptosis/immunology , Lupus Erythematosus, Systemic/immunology , Phagocytes/metabolism , Phagocytosis/immunology , Animals , Antibody Formation , Antibody-Dependent Cell Cytotoxicity , Antigens, Nuclear/immunology , Complement System Proteins/metabolism , DNA Fragmentation , Humans , Immunity, Cellular , Necrosis , Phagocytes/immunology , Phagocytes/pathology
10.
Lung Cancer ; 22(3): 243-50, 1998 Dec.
Article in English | MEDLINE | ID: mdl-10048477

ABSTRACT

The efficacy and toxicity profile of gemcitabine was evaluated in this phase II study of chemonaive patients with locally advanced and metastatic non-small cell lung cancer (NSCLC). Eighty patients (62 males, 18 females) were entered into this study. The disease stage was IIIA in ten patients, IIIB in 32, and IV in 38 patients. The median age was 61 (range 41 - 78). Karnofsky performance status was > or = 80 in 88% of patients. All patients were chemonaive, but five patients had received prior radiotherapy and 34 patients had undergone prior surgery. Gemcitabine 1250 mg/m2 was given as a 30-min intravenous infusion on days 1, 8, and 15 of a 28-day cycle. Patients received up to nine cycles (median three cycles). Of 872 doses 815 (93%) were administered without dose delay or modification. Of the 80 patients enrolled, 76 were evaluable for efficacy analysis, and 16 patients had a partial response for an overall response rate of 21.1% (95% CI, 11.9-30.3%). A further 47 patients (61.8%) had stable disease. Partial responses were seen in eight of 41 stage III patients (19.5%) and in eight of 35 stage IV patients (22.9%). The median time to progressive disease was 4.6 months. Median survival for all 80 patients was 7.1 months. Haematological toxicity was mild with grade 3 4 neutropenia in 6.3% of patients, grade 3 thrombocytopenia in 3.8% of patients, and grade 3 anaemia in 2.5% of patients. Grade 3 non-laboratory toxicity was: somnolence (1.3% of patients), infection (1.3%), nausea and vomiting (6.4%) and dyspnoea (5.1%). This study confirms that single-agent gemcitabine is active in advanced NSCLC and its well-tolerated safety profile makes it particularly suited to outpatient use.


Subject(s)
Adenocarcinoma/drug therapy , Antimetabolites, Antineoplastic/administration & dosage , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Squamous Cell/drug therapy , Deoxycytidine/analogs & derivatives , Lung Neoplasms/drug therapy , Adenocarcinoma/secondary , Adult , Aged , Antimetabolites, Antineoplastic/adverse effects , Carcinoma, Large Cell/drug therapy , Carcinoma, Large Cell/secondary , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Squamous Cell/secondary , Deoxycytidine/administration & dosage , Deoxycytidine/adverse effects , Drug Administration Schedule , Female , Humans , Karnofsky Performance Status , Lung Neoplasms/pathology , Male , Middle Aged , Treatment Outcome , Gemcitabine
11.
J Am Coll Cardiol ; 7(3): 509-17, 1986 Mar.
Article in English | MEDLINE | ID: mdl-3950230

ABSTRACT

The severity of aortic stenosis was evaluated by Doppler echocardiography in 48 adults (mean age 67 years) undergoing cardiac catheterization. Maximal Doppler systolic gradient correlated with peak to peak pressure gradient (r = 0.79, y = 0.63x + 25.2 mm Hg) and mean Doppler gradient correlated with mean pressure gradient (r = 0.77, y = 0.59x + 10.0 mm Hg) by manometry. The transvalvular pressure gradient is flow dependent, however, and associated left ventricular dysfunction was common in our patients (33%). Thus, of the 32 patients with an aortic valve area less than or equal to 1.0 cm2 at catheterization, 6 (19%) had a peak Doppler gradient less than 50 mm Hg. To take into account the influence of volume flow, aortic valve area was calculated as stroke volume, measured simultaneously by thermodilution, divided by the Doppler systolic velocity integral in the aortic jet. Aortic valve areas calculated by this method were compared with results at catheterization in the total group (r = 0.71). Significant aortic insufficiency was present in 71% of the population. In the subgroup without significant coexisting aortic insufficiency, closer agreement of valve area with catheterization was noted (n = 14, r = 0.91, y = 0.83x + 0.24 cm2). Transaortic stroke volume can be determined noninvasively by Doppler echocardiographic measures in the left ventricular outflow tract, just proximal to the stenotic valve. Aortic valve area can then be calculated as left ventricular outflow tract cross-sectional area times the systolic velocity integral of outflow tract flow, divided by the systolic velocity integral in the aortic jet.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Aortic Valve Stenosis/diagnosis , Aortic Valve/physiopathology , Echocardiography , Adult , Aged , Aortic Valve Stenosis/physiopathology , Blood Flow Velocity , Blood Pressure , Cardiac Catheterization , Cardiac Output , Echocardiography/methods , Female , Humans , Male , Middle Aged
13.
Circulation ; 63(6): 1417-21, 1981 Jun.
Article in English | MEDLINE | ID: mdl-7226488

ABSTRACT

Five patients who underwent cardiopulmonary resuscitation (CPR) were studied by two-dimensional echocardiography to assess valve motion. The mitral valve remained open throughout the entire compression-release cycle of CPR. The aortic valve opened during the compression phase of CPR and closed during the release phase. The pulmonic valve moved toward the closed position during the compression phase and the valve leaflets opened during release. Tricuspid valve leaflets never completely apposed, even during maximum chest compression, and they were widely open during release. Left ventricular dimensions did not change appreciably during CPR. These findings support the theory that forward blood flow during CPR depends on a generalized increase in intrathoracic pressure and not on direct compression of the heart itself. The left heart appears to act as a conduit for passage of blood, and mitral valve closure is not necessary for forward blood flow during CPR.


Subject(s)
Echocardiography , Heart Valves/physiopathology , Motion , Resuscitation , Adult , Aortic Valve/physiopathology , Blood Flow Velocity , Female , Heart Ventricles/physiopathology , Hemodynamics , Humans , Male , Middle Aged , Mitral Valve/physiopathology , Pulmonary Valve/physiopathology , Tricuspid Valve/physiopathology
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