Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcyReceptors

Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcyRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcyRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcyR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an in vitro vasculitis model. Dimeric FcyR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcyR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcyRs

Purification of a post-synaptic neurotoxic phospholipase A2 from Naja naja venom and its inhibition by a glycoprotein from Withania somnifera

A post-synaptic neurotoxic phospholipase A2 (PLA2) has been purified from Indian cobra Naja naja venom. It was associated with a peptide in the venom. The association was disrupted using 8 M urea. It is denoted to be a basic protein by its behavior on both ion exchange chromatography and electrophoresis. It is toxic to mice, LD50 1.9 mg/kg body weight (ip). It is proved to be post-synaptic PLA2 by chymographic experiment using frog nerve-muscle preparation. A glycoprotein, (WSG) was isolated from a folk medicinal plant Withania somnifera. The WSG inhibited the phospholipase A2 activity of NN-XIa-PLA2, isolated from the cobra venom, completely at a mole-to-mole ratio of 1:2 (NN-XIa-PLA2: WSG) but failed to neutralize the toxicity of the molecule. However, it reduced the toxicity as well as prolonged the death time of the experimental mice approximately 10 times when compared to venom alone. The WSG also inhibited several other PLA2 isoforms from the venom to varying extent. The interaction of the WSG with the PLA2 is confirmed by fluorescence quenching and gel-permeation chromatography. Chemical modification of the active histidine residue of PLA2 using p-brophenacyl bromide resulted in the loss of both catalytic activity as well as neurotoxicity of the molecule. These findings suggest that the venom PLA2 has multiple sites on it; perhaps some of them are overlapping. Application of the plant extract on snakebite wound confirms the medicinal value associated with the plant

A glycoprotein from a folk medicinal plant, Withania somnifera, inhibits hyaluronidase activity of snake venoms

Venom hyaluronidases help in rapid spreading of the toxins by destroying the integrity of the extra-cellular matrix of the tissues in the victims. A hyaluronidase inhibitor (WSG) is purified from a folk medicinal plant, Withania somnifera. The glycoprotein inhibited the hyaluronidase activity of cobra (Naja naja) and viper (Daboia russelii) venoms, which was demonstrated by zymogram assay and staining of the skin tissues for differential activity. WSG completely inhibited the activity of the enzyme at a concentration of 1:1 w/w of venom to WSG. Thus we are able to demonstrate that the glycoprotein inhibits hyaluronidase activity of the venoms. External application of the plant extract as an antidote in rural parts of India to snakebite victims appears to have a scientific basis

Fluorinated Caffeic Acid Phenethyl Ester: a Novel Anti-osteogenic molecule to Attenuate Excessive Bone Damage during Autoimmune Arthritis

Caffeic Acid Phenethyl Ester (CAPE), an antioxidant flavonoid isolated from hives of honeybee, is reported to exert its anti-osteogenic function by inhibiting the NF-kB activation. It also disrupts the microtubule network created by osteoclasts, which wo

Expression of membrane anchored cytokines and B7-1 alters tumor microenvironment and induces protective antitumor immunity in a murine breast cancer model

Many studies have shown that the systemic administration of cytokines or vaccination with cytokine-secreting tumors augments an antitumor immune response that can result in eradication of tumors. However, these approaches are hampered by the risk of systemic toxicity induced by soluble cytokines. In this study, we have evaluated the efficacy of 4TO7, a highly tumorigenic murine mammary tumor cell line, expressing glycosyl phosphatidylinositol (GPI)-anchored form of cytokine molecules alone or in combination with the costimulatory molecule B7-1 as a model for potential cell or membrane-based breast cancer vaccines. We observed that the GPI-anchored cytokines expressed on the surface of tumor cells greatly reduced the overall tumorigenicity of the 4TO7 tumor cells following direct live cell challenge as evidenced by transient tumor growth and complete regression within 30 days post challenge. Tumors co-expressing B7-1 and GPI-IL-12 grew the least and for the shortest duration, suggesting that this combination of immunostimulatory molecules is most potent. Protective immune responses were also observed following secondary tumor challenge. Further, the 4TO7-B7-1/GPI-IL-2 and 4TO7-B7-1/GPI-IL-12 transfectants were capable of inducing regression of a wild-type tumor growing at a distant site in a concomitant tumor challenge model, suggesting the tumor immunity elicited by the transfectants can act systemically and inhibit the tumor growth at a distant site. Additionally, when used as irradiated whole cell vaccines, 4TO7-B7-1/GPI-IL-12 led to a significant inhibition in tumor growth of day 7 established tumors. Lastly, we observed a significant decrease in the prevalence of myeloid-derived suppressor cells and regulatory T-cells in the tumor microenvironment on day 7 post challenge with 4TO7-B7-1/GPI-IL-12 cells, which provides mechanistic insight into antitumor efficacy of the tumor-cell membrane expressed IL-12. These studies have implications in designing membrane-based therapeutic vaccines with GPI-anchored cytokines for breast cancer. © 2013 Elsevier Ltd

Differential Role of Lipocalin 2 During Immune Complex-Mediated Acute and Chronic Inflammation in Mice

OBJECTIVE: Lipocalin 2 (LCN-2) is an innate immune protein that is expressed by a variety of cells and is highly up-regulated during several pathologic conditions, including immune complex (IC)-mediated inflammatory/autoimmune disorders. However, the function of LCN-2 during IC-mediated inflammation is largely unknown. Therefore, this study was undertaken to investigate the role of LCN-2 in IC-mediated diseases. METHODS: The up-regulation of LCN-2 was determined by enzyme-linked immunosorbent assay in 3 different mouse models of IC-mediated autoimmune disease: systemic lupus erythematosus, collagen-induced arthritis, and serum-transfer arthritis. The in vivo role of LCN-2 during IC-mediated inflammation was investigated using LCN-2-knockout mice and their wild-type littermates. RESULTS: LCN-2 levels were significantly elevated in all 3 of the autoimmune disease models. Further, in an acute skin inflammation model, LCN-2-knockout mice exhibited a 50% reduction in inflammation, with histopathologic analysis revealing notably reduced immune cell infiltration as compared to wild-type mice. Administration of recombinant LCN-2 to LCN-2-knockout mice restored inflammation to levels observed in wild-type mice. Neutralization of LCN-2 using a monoclonal antibody significantly reduced inflammation in wild-type mice. In contrast, LCN-2-knockout mice developed more severe serum-induced arthritis compared to wild-type mice. Histologic analysis revealed extensive tissue and bone destruction, with significantly reduced neutrophil infiltration but considerably more macrophage migration, in LCN-2-knockout mice compared to wild-type mice. CONCLUSION: These results demonstrate that LCN-2 may regulate immune cell recruitment to the site of inflammation, a process essential for the controlled initiation, perpetuation, and resolution of inflammatory processes. Thus, LCN-2 may present a promising target in the treatment of IC-mediated inflammatory/autoimmune diseases

Hypothetical model of inhibition of IC-mediated, NO-induced apoptotic pathway in HUVECs by decoy FcγR-Igs.

<p>Circulating ICs deposits in the blood vessels during IC-mediated inflammatory disorders. Effector cells upon binding to the IC get activated. These cells result in the secretion of pro-inflammatory factors like cytokines and toxic superoxide radicals such as nitric oxide in copious amounts. Nitric oxide ability to interact with cellular components in the endothelium results in triggering apoptotic signaling pathway and subsequently cell death and tissue damage. Decoy FcγR-Igs competes with cell surface FcγRs expressed on effector cells and block the access to the ICs deposits in various organs including blood vessels. This will eventually inhibit effector cell mediated endothelial inflammation and damage observed in autoimmune vasculitis.</p

The Pharmacogenetics and Inhibitor Risk (PIR) Study: Establishing the Spectrum of Factor (F)VIII Gene (F8) Mutations in African-American Hemophilia A Patients

Abstract The development of neutralizing antibodies against plasma-derived or recombinant (r) molecules of wildtype (wt) factor (F)VIII protein is the most serious complication of replacement therapy for hemophilia A (hA) patients. Although the pathogenesis of these antibodies, termed inhibitors, is complex and poorly understood, ethnicity, a recently established risk determinant, is clearly involved since African-American (AA) patients experience this complication ~2-fold more often than Caucasians. In a previous study -- in which the FVIII genes (F8) from 137 unrelated healthy people, representing 7 ethnic groups, were resequenced -- we identified 4 common nonsynonymous single nucleotide polymorphisms (nsSNPs) and thereby demonstrated that FVIII is not a monomorphic protein in non-hemophiliacs. Interestingly, 5 of the 6 distinct wt proteins encoded by the allelic combinations or haplotypes (H) of these nsSNPs, designated H1, H2, ..., H5, are expressed by AA’s compared to only 2 in Caucasians (H1 and H2). Because H3, H4 and H5 are 1) partially defined by AA-restricted minor alleles of R484H and M2238V, 2 nsSNPs that substitute amino acids in the A2 and C2 major B-cell inhibitor epitopes, 2) represent the wt FVIII protein in ~27% of AA’s, and 3) differ from the rFVIII proteins used clinically, we designed the PIR study -- the 1st cross-sectional study of AA hA patients -- to determine if pharmacogenetic factors contribute to their greater inhibitor risk. Because the strongest known risk factor for inhibitors is the F8 mutation type, we are resequencing the known functional regions of F8 in the 1st 50 enrolled PIR subjects, out of the 223 total AA hA patients treated at the six participating Region IV South hemophilia treatment centers, to test the plausible alternative hypothesis that the higher inhibitor incidence is due to a different spectrum of molecular abnormalities than has been found in other ethnic subgroups of patients already examined at the DNA sequence level. We have 1) determined the plasma FVIII-activity and -antigen levels, 2) identified the causative mutation in 18 patients, several of which are novel, and 3) are currently performing RT-PCR analyses for detecting the common inversions in introns 1 and 22. Once completed, we will statistically compare the prevalence of AA F8 mutation subtypes to that in hA patients from other ethnic subgroups (detailed in the HAMSTeRS database), and the proportion of AA FVIII deficiencies that are 1) severe, moderate and mild, and 2) CRM-positive and -negative

Immune Complex-Induced, Nitric Oxide-Mediated Vascular Endothelial Cell Death by Phagocytes Is Prevented with Decoy FcγReceptors

<div><p>Autoimmune vasculitis is an endothelial inflammatory disease that results from the deposition of immune-complexes (ICs) in blood vessels. The interaction between Fcgamma receptors (FcγRs) expressed on inflammatory cells with ICs is known to cause blood vessel damage. Hence, blocking the interaction of ICs and inflammatory cells is essential to prevent the IC-mediated blood vessel damage. Thus we tested if uncoupling the interaction of FcγRs and ICs prevents endothelium damage. Herein, we demonstrate that dimeric FcγR-Igs prevented nitric oxide (NO) mediated apoptosis of human umbilical vein endothelial cells (HUVECs) in an <i>in vitro</i> vasculitis model. Dimeric FcγR-Igs significantly inhibited the IC-induced upregulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release by murine monocytic cell line. However, FcγR-Igs did not affect the exogenously added NO-induced upregulation of pro-apoptotic genes such as Bax (15 fold), Bak (35 fold), cytochrome-C (11 fold) and caspase-3 (30 fold) in HUVECs. In conclusion, these data suggest that IC-induced NO could be one of the major inflammatory mediator promoting blood vessel inflammation and endothelial cell death during IC-mediated vasculitis which can be effectively blocked by dimeric decoy FcγRs.</p></div

Immune-complex induced nitric oxide secretion is prevented by dimeric FcγR-Ig molecules.

<p><b>(A)</b> RAW 264.7 cells were cultured with different concentrations (0–300μg/ml) of soluble ICs in serum free RPMI1640 medium for 12 and 24 hrs. Untreated RAW264.7 cells and cells treated with antigen, antibody and 2.4G2 mAb served as specificity controls. The culture medium was collected at specified time points, and the nitrite concentration was determined using the Griess reagent. <b>(B)</b> RAW 267.4 cells were cultured with 100 and 200μg/ml of soluble ICs for different time points (0–24 hr). The culture medium was collected at specified time points, and the nitrite concentration was determined using the Griess reagent. <b>(C)</b> CD16A-Ig and CD32A-Ig blocked the binding of ICs to RAW 264.7 cells thereby inhibited the NO production. In all the experiments RAW 264.7 cells pretreated with 2.4G2 mAb, antigen alone, antibody alone, cells treated with medium alone served as specificity controls. Data shown are the average of three individual experiments; each experiment was carried in triplicate. P < 0.05 considered as significant (*) and P< 0.005 as highly significant (**), NS: non-significant.</p