Correlation between antibodies to bisphenol A, its target enzyme protein disulfide isomerase and antibodies to neuron-specific antigens.

Evidence continues to increase linking autoimmunity and other complex diseases to the chemicals commonly found in our environment. Bisphenol A (BPA) is a synthetic monomer used widely in many forms, from food containers to toys, medical products and many others. The potential for BPA to participate as a triggering agent for autoimmune diseases is likely due to its known immunological influences. The goal of this research was to determine if immune reactivity to BPA has any correlation with neurological antibodies. BPA binds to a target enzyme called protein disulfide isomerase (PDI). Myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG) are neuronal antigens that are target sites for neuroinflammation and neuroautoimmunity. We determined the co-occurrence of anti-MBP and anti-MOG antibodies with antibodies made against BPA bound to human serum albumin in 100 healthy human subjects. Correlation between BPA to PDI, BPA to MOG, BPA to MBP, PDI to MBP and PDI to MOG were all highly statistically significant (P < 0.0001). The outcome of our study suggests that immune reactivity to BPA-human serum albumin and PDI has a high degree of statistical significance with substantial correlation with both MBP and MOG antibody levels. This suggests that BPA may be a trigger for the production of antibodies against PDI, MBP and MOG. Immune reactivity to BPA bound to human tissue proteins may be a contributing factor to neurological autoimmune disorders. Further research is needed to determine the exact relationship of these antibodies with neuroautoimmunities. Copyright © 2016 The Authors Journal of Applied Toxicology Published by John Wiley & Sons Ltd.

Generation and characterization of fully human monoclonal antibodies against human Orai1 for autoimmune disease.

Calcium entry into T cells following antigen stimulation is crucial for nuclear factor of activated T cells (NFAT)-mediated T cell activation. The movement of calcium is mediated by calcium release-activated calcium (CRAC) channels. There are two key components of this channel: Orai1 is the pore-forming subunit located in the plasma membrane, and stromal interaction molecule 1 (STIM1) functions as a Ca(2+) sensor in the endoplasmic reticulum. A subset of human patients carry mutations in either STIM1 or Orai1 that affect protein function or expression, resulting in defective store-operated Ca(2+) influx and CRAC channel function, and impaired T cell activation. These patients suffer from a hereditary form of severe combined immune deficiency syndrome, highlighting the importance of the CRAC channel for T lymphocyte function in humans. Since autoreactive T cells play an important role in the development of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and organ transplantation, Orai1 becomes an attractive therapeutic target for ameliorating autoimmune disease. We developed a novel approach to inhibiting CRAC function by generating high-affinity fully human monoclonal antibodies to human Orai1. These antibodies inhibited ICRAC current, store-operated Ca(2+) influx, NFAT transcription, and cytokine release. These fully human antibodies to human Orai1 may represent a novel therapeutic approach for the treatment of autoimmunity.

Recombinant truncated AniA of pathogenic Neisseria elicits a non-native immune response and functional blocking antibodies.

AniA of the pathogenic Neisseria is glycosylated in its C-terminal repeat region by the pilin glycosylation (pgl) pathway. AniA appears to be unique among bacterial nitrite reductases as it contains an N-terminal extension that includes a lipid modification site as well as a C-terminal extension that is glycosylated. Immunising with various glycoforms of the AniA protein demonstrated a strong humoral immune response to the basal monosaccharide. In addition, when animals were immunised with a truncated form of AniA, completely lacking the glycosylated C-terminal region, the antibody response was directed against AniA regardless of the glycosylation state of the protein. Immuno-SEM confirmed that AniA is expressed on the cell surface in Neisseria gonorrhoeae. Antisera generated against a truncated, non-glycosylated, recombinant form of the AniA protein are capable of blocking nitrite reductase function in a whole cell assay. We propose that recombinant modified AniA has potential as a vaccine antigen for N. gonorrhoeae.

The Tim3-galectin 9 pathway induces antibacterial activity in human macrophages infected with Mycobacterium tuberculosis.

T cell Ig and mucin domain 3 (Tim3) is an inhibitory molecule involved in immune tolerance, autoimmune responses, and antiviral immune evasion. However, we recently demonstrated that Tim3 and Galectin-9 (Gal9) interaction induces a program of macrophage activation that results in killing of Mycobacterium tuberculosis in the mouse model of infection. In this study, we sought to determine whether the Tim3-Gal9 pathway plays a similar role in human pulmonary TB. We identified that pulmonary TB patients have reduced expression of Tim3 on CD14(+) monocytes in vivo. By blocking Tim3 and Gal9 interaction in vitro, we show that these molecules contribute to the control of intracellular bacterial replication in human macrophages. The antimicrobial effect was partially dependent on the production of IL-1ß. Our results establish that Tim3-Gal9 interaction activates human M. tuberculosis -infected macrophages and leads to the control of bacterial growth through the production of the proinflammatory cytokine IL-1ß. Data presented in this study suggest that one of the potential pathways activated by Tim3/Gal9 is the secretion of IL-1ß, which plays a crucial role in antimicrobial immunity by modulating innate inflammatory networks.

Balance of infection-enhancing and neutralizing antibodies induced by a dengue tetravalent DNA vaccine in a mouse model.

Most dengue vaccines currently under development are designed to induce neutralizing antibodies. Although neutralizing antibodies are believed to be protective, most of the antibody species that have neutralizing activities also have infection-enhancing activities at subneutralizing doses. Thus, the balance of neutralizing and enhancing activities induced by a dengue tetravalent vaccine was analyzed in this study in a DNA vaccine model using mice. The balance was measured using semi-adherent K562 cells in an assay system we previously developed. This system detected enhancing activities in sera from mice immunized with the vaccine when complement was absent from the virus-antibody mixture in this assay. Mice passively transferred with vaccinated mouse sera or monoclonal antibodies that possessed neutralizing activities as determined on Vero cells, also showed enhancing activities in their sera during the course of antibody waning in the absence of complement. However, these enhancing activities were abolished or altered to neutralizing activities in the presence of complement in our assay system, except in mice passively transferred with a monoclonal antibody of isotype IgG1. Thus, enhancing activities were not shown in mice immunized with a DNA vaccine as far as complement was included in our in vitro assay system.

TRP channels and cancer: new targets for diagnosis and chemotherapy.

The Transient Receptor Potential (TRP) channels family consists of seven different subfamilies, namely TRPC (Canonical), TRPV (Vanilloid), TRPM (Melastatin), TRPML (Mucolipin), TRPP (Polycystin), and TRPA (Ankyrin transmembrane protein) and TRPN (NomPC-like) that are related to several physiological and pathological processes. Recent years have witnessed an increased interest of research into the connection between TRP channels and cancer, leading to the discovery of tumor-related functions such as regulation of proliferation, differentiation, apoptotis, angiogenesis, migration and invasion during cancer progression. Among the TRP families, TRPCs, TRPMs and TRPVs are mainly related to malignant growth and progression. Depending on the type and stage of the cancer, regulation of TRPs mRNA and protein expression have been reported; these changes may regulate ion-dependent cell proliferation and resistance of cancer cells to apoptotic-induced cell death with consequent cancer promoting effects and resistance to chemotherapic treatments. Considerable efforts have been made to fight cancer cells and targeted therapy seems to be the most promising strategy: in this regard, ion channels belonging to the TRP channel superfamily could play an important role. Aim of this review is to summarize data reported so far on the expression and the functional role of TRP channels during cancer growth and progression, and the relationship with clinico-pathological markers. Moreover, the feasibility of TRP channels as target of chemotherapy and the different approaches by which these channels can be targeted will be analyzed in detail. Deeper investigations are required to understand the role TRP channels in cancer in order to develop further knowledge of TRP proteins as valuable diagnostic and/or prognostic markers, as well as targets for pharmaceutical intervention and targeting.

Deficiency of prohormone convertase dPC2 (AMONTILLADO) results in impaired production of bioactive neuropeptide hormones in Drosophila.

Peptide hormones synthesized by secretory neurons in the CNS are important regulators of physiology, behavior, and development. Like other neuropeptides, they are synthesized from larger precursor molecules by a specific set of enzymes. Using a combination of neurogenetics, immunostainings, and direct mass spectrometric profiling, we show that the presence of Drosophila prohormone convertase 2 encoded by the gene amontillado (amon) is a prerequisite for the proper processing of neuropeptide hormones from the major neurohemal organs of the CNS. A loss of amon correlates with a loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs. Neuropeptide hormone signals were still detectable in the adult corpora cardiaca of older amon-deficient flies which were amon heat-shock-rescued until eclosion. A semiquantification by direct peptide profiling using stable isotopic standards showed, however, that their neuropeptide hormone levels are strongly reduced. Targeted expression of GFP under the control of amon regulatory regions revealed a co-localization with the investigated peptide hormones in secretory neurons of the brain and ventral nerve cord. The lack of AMON activity resulted in a deficiency of L3 larva to enter the wandering phase. In conclusion, our findings provide the first direct evidence that AMON is a key enzyme in the production of neuropeptides in the fruitfly.

Delayed allergic reaction to natalizumab associated with early formation of neutralizing antibodies.

BACKGROUND: Natalizumab is a new therapeutic option for relapsing-remitting multiple sclerosis. As with other antibody therapies, hypersensitivity reactions have been observed. In the Natalizumab Safety and Efficacy in Relapsing-Remitting Multiple Sclerosis (AFFIRM) trial, infusion-related hypersensitivity reactions developed in 4% of patients, usually within 2 hours after starting the infusion. OBJECTIVE: To report a significant, delayed, serum sickness-like, type III systemic allergic reaction to natalizumab. DESIGN: Case report describing clinical follow-up and the serial measurement of antinatalizumab antibodies. PATIENT: A 23-year-old man with relapsing-remitting multiple sclerosis developed a fever, arthralgias, urticarial exanthema, and a swollen lower lip during several days after his second infusion of natalizumab. RESULTS: The patient developed a delayed, serum sickness-like, type III systemic allergic reaction to natalizumab. Five weeks after initiation of this therapy, he tested positive for antinatalizumab antibodies and exhibited persistent antibody titers 8 and 12 weeks later. His symptoms completely resolved with a short course of oral glucocorticosteroids. CONCLUSION: Clinicians and patients should be alert not only to immediate but also to significantly delayed substantial allergic reactions to natalizumab.

Lipid-lowering effects of anti-angiopoietin-like 4 antibody recapitulate the lipid phenotype found in angiopoietin-like 4 knockout mice.

We used gene knockout mice to explore the role of Angiopoietin-like-4 (Angptl4) in lipid metabolism as well as to generate anti-Angptl4 mAbs with pharmacological activity. Angptl4 -/- mice had lower triglyceride (TG) levels resulting both from increased very low-density lipoprotein (VLDL) clearance and decreased VLDL production and had modestly lower cholesterol levels. Also, both Angptl4 -/- suckling mice and adult mice fed a high-fat diet showed reduced viability associated with lipogranulomatous lesions of the intestines and their draining lymphatics and mesenteric lymph nodes. Treating C57BL/6J, ApoE -/-, LDLr -/-, and db/db mice with the anti-Angptl4 mAb 14D12 recapitulated the lipid and histopathologic phenotypes noted in Angptl4 -/- mice. This demonstrates that the knockout phenotype reflects not only the physiologic function of the Angptl4 gene but also predicts the pharmacologic consequences of Angptl4 protein inhibition with a neutralizing antibody in relevant models of human disease.

Macrophage colony-stimulating factor enhances rituximab-dependent cellular cytotoxicity by monocytes.

Recent studies suggest that monocytes are the dominant effectors by which rituximab induces cell death in B-cell lymphoma. Because macrophage colony-stimulating factor (M-CSF) can enhance the cytotoxicity of monocytes, the authors examined whether this growth factor can enhance their ability to kill lymphoma cells in vitro. Monocytes derived from a healthy volunteer were cultured for 48 h in the presence or absence of M-CSF. Monocytes stimul ated with M-CSF were significantly more cytotoxic to Daudi B-cell lymphomas than unstimulated monocytes. Flow cytometry revealed that M-CSF increased monocyte expression of Fcgamma receptors III and I by 1.6- and 1.5-fold, whereas the expression of Fcgamma receptor II remained unchanged. These results suggest that pretreatment with M-CSF can improve the therapeutic efficacy of rituximab against intractable CD20(+) lymphoma.

Recombinant gp120 vaccine-induced antibodies inhibit clinical strains of HIV-1 in the presence of Fc receptor-bearing effector cells and correlate inversely with HIV infection rate.

Nonneutralizing Abs may play a role in protecting animals and humans from lentiviral infections. We explored the Ab-dependent, cell-mediated virus inhibition (ADCVI) Ab response to recombinant gp120 (rgp120) vaccination in sera from 530 participants in the Vax 004 trial. Serum ADCVI activity was measured against a clinical R5 strain of HIV-1 using peripheral blood mononuclear effector cells from healthy donors. The level of vaccine-induced ADCVI activity correlated inversely with the rate of acquiring HIV infection following vaccination, such that for every 10% increase in ADCVI activity, there was a 6.3% decrease in the hazard rate of infection (p=0.019). Some vaccinated individuals also mounted an ADCVI response against two other clinical R5 strains of HIV-1. However, ADCVI activity correlated poorly with neutralizing or CD4-gp120-blocking Ab activity measured against laboratory strains. Finally, the degree to which the ADCVI Ab response predicted the rate of infection was influenced by polymorphisms at the FcgammaRIIa and FcgammaRIIIa gene loci. These data indicate that rgp120 vaccination can elicit Abs with antiviral activity against clinical strains of HIV-1. However, such activity requires the presence of FcR-bearing effector cells. Our results provide further evidence that ADCVI may play a role in preventing HIV infection.

Plasmodium falciparum: immunization with MSP1-42 induced non-inhibitory antibodies that have no blocking activities but enhanced the potency of inhibitory anti-MSP1-42 antibodies.

Hyperimmunization with Plasmodium falciparum MSP1-42 could induce antibodies that have little or no parasite growth inhibitory activities. These antisera had no blocking activities as determined by their ability to interfere with the in vitro activities of growth inhibitory anti-MSP1-42 sera. Equally important, they enhanced the potency of growth inhibitory anti-MSP1-42 sera.

TNFalpha kinoid vaccination-induced neutralizing antibodies to TNFalpha protect mice from autologous TNFalpha-driven chronic and acute inflammation.

The proinflammatory cytokine TNFalpha is a potent mediator of septic shock and a therapeutic target for chronic inflammatory pathologies including rheumatoid arthritis and Crohn's disease. As an alternative to anti-human TNFalpha (hTNFalpha) mAbs and other hTNFalpha blocker approved drugs, we developed an active anti-hTNFalpha immunotherapy, based on a vaccine comprised of a keyhole limpet hemocyanin-hTNFalpha heterocomplex immunogen (hTNFalpha kinoid) adjuvanted in incomplete Freund's adjuvant. In mice transgenic for hTNFalpha (TTg mice), hTNFalpha kinoid vaccination elicited high titers of Abs that neutralized hTNFalpha bioactivities but did not result in a cellular response to hTNFalpha. The vaccine was safe and effective in two experimental models. Kinoid-immunized but not control TTg mice resisted hTNFalpha-driven shock in one model and were prevented from spontaneous arthritis, inflammatory synovitis, and articular destruction in a second model. These data demonstrate an anti-cytokine induction of autoimmune protection against both acute and chronic hTNFalpha exposure. They show that active vaccination against a human cytokine can be achieved, and that the immune response can be effective and safe.

Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae.

PURPOSE: This study describes the soluble production, purification, and functional testing of an anti-transferrin receptor single-chain antibody (OX26 scFv) using the yeast Saccharomyces cerevisiae. METHODS: The yeast secretion apparatus was optimized by modulating expression temperature, the folding environment of the endoplasmic reticulum, and gene dosage. Secreted scFv was purified using immobilized metal affinity chromatography, and tested for binding and internalization into the RBE4 rat brain endothelial cell line. RESULTS: Secretion of OX26 scFv was optimal when expression was induced at 20 degrees C. Co-overexpression of heavy chain binding protein and protein disulfide isomerase elevated scFv expression levels by 10.4 +/- 0.3-fold. Optimization of scFv gene dosage increased secretion by 7.1 +/- 0.2-fold, but the overall benefits of binding protein and protein disulfide isomerase overexpression were diminished. Purified OX26 scFv yields of 0.5 mg/L secreted protein were achieved, and the scFv was actively internalized into RBE4 cells with a pattern similar to that observed with intact OX26 monoclonal antibody. CONCLUSIONS: The optimized S. cerevisiae expression system is amenable to production of soluble and active brain targeting OX26 scFv, and the yeast-produced scFv has potential for the targeting and delivery of small molecules, proteins, or drug carriers across the blood-brain barrier (BBB).

Inhibition of lupus disease by anti-double-stranded DNA antibodies of the IgM isotype in the (NZB x NZW)F1 mouse.

OBJECTIVE: In systemic lupus erythematosus (SLE), immune complexes (ICs) containing pathogenic IgG anti-double-stranded DNA (anti-dsDNA) autoantibodies are deposited in renal capillaries and initiate glomerulonephritis (GN) by the activation of complement and effector cells. In contrast, it has been demonstrated that the presence of IgM anti-dsDNA antibodies correlates negatively with the development of GN in SLE. The aim of this study was to determine whether anti-dsDNA antibodies of the IgM isotype protect against IC-mediated organ damage in SLE. METHODS: Lupus-prone (NZB x NZW)F(1) mice (females) were treated with murine monoclonal IgM anti-dsDNA antibodies. Treatment was delivered by subcutaneous injection at a dosage of 100 mug/week starting at 16 weeks of age (prophylactic) or at 24 weeks of age (therapeutic). RESULTS: Mice treated with IgM anti-dsDNA exhibited a delayed onset of proteinuria and a reduced degree of renal pathology, which resulted in significantly improved survival as compared with control mice. Serum concentrations of IgG anti-dsDNA antibodies were not significantly modified. However, glomerular deposition of ICs was markedly reduced in both treatment protocol groups. In contrast, higher amounts of IgG and IgM and increased expression of Fcgamma receptor were demonstrated in liver sections from the treated mice compared with the untreated mice, suggesting an enhanced clearance of soluble ICs from phagocytic cells of the reticuloendothelial system. CONCLUSION: These data demonstrate the efficacy of IgM anti-dsDNA treatment in inhibiting the pathologic changes of lupus in (NZB x NZW)F(1) mice. Lower glomerular IC deposition is associated with a reduced inflammatory response and impaired organ damage. The reduced frequency of GN in SLE patients who have IgM anti-dsDNA antibodies may therefore reflect a disease-modifying effect of this class of autoantibodies that has potential therapeutic implications. Our findings should encourage the development of new therapeutic modalities using IgM anti-dsDNA antibodies in humans with SLE.

Monoclonal antibodies to the thyrotropin receptor.

The thyrotropin receptor (TSHR) is a seven transmembrane G-protein linked glycoprotein expressed on the thyroid cell surface and which, under the regulation of TSH, controls the production and secretion of thyroid hormone from the thyroid gland. This membrane protein is also a major target antigen in the autoimmune thyroid diseases. In Graves' disease, autoantibodies to the TSHR (TSHR-Abs) stimulate the TSHR to produce thyroid hormone excessively. In autoimmune thyroid failure, some patients exhibit TSHR-Abs which block TSH action on the receptor. There have been many attempts to generate human stimulating TSHR-mAbs, but to date, only one pathologically relevant human stimulating TSHR-mAb has been isolated. Most mAbs to the TSHR have been derived from rodents immunized with TSHR antigen from bacteria or insect cells. These antigens lacked the native conformation of the TSHR and the resulting mAbs were exclusively blocking or neutral TSHR-mAbs. However, mAbs raised against intact native TSHR antigen have included stimulating mAbs. One such stimulating mAb has demonstrated a number of differences in its regulation of TSHR post-translational processing. These differences are likely to be reflective of TSHR-Abs seen in Graves' disease.

Antibody against cholesteryl ester transfer protein (CETP) elicited by a recombinant chimeric enzyme vaccine attenuated atherosclerosis in a rabbit model.

The recombinant chimeric enzyme of AnsB-TTP-CETPC, which comprised asparagianse, tetanus toxin helper T-cell epitope and CETP B-cell epitope, was used to vaccinate New Zealand white rabbits in alum adjuvant. After anti-CETP antibodies were successfully produced, rabbits were fed with a high cholesterol diet for fifteen weeks until atherosclerotic lesions formed in arteries. The results showed that after CETP was inhibited by anti-CETP antibodies the fraction of plasma cholesterol in HDL increased and the fraction of plasma cholesterol in LDL decreased in rAnsB-TTP-CETPC immunized rabbits. The average size of aorta atherosclerotic plaques in rabbits treated with rAnsB-TTP-CETPC was 42.3% less than in rabbits treated with OVA (neutral control), or 47.6% less than in rabbits treated with rHSP 65 (negative control). The average thickness of hyperplastic coronary artery in rAnsB-TTP-CETPC immunized rabbits was 159+/-12 microm, which was significantly lower than in rHSP 65 immunized rabbits (187+/-15 microm) or OVA immunized rabbits (248+/-18 microm). The data reported here demonstrated that rAnsB-TTP-CETPC could significantly attenuate the development of atherosclerosis in rabbits fed with high cholesterol diet, and might be developed to an anti-atherosclerosis vaccine in the future.

Targeting fibroblast growth factor-inducible-14 signaling protects from chronic relapsing experimental autoimmune encephalomyelitis.

The TNF-related weak inducer of apoptosis (TWEAK) is a TNF family member mediating proinflammatory effects by its receptor fibroblast growth factor-inducible-14 (Fn14). We studied the role of TWEAK/Fn14 in experimental autoimmune encephalomyelitis (EAE) by protein vaccination with TWEAK and Fn14 and recombinant TWEAK-DNA, respectively. TWEAK-DNA vaccination worsened the clinical course of EAE and increased central nervous system (CNS) inflammation. TWEAK increased the secretion of CCL2 [monocyte chemotactic protein-1 (MCP-1)] by CNS endothelial cells and astrocytes in vitro, suggesting CCL2 as a critical mediator of TWEAKs proinflammatory effects. Vaccination with the extracellular domain of TWEAK or with Fn14 resulted in the induction of specific inhibitory antibodies and an amelioration of EAE signs in two different models in rats and mice. Spinal cord inflammatory infiltrates were significantly diminished. Purified IgG from TWEAK- or Fn14-vaccinated rats prevented TWEAK-induced production of CCL2 by endothelial cells. Blocking Fn14 signaling represents a novel approach with potential for the treatment of CNS autoimmunity.

Active immunization against murine TNFalpha peptides in mice: generation of endogenous antibodies cross-reacting with the native cytokine and in vivo protection.

New lines of treatment targeting cytokines have been successfully developed recently and are now widely used in therapy. They are based on passive administration of cytokine inhibitors either soluble receptors or mAbs and the major example is TNFalpha in rheumatoid arthritis (RA). Since a few years, our group has developed a novel alternative approach targeting cytokines by using active immunization against biologically inactive but immunogenic cytokine derivatives. In the present work, we present a new aspect of this research, based on immunization against specific cytokine peptides chosen by molecular modelling. We could elicit a significant humoral response against four TNFalpha peptides by active immunization, and show that the Abs generated cross-reacted with the native cytokine with good titers as determined by ELISA. Interestingly, during coimmunization experiments with couples of peptides, one showed a clear immunodominant effect over the other. Overall, we could not show the neutralization of TNFalpha biological activity in vitro by the immunized sera, but it seems that it is not a prerequisite to observe clinical efficacy. Indeed, using the LPS/galactosamine-induced shock, we could demonstrate that one of the four peptides tested conferred a clinical protection. These results validate the feasibility and efficacy of active immunization against cytokine peptides, and confirm that active immunization against cytokines could represent in the future an alternative to passive immunization in many diseases.