Accumulation Of IgG Research Articles

Novel mitoNEET ligand NL-1 improves therapeutic outcomes in an aged rat model of cerebral ischemia/reperfusion injury

Cerebral ischemic stroke is a leading cause of mortality and disability worldwide. Currently, there are a lack of drugs capable of reducing neuronal cell loss due to ischemia/reperfusion-injury after stroke. Previously, we identified mitoNEET, a [2Fe-2S] redox mitochondrial protein, as a putative drug target for ischemic stroke. In this study, we tested NL-1, a novel mitoNEET ligand, in a preclinical model of ischemic stroke with reperfusion using aged female rats. Using a transient middle cerebral artery occlusion (tMCAO), we induced a 2 h ischemic injury and then evaluated the effects of NL-1 treatment on ischemic/reperfusion brain injury at 24 and 72 h. Test compounds were administered at time of reperfusion via intravenous dosing. Results of the study demonstrated that NL-1 (10 mg/kg) treatment markedly improved survival and reduced infarct volume and hemispheric swelling in the brain as compared aged rats treated with vehicle or a lower dose of NL-1 (0.25 mg/kg). Interestingly, the protective effect of NL-1 was significantly improved when encapsulated in PLGA nanoparticles, where a 40-fold lesser dose (0.25 mg/kg) of NL-1 produced an equivalent effect as the 10 mg/kg dose. Evaluation of changes in blood-brain barrier permeability and lipid peroxidation corroborated the protective actions of NL-1 (10 mg/kg) or NL-1 NP treatment demonstrated a reduced accumulation of parenchymal IgG, decreased levels of 4-hydroxynonenal (4-HNE) and a decreased TUNEL positive cells in the brains of aged female rats at 72 h after tMCAO with reperfusion. Our studies indicate that targeting mitoNEET following ischemia/reperfusion-injury is a novel drug target pathway that warrants further investigation.

Inter-Leaflet Phospholipid Exchange Impacts the Ligand Density Available for Protein Binding at Supported Lipid Bilayers.

Phospholipid bilayers formed at solid-liquid interfaces have garnered interest as mimics of cell membranes to model association reactions of proteins with lipid bilayer-tethered ligands. Despite the importance of understanding how ligand density in a lipid bilayer impacts the protein-ligand association response, relating the ligand-modified lipid fraction to the absolute density of solution-accessible ligands in a lipid bilayer remains a challenge in interfacial quantitative analysis. In this work, confocal Raman microscopy is employed to quantify the association of anti-biotin IgG with a small fraction of biotinylated lipids dispersed in either gel-phase or liquid-crystalline supported lipid bilayers deposited on the interior surfaces of wide-pore silica surfaces. We examine the question of whether inter-leaflet lipid translocation contributes to the population of solution-accessible biotin ligands on the distal leaflet of a supported lipid bilayer by comparing their protein accumulation response with ligands dispersed in lipid monolayers on nitrile-derivatized silica surfaces. The binding of the antibody to biotin ligands dispersed in gel-phase bilayers exhibited an equivalent biotin coverage response as the accumulation of IgG onto gel-phase monolayers, indicating that gel-phase bilayer symmetry was preserved. This result contrasts with the ∼60% greater anti-biotin capture observed at fluid-phase bilayers compared to fluid-phase monolayers prepared at equivalent biotin fractions. This enhanced protein capture is attributed to biotin-capped lipids being transferred from the surface-associated proximal leaflet of the bilayer to the solution-exposed distal leaflet by the inter-leaflet exchange or lipid flip-flop, a facile process in fluid-phase supported lipid bilayers. The results suggest caution in interpreting the results of quantitative studies of protein binding to lipid-tethered ligands dispersed in fluid-phase phospholipid bilayers.

Intravenous immunoglobulin-induced eczematous eruption in autoimmune neuromuscular diseases

Intravenous immunoglobulin (IVIg) have been administrated for the long time in patients with several autoimmune neuromuscular diseases. Eczematous eruption has been described as IVIg-induced adverse effect. The purpose of this study is to clarify the incidence and characteristic of IVIg-induced eczematous eruption in autoimmune neuromuscular disease. We retrospectively collected the data from 92 patients with autoimmune neuromuscular diseases, including 35 patients with chronic inflammatory demyelinating polyneuropathy (CIDP), 8 patients with multifocal motor neuropathy (MMN), 25 patients with myositis, 15 patients with Guillain-Barré syndrome (GBS), and 9 patients with myasthenia gravis (MG), who have administrated IVIg in Yamaguchi University Hospital. There are 10 patients (6 CIDP/4 MMN), who had an eczematous skin reaction after IVIg infusion. The frequencies of IVIg-induced eczematous eruption were significantly higher in patients with multifocal acquired demyelinating sensory and motor (MADSAM) and MMN than in patients with GBS, myositis, and MG. In addition, corticosteroids or immunosuppressive drugs had been administrated before IVIg treatment more frequently in patients with myositis and MG than in those with MADSAM and MMN. MADSAM or MMN patients had more frequently IVIg-induced eczematous eruption than other autoimmune neuromuscular diseases. Pathophysiology of MADAM and MMN is considered to be cell-mediated immunity against the peripheral nerve and the accumulation of IgG in both epidermis and dermis of the hand after IVIg may induce the infiltration of inflammatory cells around the vessels in the skin, causing eczematous eruption in MADSAM and MMN.

Blockade of the Neonatal Fc Receptor (FcRn) Represents an Effective Mechanism for the Removal of Pathogenic Autoantibodies in Primary Immune Thrombocytopenia

Introduction: The neonatal Fc receptor, FcRn is ubiquitously expressed, & is responsible for maintaining the half-life of IgG & albumin, by rescuing these proteins from intracellular lysosomal degradation. Blockade of the interaction of FcRn with IgG would be expected to prevent salvage of both non-pathogenic and pathogenic IgG. Patients with primary immune thrombocytopenia (ITP) have autoantibodies against platelet membrane proteins such as the cell adhesion receptor CD61/CD41 (GPIIb/IIIa). Rozanolixizumab (a high affinity IgG4 monoclonal antibody (mAb) that specifically inhibits IgG binding to FcRn) is currently being evaluated in phase 2 clinical studies in patients with ITP. The aim of the current study was to understand the in vitro properties of rozanolixizumab & to explore the in vivo effects of a surrogate anti-mouse FcRn mAb (murinized “4464”) in a mouse model of ITP.Methods: The affinity of rozanolixizumab for FcRn was tested using surface plasmon resonance technology (Biacore), with the antibody captured by an anti-Fc immobilized on the sensor chip & soluble FcRn in solution, whilst the affinity of fluorescently labelled rozanolixizumab for human FcRn expressed on the surface of Madin-Darby canine kidney (MDCK) cells was measured by flow cytometry. Recycling & transcytosis of human IgG (hIgG) was also assessed in FcRn-transfected MDCK cells. For IgG tracking experiments, endothelial & epithelial cell lines were incubated with an AF647-conjugated IgG for 24 hours in the presence or absence of rozanolixizumab. Prior to imaging, nuclei were labelled using Hoechst at 37°C & cells were then visualized by fluorescence microscopy. Phagocytosis of anti-human MHC I (W6/32)-coated platelets by THP-1 cells was assessed by flow cytometry at 1 hour. The effect of rozanolixizumab on IgG recycling in vivo was assessed in the human FcRn-transgenic (hFcRn-Tg) mouse: hIgG at 500mg/kg was administered IV to mice on day 1 & dosed with antibodies on day 2; serial bleeds were then taken at multiple time points post antibody dosing & levels of hIgG, albumin & rozanolixizumab were quantified using LC-MS/MS. For the induction of ITP in mice, animals received an anti-CD41 antibody (MWReg30), delivered by osmotic minipump (~1µg of antibody delivered every 24 hours) & the anti-mouse FcRn mAb (4464) was administered at 30mg/kg either on days 0, 2 & 4 (prophylactic dosing) or on days 3 & 5 (therapeutic dosing); IVIg (1g/kg) was also evaluated & control mice received either PBS or an isotype-matched control mAb (101.4). Daily blood samples were taken to assess platelet numbers.Results: Rozanolixizumab has a high affinity for FcRn in a protein-protein (Biacore) assay with a KD of ~25pM at both pH6.0 & pH7.4; in the cell-based assay, the affinity was 1nM at both pH's. Rozanolixizumab efficiently inhibited the recycling & transcytosis of IgG in human FcRn-transfected MDCK cells (IC50 0.4nM & 1.1nM, respectively). Confocal microscopy experiments showed that there was an increased accumulation of IgG in cells in the presence of rozanolixizumab, relative to an isotype control mAb, & that the IgG was co-localized in lysosomes where it appeared to be degraded. FcRn has been postulated to play a direct role in phagocytosis, but the phagocytosis of opsonized platelets by THP-1 cells was unaffected by blockade with rozanolixizumab. Treatment with rozanolixizumab resulted in a dose-dependent accelerated clearance of hIgG in the hFcRn-Tg mouse; hIgG levels in all treated groups were significantly lower vs PBS controls from 24 hours post-dosing until the end of the experiment 8 days post dosing (p<0.01). No effect on albumin levels occurred in these mice. In the mouse model of ITP, both prophylactic & therapeutic treatment regimens with 4464 resulted in an increased number of platelets compared to control animals that received either PBS or 101.4. Animals that received 4464 from day 0 had significantly greater platelet numbers than control animals on days 4 & 5 (p<0.05); therapeutic dosing gave a significantly greater platelet number than control animals on day 5 (p<0.05).Conclusion: Rozanolixizumab is a high affinity mAb that inhibits the IgG recycling function of FcRn in vitro &in vivo . An anti-mouse FcRn mAb demonstrated efficacy in a mouse model of ITP. These data support the evaluation of rozanolixizumab in patients with ITP, & a phase 2 clinical study is currently ongoing in this patient population. DisclosuresEddleston:UCB Pharma: Employment. Greenslade:UCB Pharma: Employment. Qureshi:UCB Pharma: Employment. West:UCB Pharma: Employment. Atherfold:UCB Pharma: Employment. Amirkhosravi:UCB Pharma: Research Funding. Desai:UCB Pharma: Research Funding. Rivera-Amaya:UCB Pharma: Research Funding. Smith:UCB Pharma: Consultancy, Equity Ownership. Shock:UCB Pharma: Employment.

Thermovaccination: Thermoheliox as an Immune Response Stimulant. Kinetics of Antibodies and C-Reactive Protein Synthesis in Coronaviral Infection.

The high efficiency of using thermoheliox (inhalation with a high-temperature mixture of helium and oxygen) in the treatment of patients affected by COVID-19 was shown. The dynamics of accumulation of IgG, IgM, and C-reactive protein (CRP) in patients with coronavirus infection in the “working” and control groups was studied experimentally. It was shown that thermoheliox intensifies the synthesis of IgG, IgM, and CRP antibodies, while eliminating the induction period on the kinetic curves of the synthesis of specific antibodies in the IgG form and transfers the synthesis of CRP to a fast phase. The results of experiments confirm the previously obtained data based on the analysis of the kinetic model of the development of coronaviral infection in the human body.

Blood-brain barrier permeability towards small and large tracers in a mouse model of osmotic demyelination syndrome

During osmotic demyelination syndrome (ODS), myelin and oligodendrocyte are lost according to specific patterns in centro- or extra-pontine regions. In both experimental model of ODS and human cases, brain lesions are locally correlated with the disruption of the blood brain-barrier (BBB). The initiation, the degree and the duration of blood-brain barrier (BBB) opening as well as its contribution to brain damages are still a matter of debate. Using a panel of intravascular tracers from low- to high- molecular weight (from 0.45 kDa 150 kDa), we have assessed the BBB permeability at different timings of ODS induced experimentally in mice. ODS was mimicked according to a protocol of rapid correction of a chronic hyponatremia. We demonstrated that BBB leakage towards smallest tracers Lucifer Yellow (0.45 kDa) and Texas Red-dextran (3 kDa) was delayed by 36 h compared to the first clues of oligodendrocyte loss (occurring 12 h post-correction of hyponatremia). At 48 h post-correction and concomitantly to myelin loss, BBB was massively disrupted as attested by accumulation of Evans Blue (69 kDa) and IgG (150 kDa) in brain parenchyma. Analysis of BBB ultrastructure verified that brain endothelial cells had minimal alterations during chronic hyponatremia and at 12 h post-correction of hyponatremia. However, brain endothelium yielded worsened alterations at 48 h, such as enlarged vesicular to tubular-like cytoplasmic profiles of pinocytosis and/or transcytosis, local basal laminae abnormalities and sub-endothelial cavities. The protein expressions of occludin and claudin-1, involved in inter-endothelial tight junctions, were also downregulated at 48 h post-correction of hyponatremia. Our results revealed that functional BBB opening occured late in pre-established ODS lesions, and therefore was not a primary event initiating oligodendrocyte damages in the mouse model of ODS.

Neural epidermal growth factor-like 1 protein (NELL-1) associated membranous nephropathy

Membranous nephropathy is characterized by deposition of immune complexes along the glomerular basement membrane. PLA2R and THSD7A are target antigens in 70% and 1-5% of primary membranous nephropathy cases, respectively. In the remaining cases, the target antigen is unknown. Here, laser microdissection of glomeruli followed by mass spectrometry was used to identify novel antigen(s) in PLA2R-negative membranous nephropathy. An initial pilot mass spectrometry study in 35 cases of PLA2R-negative membranous nephropathy showed high spectral counts for neural tissue encoding protein with EGF-like repeats, NELL-1, in six cases. Mass spectrometry failed to detect NELL-1 in 23 PLA2R-associated membranous nephropathy and 88 controls. NELL-1 was localized by immunohistochemistry, which showed bright granular glomerular basement membrane staining for NELL-1 in all six cases. Next, an additional 23 NELL-1 positive cases of membranous nephropathy were identified by immunohistochemistry in a discovery cohort of 91 PLA2R-negative membranous nephropathy cases, 14 were confirmed by mass spectrometry. Thus, 29 of 126 PLA2R-negative cases were positive for NELL-1. PLA2R-associated membranous nephropathy and controls stained negative for NELL-1. We then identified five NELL-1 positive cases of membranous nephropathy out of 84 PLA2R and THSD7A-negative cases in two validation cohorts from France and Belgium. By confocal microscopy, both IgG and NELL-1 co-localized to the glomerular basement membrane. Western blot analysis showed reactivity to NELL-1 in five available sera, but no reactivity in control sera. Clinical and biopsy findings of NELL-1 positive membranous nephropathy showed features of primary membranous nephropathy. Thus, a subset of membranous nephropathy is associated with accumulation and co-localization of NELL-1 and IgG along the glomerular basement membrane, and with anti-NELL-1 antibodies in the serum. Hence, NELL-1 defines a distinct type of primary membranous nephropathy.

Bevacizumab-associated glomerular microangiopathy

Bevacizumab is a humanized monoclonal IgG1 antibody, which neutralizes vascular endothelial growth factor and is used for treating multiple cancer types. As a known and frequent adverse event, this therapy can lead to renal damage including proteinuria and nephrotic syndrome. In a retrospective approach, we analyzed 17 renal biopsies from patients receiving bevacizumab treatment. We observed a distinctive histopathological pseudothrombotic pattern different from the previously reported thrombotic microangiopathy. Since this pattern includes some features similar to acute and chronic thrombotic microangiopathy, focal segmental glomerulosclerosis and cryoglobulinemic membranoproliferative glomerulonephritis, biopsies with these diagnoses were included for comparison. Clinical, laboratory, light microscopic, immunohistochemical (including a proximity ligation assay), proteomic and electron microscopic features were assessed. Nephrotic syndrome was present in 15 of the 17 bevacizumab-treated patients. All 17 displayed a patchy pattern of variably PAS-positive hyaline pseudothrombi occluding markedly dilated glomerular capillaries in their biopsies. Mass spectrometry-based proteome analysis revealed a special protein pattern demonstrating some features of thrombotic microangiopathy and some of cryoglobulinemic glomerulonephritis, including a strong accumulation of IgG in the pseudothrombi. Proximity ligation assay did not show interaction of IgG with C1q, arguing for accumulation without classic pathway complement activation. In contrast to thrombi in thrombotic microangiopathy cases, the hyaline pseudothrombi did not contain clusters of CD61-positive platelets. Electron microscopy of bevacizumab cases did not show fibrin polymers or extensive loss of podocyte foot processes. Even though cases of bevacizumab-associated microangiopathy share some features with thrombotic microangiopathy, its overall histopathological pattern is quite different from acute or chronic thrombotic microangiopathy cases. We conclude that bevacizumab therapy can lead to a unique hyaline occlusive glomerular microangiopathy, likely arising from endothelial leakage followed by subendothelial accumulation of serum proteins. It can be diagnosed by light microscopy and is an important differential diagnosis in cancer patients with nephrotic syndrome.

Differential trafficking of albumin and IgG facilitated by the neonatal Fc receptor in podocytes in vitro and in vivo.

Proteinuria is strongly associated with kidney disease progression but the mechanisms underlying podocyte handling of serum proteins such as albumin and IgG remain to be elucidated. We have previously shown that albumin and IgG are transcytosed by podocytes in vitro. In other epithelial cells, the neonatal Fc receptor (FcRn) is required to salvage albumin and IgG from the degradative pathway thereby allowing these proteins to be transcytosed or recycled. Here we directly examine the role of FcRn in albumin and IgG trafficking in podocytes by studying handling of these proteins in FcRn knockout (KO) podocytes in vitro and in a podocyte-specific FcRn knockout mice in vivo. In vitro, we find that knockout of FcRn leads to IgG accumulation in podocytes but does not alter albumin trafficking. Similarly, in vivo, podocyte-specific knockout of FcRn does not result in albumin accumulation in podocytes in vivo as measured by mean albumin fluorescence intensity whereas these mice demonstrate significant intraglomerular accumulation of IgG over time. In addition we find that podocyte-specific FcRn KO mice demonstrate mesangial expansion as they age and activation of mesangial cells as demonstrated by increased expression of α-smooth muscle actin. Taken together, these results suggest that trafficking pathways for albumin and IgG differ in podocytes and that sustained disruption of trafficking of plasma proteins alters glomerular structure.

Omics-Based Approach Reveals Complement-Mediated Inflammation in Chronic Lymphocytic Inflammation With Pontine Perivascular Enhancement Responsive to Steroids (CLIPPERS).

Chronic lymphocytic inflammation with pontine perivascular enhancement responsive to steroids (CLIPPERS) is a rare syndrome with relapsing brainstem/cerebellar symptoms. To examine the pathogenic processes and investigate potential biomarkers, we analyzed combined materials of brain and cerebrospinal fluid (CSF) by comprehensive methodologies. To identify major pathways of perivascular inflammation in CLIPPERS, we first compared the CSF proteome (n = 5) to a neurodegenerative condition, Alzheimer's disease (AD, n = 5). Activation of complement was confirmed by immunohistochemistry (IHC) on CLIPPERS brain samples (n = 3) and by ELISA in the CSF. For potential biomarkers, we used biomarker arrays, and compared inflammatory and vessel-associated proteins in the CSF of CLIPPERS (n = 5) with another inflammatory relapsing CNS disease, multiple sclerosis (RMS, n = 9) and healthy subjects (HS, n = 7). Two hundred and seven proteins in the CSF discriminated CLIPPERS from AD. The complement cascade, immunoglobulins, and matrix proteins were among the most frequently represented pathways. Pathway analysis of upstream regulators suggested the importance of vascular cell adhesion protein 1 (VCAM1), IFN-γ, interleukin (IL)-1, and IL-10. Differential regulation of more than 10 complement proteins of the 3 complement pathways in the CSF pointed to the role of complement activation. IHC on brain samples confirmed the perivascular complement activation, i.e., deposition of C3bc, C3d, and the terminal C5b-9 complement complex that partially overlapped with accumulation of IgG in the vessel wall. Besides endothelial cell damage, reactivity to smooth muscle actin was lost in the walls of inflamed vessels, but the glia limitans was preserved. The semi-quantitative array indicated that increased level of IL-8/CXCL8 (p < 0.05), eotaxin/CCL11 (p < 0.01), and granulocyte colony-stimulating factor (p < 0.05) in CSF could distinguish CLIPPERS from HS. The quantitative array confirmed elevated concentration of IL-8/CXCL8 and eotaxin/CCL11 compared to HS (p < 0.05, respectively) besides increased levels of ICAM-1 (p < 0.05) and VCAM-1 (p < 0.001). The increased concentration of VCAM-1 were able to differentiate CLIPPERS from RMS (p < 0.01), and a trend of elevated levels of ICAM-1 and IL-8/CXCL8 compared to RMS was also observed (p = 0.06, respectively). Complement activation, IgG deposition, and alterations of the extracellular matrix may contribute to inflammation in CLIPPERS. VCAM1, ICAM1, and IL-8 in the CSF may differentiate CLIPPERS from RMS.

Delayed onset of autoreactive antibody production and M2-skewed macrophages contribute to improved survival of TACI deficient MRL-Fas/Lpr mouse

Anti-B cell activating factor belonging to TNF-family (BAFF) antibody therapy is indicated for the treatment of patients with active systemic lupus erythematosus (SLE). We hypothesized that the BAFF receptor, transmembrane activator and calcium-modulator and cyclophilin interactor (TACI) may be responsible for the generation of antibody secreting plasma cells in SLE. To test this hypothesis, we generated TACI deficient MRL-Fas/Lpr (LPR-TACI−/−) mouse. TACI deficiency resulted in improved survival of MRL-Fas/Lpr mice and delayed production of anti-dsDNA and anti-SAM/RNP antibodies. There was also a delay in the onset of proteinuria and the accumulation of IgG and inflammatory macrophages (Mϕs) in the glomeruli of young LPR-TACI−/− mice compared to wild-type mice. Underscoring the role of TACI in influencing Mϕ phenotype, the transfer of Mϕs from 12-week-old LPR-TACI−/− mice to age-matched sick wild-type animals led to a decrease in proteinuria and improvement in kidney pathology. The fact that, in LPR-TACI−/− mouse a more pronounced delay was in IgM and IgG3 autoreactive antibody isotypes and the kinetics of follicular helper T (Tfh) cell-development was comparable between the littermates suggest a role for TACI in T cell-independent autoantibody production in MRL-Fas/Lpr mouse prior to the onset of T cell-dependent antibody production.

Abstract B08: Biomimetic Culture Platform for Ex Vivo Preservation of Patient-Derived Multiple Myeloma Cells

Abstract Multiple myeloma (MM) is an incurable B-cell malignancy with a typical survival of 5 to 7 years, and is characterized by: (1) the accumulation of IgG (κ or λ) producing monoclonal plasma cells in bone marrow (BM) and (2) the progression of metastatic osteolytic lesions in bones. We have developed a new approach for the extended culture (&amp;gt; 3 weeks) of difficult-to-preserve primary patient multiple myeloma cells (PMMCs) present in bone marrow aspirates, using an osteoblast (OSB)-derived 3D tissue scaffold constructed in a perfused microfluidic environment [1]. In contrast, the ex vivo viability of PMMCs cannot be preserved using conventional culture techniques beyond a few days. This biomimetic model was used to show, for the first time, that: (1) the adhesion of PMMCs to OSB, mediated by osteoblastic N-cadherin, and the long-term viability of OSBs were critical factors in maintaining the ex vivo viability and proliferative capacity of PMMCs; (2) perfusion flow enhances the survival of OSBs, and thus increases the long-term viability of PMMCs; and noteworthy, (3) PMMCs can acquire drug-resistance due to the adhesive interactions with OSBs, a phenomenon called cell adhesion-mediated drug-resistance (CAM-DR) [2]. These findings support that the in vitro model is capable of recapitulating critical MM tumor microenvironment associated with the survival and drug-resistance of PMMCs. Upon further development, our ex vivo model may provide a new means of selecting the best therapy for an individual patient based on the ex vivo response of his or her PMMCs to drug treatment options as well as novel immunotherapeutic treatments such as chimeric antigen receptor (CAR) T-cell therapy. This precision medicine approach is expected to provide a transformative means of: (1) reducing ineffective or unnecessary therapies; (2) minimizing excess toxicity and costs; and (3) inhibiting the development of tumor cells' cross-resistance to additional drugs, as a critical part of the long-term care of MM patients. Also, we anticipate that our approach can be used for: (1) preclinical evaluation of new therapeutics, (2) studying the development of drug-resistant PMMC subpopulations, (3) identifying novel therapeutic targets, and (4) developing ex vivo models of breast and prostate cancers that metastasize to BM via similar mechanisms. Currently, we are in the early stage of prospectively correlating the ex vivo chemosensitivity and resistance of PMMCs with short-term clinical response, as a means of validating the predictive capability of our ex vivo MM culture platform. We also plan to incorporate the 3D-reconstructed network of primary human osteocytes [3] to study interactions of osteocytes with PMMCs and other BMMCs including immune cells.

Control of lupus by novel tryptophan metabolite 5-methoxytryptophan. (THER5P.907)

Abstract Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by the presence of pathogenic autoantibodies which potentially drive immune-complex related inflammation in various tissues and organs. We previously identified that 5-methoxytryptophan (5-MTP), a soluble factor released by fibroblasts, provides endogenous control of proinflammatory cytokine-induced cyclooxgenase-2 expression. However, its effect on controlling systemic inflammation and systemic inflammatory disorders such as SLE remains unclear. Clinical studies by measuring serum 5-MTP concentrations found that a considerable amount of 5-MTP was detected in healthy subjects and was significantly reduced in SLE patients. Mouse models of experimental lupus-like disease induced by pristine shown that exogenous supplementation of 5-MTP not only prevented serum anti-dsDNA IgG production but also rescued the loss of body weight. Notably, 5-MTP administrations rescued kidney by reducing the accumulation of IgG. The beneficial effect of 5-MTP was correlated with inhibition of IL-6 production in bone-marrow derived dendritic cells treated with TLR7 ligand, which is a stimulator of lupus progression. Additionally, 5-MTP also suppressed Th1 cell while increased regulatory T cells which play an essential role in preventing autoimmunity. These results that 5-MTP is a novel circulating anti-inflammatory molecule which protects against excessive systemic inflammatory responses in animal models.

IgG accumulates in inhibitory hippocampal neurons of experimental antiphospholipid syndrome

Mice immunized with β2-glycoprotein I (β2GPI) are an experimental model of the antiphospholipid syndrome (eAPS) displaying elevated titers of antiphospholipid antibodies (aPL). We presently studied whether the behavioral hyperactivity in eAPS mice is associated with in vivo binding and accumulation of IgG in the brain. At 6 weeks post immunization eAPS mice had significantly higher levels of aPL (1.32 ± 0.28 and 0.02 ± 0.01 AU, p < 0.001 by t-test) compared to adjuvant immunized controls, as measured by ELISA. Significant hyperactivity in a staircase test in the eAPS mice compared to controls was found in stair-climbing (18.4 ± 0.9 and 12.0 ± 1.7, respectively) and rearing measures (23.5 ± 2.1 and 12.5 ± 1.9, p < 0.01 by t-test). Immunofluorescence staining in eAPS mice revealed significant in vivo accumulation of IgG in cortical and hippocampal neurons which was not seen in controls. Staining for IgG was markedly intense in inhibitory interneurons co-stained for GAD67 in the hippocampus of eAPS mice. The integrity of the blood brain barrier (BBB) evaluated by injection of Evans blue (EB) was impaired in eAPS and adjuvant immunized mice compared to naïve mice. Electrophysiological recordings in hippocampal brain slices showed altered response to paired pulse stimulation as well as dysregulation of carbachol-induced γ- oscillations in eAPS mice compared to control. Penetration into the brain and direct interaction of aPL with inhibitory interneurons in the hippocampus may explain the hyperactive behavior of the eAPS mice. A direct role of aPL in causing CNS dysfunction points to these antibodies as an important therapeutic target in APS.

Serum TRPM1 Autoantibodies from Melanoma Associated Retinopathy Patients Enter Retinal ON-Bipolar Cells and Attenuate the Electroretinogram in Mice

Melanoma-associated retinopathy (MAR) is a paraneoplastic syndrome associated with cutaneous malignant melanoma and the presence of autoantibodies that label neurons in the inner retina. The visual symptoms and electroretinogram (ERG) phenotype characteristic of MAR resemble the congenital visual disease caused by mutations in TRPM1, a cation channel expressed by both melanocytes and retinal bipolar cells. Four serum samples from MAR patients were identified as TRPM1 immunoreactive by 1. Labeling of ON-bipolar cells in TRPM1+/+ but not TRPM1−/− mouse retina, 2. Labeling of TRPM1-transfected CHO cells; and 3. Attenuation of the ERG b-wave following intravitreal injection of TRPM1-positive MAR IgG into wild-type mouse eyes, and the appearance of the IgG in the retinal bipolar cells at the conclusion of the experiment. Furthermore, the epitope targeted by the MAR autoantibodies was localized within the amino-terminal cytoplasmic domain of TRPM1. Incubation of live retinal neurons with TRPM1-positive MAR serum resulted in the selective accumulation of IgG in ON-bipolar cells from TRPM1+/+ mice, but not TRPM1−/− mice, suggesting that the visual deficits in MAR are caused by the uptake of TRPM1 autoantibodies into ON-bipolar cells, where they bind to an intracellular epitope of the channel and reduce the ON-bipolar cell response to light.

Accumulation of Fetal IgG in Immunoglobulin Injection into the Fetal Abdominal Cavity Is Proven

Objective: There have been a number of studies on immunoglobulin injection into fetuses or mothers during pregnancy for the treatment of congenital cytomegalovirus infection. However, no study has examined the effect of injected immunoglobulin on fetal hemodynamics. In this study, we examined the effect of immunoglobulin injection on fetal hemodynamics by retrospectively measuring the concentrations of several IgG subclasses in stored umbilical cord blood sera collected during fetal therapy. Methods: Five patients who underwent immunoglobulin injection into the fetal abdominal cavity (IFAC) as a fetal therapy during pregnancy were included in this study. Frozen-stored umbilical venous blood samples collected from these patients during IFAC were measured for serum concentrations of each IgG subclass. Results: The largest change was observed in the IgG2 concentration, with a mean increase of 221% following IFAC. The IgG4 concentration also showed a mean increase of 63%. In contrast, the concentration of IgG1, which has the strongest physiological activity of all IgG subclasses examined, only exhibited an overall mean increase of 1.4%. Conclusion: Our results confirmed that immunoglobulins are incorporated into the fetal circulation following IFAC.

Association of Fibronectin With Hypogalactosylated Immunoglobulin G in Gingival Crevicular Fluid in Periodontitis

Fibronectin (FN) can bind to immunoglobulins (Ig), influencing both the normal clearance and abnormal deposition of circulating immune complexes. This study focuses on the possible interaction between FN and IgG present in gingival crevicular fluid (GCF) of periodontitis patients and periodontally healthy controls, with the aim to determine whether such interaction may be connected with the glycosylation profile of IgG and, thus, consequentional in accumulation of proinflammatory IgG in periodontal pockets. The study included 30 patients with initial or advanced periodontitis, and 13 periodontally healthy subjects. GCF IgG was purified and analyzed for the presence of FN and its fragments and for galactose expression. IgG isolated from GCF contained FN, which was bound to the IgG heavy chains. IgG from GCF of advanced periodontitis patients contained high levels of hypogalactosylated IgG, and bound more FN than IgG from GCF of the other two groups, which contained low levels of this glycoform. FN was in a degraded form in GCF from all studied patients, and a fragment of 48- to 53-kDa molecular mass seemed to be the sole one linked to IgG. IgG and the FN fragment of 48 to 53 kDa in GCF of periodontitis patients and periodontally healthy subjects are physically connected. This fragment was linked to the heavy chains of IgG and the reaction seemed to be particularly intensive with IgG from advanced periodontitis, which contained significantly less galactose in its sugar chains.

Tumor Necrosis Factor-α Does Not Mediate Diabetes-Induced Vascular Inflammation in Mice

Vascular inflammation is a key feature of both micro- and macrovascular complications in diabetes. Several lines of evidence have implicated the cytokine tumor necrosis factor (TNF) alpha as an important mediator of inflammation in diabetes. In the present study we evaluated the role of TNF alpha in streptozotocin (STZ)-induced diabetes on vascular inflammation in C57BL/6 wild-type and apoE-/- mice. Diabetes increased the expression of vascular cell adhesion molecule (VCAM)-1 in cerebral arteries 150 m in diameter as well as the macrophage accumulation in aortic root atherosclerotic plaques in apoE-/- mice. A more pronounced vascular inflammatory response was observed in diabetic TNF alpha-deficient apoE-/- mice. These mice were also characterized by increased accumulation of IgG and IgM autoantibodies in atherosclerotic lesions. Diabetes also increased VCAM-1 expression and plaque formation in apoE-competent TNF alpha -/- mice, whereas no such effects were observed in C57BL/6 wild-type mice. The present findings suggest that TNF alpha does not mediate diabetic-induced vascular inflammation in mice and reveal an unexpected protective role for TNF alpha. These effects are partly attributable to a direct antiinflammatory role of TNF alpha, but may also reflect a defective development of the immune system in these mice.

Group G Streptococcal IgG Binding Molecules FOG and Protein G Have Different Impacts on Opsonization by C1q

Recent epidemiological data on diseases caused by beta-hemolytic streptococci belonging to Lancefield group C and G (GCS, GGS) underline that they are an emerging threat to human health. Among various virulence factors expressed by GCS and GGS isolates from human infections, M and M-like proteins are considered important because of their anti-phagocytic activity. In addition, protein G has been implicated in the accumulation of IgG on the bacterial surface through non-immune binding. The function of this interaction, however, is still unknown. Using isogenic mutants lacking protein G or the M-like protein FOG (group G streptococci), respectively, we could show that FOG contributes substantially to IgG binding. A detailed characterization of the interaction between IgG and FOG revealed its ability to bind the Fc region of human IgG and its binding to the subclasses IgG1, IgG2, and IgG4. FOG was also found to bind IgG of several animal species. Surface plasmon resonance measurements indicate a high affinity to human IgG with a dissociation constant of 2.4 pm. The binding site was localized in a central motif of FOG. It has long been speculated about anti-opsonic functions of streptococcal Fc-binding proteins. The presented data for the first time provide evidence and, furthermore, indicate functional differences between protein G and FOG. By obstructing the interaction between IgG and C1q, protein G prevented recognition by the classical pathway of the complement system. In contrast, IgG that was bound to FOG remained capable of binding C1q, an effect that may have important consequences in the pathogenesis of GGS infections.

Effect of membrane-bound IgG and desialysation in the interaction of monocytes with senescent erythrocytes

Determination of the erythrocyte lifespan is a complex process affected by many cellular parameters. In the present study we measured and characterised the red blood cell (RBC) membrane proteins, mainly band 3, and quantified membrane-bound IgG in senescent RBC (SeRBC) and young RBC (YRBC). We also investigated, through a functional assay, the interaction between SeRBC and peripheral blood monocytes. We applied this erythrophagocytosis assay to study the phagocytosis of desialysed RBC. The results obtained showed no changes in the protein content between SeRBC and YRBC and no differences when examining membrane proteins by SDS-PAGE. Then, considering that the accumulation of autologous IgG on RBC membrane provides a direct mechanism for the removal of SeRBC, we measured the IgG content of intact RBC using an enzyme-linked anti-immunoglobulin test finding that the number of IgG molecules bound to SeRBC was significantly higher than that observed for YRBC. The increase observed in the percentage of erythrophagocytosis with SeRBC and sensitised RBC (SRBC) confirmed the involvement of autologous IgG in the selective removal of erythrocytes. We also observed a higher percentage of monocytes with phagocytosed and adherent RBC (AM) obtained with neuraminidase-treated RBC than those obtained with YRBC. This finding suggests that a decrease in sialic acid content of SeRBC may be involved in physiological erythrophagocytosis.