Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 34
Filter
1.
Sci Rep ; 9(1): 7046, 2019 05 07.
Article in English | MEDLINE | ID: mdl-31065015

ABSTRACT

Ligand binding assays routinely employ fluorescently-labeled protein ligands to quantify the extent of binding. These ligands are commonly generated through chemical modification of accessible lysine residues, which often results in heterogeneous populations exhibiting variable binding properties. This could be remedied by quantitative, site-specific labeling. Recently, we reported on a single-step method integrating recombinant protein purification with 2-cyanobenzothiazole (CBT) condensation for labeling a proteolytically exposed N-terminal cysteine. Here, using three growth factors, we show that unlike random lysine labeling, this site-specific approach yielded homogeneous populations of growth factors that were quantitatively labeled at their N-termini and retained their binding characteristics. We demonstrate the utility of this labeling method through the development of a novel assay that quantifies the capacity of antibodies to block receptor-ligand interactions (i.e. antibody blockade). The assay uses bioluminescence resonance energy transfer (BRET) to detect binding of CBT-labeled growth factors to their cognate receptors genetically fused to NanoLuc luciferase. The ability of antibodies to block these interactions is quantified through decrease in BRET. Using several antibodies, we show that the assay provides reliable quantification of antibody blockade in a cellular context. As demonstrated here, this simple method for generating uniformly-labeled proteins has potential to promote more accurate and robust ligand binding assays.


Subject(s)
Antibodies, Blocking/analysis , Fluorescent Dyes/chemistry , Proteomics/methods , Antibodies, Blocking/metabolism , Becaplermin/genetics , Becaplermin/metabolism , Benzopyrans/chemistry , Benzothiazoles/chemistry , Cetuximab/pharmacology , Epidermal Growth Factor/genetics , Epidermal Growth Factor/metabolism , HEK293 Cells , Humans , Indoles/chemistry , Ligands , Luminescent Measurements/methods , Nitriles/chemistry , Panitumumab/pharmacology , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Reproducibility of Results , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism
2.
J Immunol Methods ; 456: 7-14, 2018 05.
Article in English | MEDLINE | ID: mdl-29427592

ABSTRACT

LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.


Subject(s)
Antibodies, Blocking/analysis , Antigens, CD/immunology , Cell Engineering , Programmed Cell Death 1 Receptor/immunology , Signal Transduction/immunology , Antibodies, Blocking/immunology , Antigens, CD/genetics , Flow Cytometry , Gene Expression Profiling , Humans , Jurkat Cells , Programmed Cell Death 1 Receptor/genetics , Real-Time Polymerase Chain Reaction , Signal Transduction/genetics , T-Lymphocytes/immunology , Tumor Cells, Cultured , Lymphocyte Activation Gene 3 Protein
3.
J Biol Chem ; 292(28): 11960-11969, 2017 07 14.
Article in English | MEDLINE | ID: mdl-28533429

ABSTRACT

FREP1 in mosquito midguts facilitates Plasmodium falciparum parasite transmission. The fibrinogen-like (FBG) domain of FREP1 is highly conserved (>90% identical) among Anopheles species from different continents, suggesting that anti-FBG antibodies may block malaria transmission to all anopheline mosquitoes. Using standard membrane-feeding assays, anti-FREP1 polyclonal antibodies significantly blocked transmission of Plasmodium berghei and Plasmodium vivax to Anopheles gambiae and Anopheles dirus, respectively. Furthermore, in vivo studies of mice immunized with FBG achieved >75% blocking efficacy of P. berghei to A. gambiae without triggering immunopathology. Anti-FBG serum also reduced >81% of P. falciparum infection to A. gambiae Finally, we showed that FBG interacts with Plasmodium gametocytes and ookinetes, revealing the molecular mechanism of its antibody transmission-blocking activity. Collectively, our data support that FREP1-mediated Plasmodium transmission to mosquitoes is a conserved pathway and that targeting the FBG domain of FREP1 will limit the transmission of multiple Plasmodium species to multiple Anopheles species.


Subject(s)
Anopheles/metabolism , Insect Proteins/therapeutic use , Malaria Vaccines/therapeutic use , Malaria, Falciparum/prevention & control , Plasmodium falciparum/immunology , Amino Acid Sequence , Animals , Anopheles/immunology , Anopheles/parasitology , Antibodies, Blocking/analysis , Conserved Sequence , Female , Germ Cells/immunology , Germ Cells/metabolism , Humans , Insect Proteins/chemistry , Insect Proteins/genetics , Insect Proteins/metabolism , Malaria Vaccines/chemistry , Malaria Vaccines/genetics , Malaria Vaccines/metabolism , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Malaria, Falciparum/transmission , Malaria, Vivax/blood , Malaria, Vivax/immunology , Malaria, Vivax/parasitology , Male , Mice , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Peptide Fragments/therapeutic use , Plasmodium berghei/growth & development , Plasmodium berghei/immunology , Plasmodium falciparum/growth & development , Plasmodium vivax/growth & development , Plasmodium vivax/immunology , Protein Interaction Domains and Motifs , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/therapeutic use , Sequence Alignment , Sequence Homology, Amino Acid , Species Specificity , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/metabolism , Vaccines, Synthetic/therapeutic use
4.
J Appl Toxicol ; 37(4): 479-484, 2017 04.
Article in English | MEDLINE | ID: mdl-27610592

ABSTRACT

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.


Subject(s)
Antibodies, Blocking/biosynthesis , Antibodies/metabolism , Benzhydryl Compounds/antagonists & inhibitors , Benzhydryl Compounds/immunology , Neurons/immunology , Phenols/antagonists & inhibitors , Phenols/immunology , Protein Disulfide-Isomerases/immunology , Adolescent , Adult , Aged , Antibodies/pharmacology , Antibodies, Blocking/analysis , Autoimmune Diseases/chemically induced , Autoimmune Diseases/immunology , Autoimmunity/drug effects , Autoimmunity/immunology , Humans , Middle Aged , Myelin Basic Protein/biosynthesis , Myelin Basic Protein/genetics , Myelin-Oligodendrocyte Glycoprotein/biosynthesis , Myelin-Oligodendrocyte Glycoprotein/genetics , Nervous System Diseases/chemically induced , Nervous System Diseases/immunology , Protein Disulfide-Isomerases/antagonists & inhibitors , Young Adult
5.
Thyroid ; 26(5): 734-40, 2016 05.
Article in English | MEDLINE | ID: mdl-26956921

ABSTRACT

BACKGROUND AND OBJECTIVE: A cell-based bioassay for the measurement of thyroid blocking autoantibodies (TBAb) has been recently reported. The analytical performance and validation of this bioassay is assessed and described. METHODS: Chinese hamster ovary cells expressing a chimeric thyrotropin receptor were treated with bovine (b) TSH and different concentrations of an immunoglobulin G (IgG) monoclonal human TBAb (K1-70). TBAb was measured as a function of luciferase activity relative to bTSH alone and expressed as percent inhibition. Results obtained in the chimeric cell line were compared with those of a wild-type cell line. Analytical performance studies were subsequently performed with the chimeric cell line only. RESULTS: Immunodepletion of K1-70 IgG by using a protein G-Sepharose column showed that positive percent inhibition in the TBAb bioassay was detectable from K1-70 IgG only. The limit of blank was determined to be 12.2%. The limit of detection was 14% inhibition, equivalent to 0.4 ng/mL K1-70, while the limit of quantitation was 22% (coefficient of variation [CV] 12%) equivalent to 0.625 ng/mL K1-70. The dynamic range was between 14 ± 3.7 (mean % inhibition ± standard deviation) and 101 ± 2.6, equivalent to 0.4-10 ng/mL K1-70. The linear range was between 22 ± 2.6 and 93 ± 0.6 inhibition, equivalent to 0.625-5 ng/mL K1-70. The upper limit of the 99th percent reference range was 34% inhibition. In two laboratories, CV values for the intra- and inter-assay precisions for K1-70 ranged from 2% to 12% and from 1.7% to 14.5%, respectively. For patient sera, the CV values for the intra- and inter-assay precisions ranged from 3% to 9% and from 3% to 11%, respectively. No interference was found when follicle-stimulating hormone, luteinizing hormone, and human chorionic gonadotrophin were tested in the TBAb bioassay. The median of % inhibition values in 40 TBAb positive sera from patients with autoimmune thyroid disease were 93.5 (range 25-103) and 92 (range 64-107) for the wild type and chimeric cell lines, respectively. Further, all 40 samples of patients with various non-thyroidal autoimmune diseases were TBAb negative. CONCLUSIONS: This TBAb bioassay exhibits excellent analytical performance and high level of reproducibility.


Subject(s)
Antibodies, Blocking/analysis , Autoantibodies/analysis , Thyroid Gland/immunology , Animals , Biological Assay , CHO Cells , Cricetulus , Humans , Immunoglobulin G/analysis , Immunoglobulins, Thyroid-Stimulating/analysis , Reproducibility of Results
6.
Article in English | MEDLINE | ID: mdl-25985825

ABSTRACT

OBJECTIVES: Allogeneic lymphocytes of paternal origin or supplied by a male third party have been used for the treatment of recurrent spontaneous abortion. Few studies, however, have examined the use of female third party lymphocytes. Our purpose was to determine whether female third party lymphocytes could be used for immunotherapy of women with recurrent spontaneous abortion. METHODS: In this retrospective non-randomised cohort-controlled study, the medical records of patients with three or more spontaneous abortions who received immunotherapy with lymphocytes from their partner, a male third party or a female third party, as well as those who received no immunotherapy, from 1996 to 2012 were reviewed. All patients were negative for mixed lymphocyte culture reaction (MLR)-blocking antibodies. Immunotherapy was performed in 302 patients in two courses, while 53 patients received no immunotherapy. RESULTS: The pregnancy rates in patients who received lymphocytes from their partners, a male third party or a female third party, and in those not immunised, were 85.6%, 87.3%, 89.7%, and 79.3%, respectively (p = 0.523);the live birth rates were 87.3%, 75.8%, 84.6%, and 40.5%, respectively (p < 0.001). CONCLUSIONS: We conclude that female third party lymphocytes can be used for immunotherapy in patients with recurrent spontaneous abortion.


Subject(s)
Abortion, Habitual/therapy , Immunotherapy/methods , Live Birth , Lymphocyte Transfusion , Adult , Antibodies, Blocking/analysis , Female , Humans , Lymphocyte Culture Test, Mixed , Male , Pregnancy , Pregnancy Rate , Retrospective Studies , Time Factors , Transplantation, Homologous , Young Adult
7.
J Immunoassay Immunochem ; 33(4): 403-13, 2012.
Article in English | MEDLINE | ID: mdl-22963489

ABSTRACT

Antibodies generated against Region II of Plasmodium vivax Duffy binding protein (PvRII) can block binding of this parasite ligand to its receptor, the Duffy antigen receptor for chemokines (DARC), and prevent erythrocyte infection by the parasite. An in vitro functional assay that can serve as an immune correlate of an antigen activity is an important tool to guide vaccine development. We describe here the development of a quantitative binding assay and its use to study immune responses against PvRII. The assay was used to test anti-PvRII mouse sera, and was found a useful tool for quantitative estimation of anti-PvRII blocking antibodies.


Subject(s)
Antibodies, Protozoan/analysis , Antibodies, Protozoan/immunology , Antigen-Antibody Reactions/immunology , Antigens, Protozoan/immunology , Protozoan Proteins/immunology , Receptors, Cell Surface/immunology , Animals , Antibodies, Blocking/analysis , Antibodies, Blocking/immunology , Antigens, Protozoan/biosynthesis , Antigens, Protozoan/isolation & purification , Binding Sites , Cells, Cultured , HEK293 Cells , Humans , Immune Sera/immunology , Ligands , Mice , Mice, Inbred BALB C , Protozoan Proteins/biosynthesis , Protozoan Proteins/isolation & purification , Rabbits , Receptors, Cell Surface/biosynthesis , Receptors, Cell Surface/isolation & purification , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification
8.
J Pediatr Endocrinol Metab ; 24(7-8): 591-4, 2011.
Article in English | MEDLINE | ID: mdl-21932608

ABSTRACT

A 5-year-old boy was presented for a growth disturbance, which was initially noted at 3 years of age. Endocrinological testing identified severe hypothyroidism, defined by the following levels: TSH 990.5 microU/mL, F-T3 0.26 pg/mL, and F-T4 0.09 ng/dL. Serum anti-thyroid peroxidase (TPO) antibodies were 158 IU/mL and serum thyroid-stimulation blocking antibodies (TSBab) levels were 82.1 IU/mL (normal range < 45.6). Thyroid scintigraphy with 99mTc showed markedly decreased uptake, and magnetic resonance imaging (MRI) revealed pituitary hyperplasia. He was diagnosed with atrophic autoimmune thyroiditis. His thyroid function and pituitary size normalized following thyroid hormone replacement therapy. We report a rare case of a young boy with atrophic thyroiditis caused by TSBab.


Subject(s)
Antibodies, Blocking/analysis , Thyroid Gland/immunology , Thyroiditis, Autoimmune/immunology , Child Development/drug effects , Child, Preschool , Growth Disorders/etiology , Growth Disorders/immunology , Hormone Replacement Therapy , Humans , Hypothyroidism/etiology , Hypothyroidism/immunology , Male , Thyroiditis, Autoimmune/drug therapy , Thyroiditis, Autoimmune/physiopathology , Thyroxine/therapeutic use , Treatment Outcome
9.
Mult Scler ; 16(1): 78-80, 2010 Jan.
Article in English | MEDLINE | ID: mdl-19995836

ABSTRACT

The Interferon Dose Escalation Assessment of Safety extension trial monitored neutralizing antibodies to interferon beta-1b in patients who currently or had previously received the double dose (500 microg) for up to 28 months. Fifteen patients entered the extension trial; five patients were neutralizing antibody-positive at the start of the trial. The present study demonstrates that when neutralizing antibodies develop in patients receiving higher doses of interferon beta-1b they tend to persist for a prolonged period, although neutralizing antibody titers tend to decrease over time and some patients may revert to neutralizing antibody-negative status.


Subject(s)
Antibodies, Blocking/analysis , Interferon-beta/therapeutic use , Multiple Sclerosis/drug therapy , Multiple Sclerosis/immunology , Adult , Disability Evaluation , Dose-Response Relationship, Drug , Female , Humans , Interferon beta-1b , Interferon-beta/administration & dosage , Male , Middle Aged , Neutralization Tests , Patient Dropouts , Treatment Outcome
10.
J Clin Pharmacol ; 50(7): 835-41, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20035038

ABSTRACT

The safety and pharmacokinetics of a single dose of the IL-1 inhibitor, rilonacept (IL-1 Trap; 160 mg, subcutaneously), was studied in a group of 6 patients with well-controlled end-stage renal disease (ESRD) who were observed for a period of 42 days following dosing. The safety of rilonacept administration was ascertained by regular monitoring of patients for adverse events, by periodic determination of a battery of standard laboratory and hematology tests, and by testing for binding and neutralizing antibodies to rilonacept. Two of the 6 patients had treatment-emergent adverse events that were moderate in intensity and unrelated to administration of rilonacept. There were no deaths, serious adverse events, or withdrawals due to adverse events. No patient developed binding or neutralizing antibodies to rilonacept by the 42nd day postdosing. Mean C(max) estimated by a noncompartmental analysis was 17.2 mg/L; t(max,) 2.80 days; terminal t(1/2), 7.63 days; and AUC(0-infinity), 199.3 d.mg/L. Comparison of these results to those obtained in a population of patients with cryopyrin-associated periodic syndromes, a group of rare, inherited, autoinflammatory disorders (mean [SD] eGFR of 73.1 [13.3] mL/min/1.73m2), shows that ESRD and related hemodialysis procedures do not prolong the elimination of rilonacept, and therefore no dose adjustment should be needed relative to individuals with normal renal function.


Subject(s)
Kidney Failure, Chronic/metabolism , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/pharmacokinetics , Adult , Algorithms , Antibodies, Blocking/analysis , Area Under Curve , Cryopyrin-Associated Periodic Syndromes/complications , Female , Half-Life , Humans , Injections, Subcutaneous , Linear Models , Male , Middle Aged , Receptors, Interleukin-1/antagonists & inhibitors , Recombinant Fusion Proteins/immunology , Renal Dialysis
11.
J Clin Endocrinol Metab ; 94(12): 4742-8, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19850692

ABSTRACT

CONTEXT: TSH receptor (TSHR) blocking antibodies (Abs) inhibit TSH-induced thyroid growth and function in some adults with chronic lymphocytic thyroiditis (CLT), but their role in the pediatric age range is unknown. OBJECTIVES: Our objectives were: 1) to determine the prevalence of TSHR blocking Abs in children and adolescents with CLT and 2) assess their functional significance both in vivo and in vitro. DESIGN AND SETTING: This was a retrospective study in a referral outpatient setting. PATIENTS: Sera from a total of 87 CLT patients and 33 controls were studied. MAIN OUTCOME MEASURES: TSHR Abs were measured by both ELISA and bioassay. RESULTS: Eight of 87 children and adolescents with CLT (9.2%), including one as young as 4 yr of age, had TSHR Abs in serum as measured by ELISA. The prevalence was significantly higher in individuals whose serum TSH concentration was 20 mU/liter or greater within 3 months of study than in less hypothyroid patients (eight of 45 vs. none of 42, P < 0.005). Conversely, TSHR Ab-positive patients were significantly more hypothyroid at diagnosis but only when the analysis was restricted to those with severe hypothyroidism was a decreased prevalence of goiter observed. IgG purified from TSHR Ab sera retained the TSH binding-inhibitory activity and TSHR Ab-positive sera inhibited TSH-induced stimulation of cAMP significantly more than normal. CONCLUSIONS: TSHR-blocking Abs contribute significantly to the severity of the hypothyroidism in some children with CLT, but as compared with adults, they appear to play less of a role in determining the presence or absence of a goiter.


Subject(s)
Antibodies, Blocking/analysis , Receptors, Thyrotropin/antagonists & inhibitors , Receptors, Thyrotropin/immunology , Thyroiditis, Autoimmune/immunology , Adolescent , Adult , Child , Child, Preschool , Chronic Disease , Enzyme-Linked Immunosorbent Assay , Female , Humans , Immunoglobulin G/analysis , Immunoglobulin G/biosynthesis , Longitudinal Studies , Male , Thyroid Function Tests , Thyroid Hormones/blood , Thyrotropin/antagonists & inhibitors
13.
Neurology ; 69(14): 1391-403, 2007 Oct 02.
Article in English | MEDLINE | ID: mdl-17761550

ABSTRACT

OBJECTIVE: To determine the incidence and clinical effects of antibodies that develop during treatment with natalizumab. METHODS: In two randomized, double-blind, placebo-controlled studies (natalizumab safety and efficacy in relapsing remitting multiple sclerosis [MS, AFFIRM] and safety and efficacy of natalizumab in combination with interferon beta-1a [INF beta]1a] in patients with relapsing remitting MS [SENTINEL]) of patients with relapsing multiple sclerosis, blood samples were obtained at baseline and every 12 weeks to determine the presence of antibodies against natalizumab. Antibodies to natalizumab were measured using an ELISA. Patients were categorized as "transiently positive" if they had detectable antibodies (>or=0.5 microg/mL) at a single time point or "persistently positive" if they had antibodies at two or more time points >or=6 weeks apart. RESULTS: In the AFFIRM study, antibodies were detected in 57 of 625 (9%) of natalizumab-treated patients: Twenty (3%) were transiently positive and 37 (6%) were persistently positive. Persistently positive patients showed a loss of clinical efficacy as measured by disability progression (p

Subject(s)
Antibodies, Blocking/blood , Antibodies, Blocking/immunology , Antibodies, Monoclonal/adverse effects , Antibodies, Monoclonal/immunology , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Multiple Sclerosis, Relapsing-Remitting/immunology , Antibodies, Blocking/analysis , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal, Humanized , Antibody Specificity/immunology , Brain/drug effects , Brain/immunology , Brain/pathology , Disability Evaluation , Double-Blind Method , Enzyme-Linked Immunosorbent Assay/methods , Flow Cytometry/methods , Humans , Interferon beta-1a , Interferon-beta/administration & dosage , Magnetic Resonance Imaging , Multiple Sclerosis, Relapsing-Remitting/physiopathology , Natalizumab , Placebo Effect , Secondary Prevention , Treatment Outcome
14.
Proc Natl Acad Sci U S A ; 103(51): 19442-7, 2006 Dec 19.
Article in English | MEDLINE | ID: mdl-17158801

ABSTRACT

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.


Subject(s)
Antibodies, Blocking/biosynthesis , Immunotherapy/methods , Inflammation/prevention & control , Joint Diseases/prevention & control , Tumor Necrosis Factor-alpha/immunology , Vaccination/methods , Analysis of Variance , Animals , Antibodies, Blocking/analysis , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Hemocyanins/immunology , Immunoblotting , Inflammation/immunology , Joint Diseases/immunology , Mice , Mice, Transgenic
15.
Clin Vaccine Immunol ; 13(10): 1111-8, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16928883

ABSTRACT

Several human mucosal fluids are known to possess an innate ability to inhibit human immunodeficiency virus type 1 (HIV-1) infection and replication in vitro. This study compared the HIV-1 inhibitory activities of several mucosal fluids, whole, submandibular/sublingual (sm/sl), and parotid saliva, breast milk, colostrum, seminal plasma, and cervicovaginal secretions, from HIV-1-seronegative donors by using a 3-day microtiter infection assay. A wide range of HIV-1 inhibitory activity was exhibited in all mucosal fluids tested, with some donors exhibiting high levels of activity while others showed significantly lower levels. Colostrum, whole milk, and whole saliva possessed the highest levels of anti-HIV-1 activity, seminal fluid, cervicovaginal secretions, and sm/sl exhibited moderate levels, and parotid saliva consistently demonstrated the lowest levels of HIV-1 inhibition. Fast protein liquid chromatography gel filtration studies revealed the presence of at least three distinct peaks of inhibitory activity against HIV-1 in saliva and breast milk. Incubation of unfractionated and fractionated whole saliva with antibodies raised against human lactoferrin (hLf), secretory leukocyte protease inhibitor (SLPI), and, to a lesser extent, MG2 (high-molecular-weight mucinous glycoprotein) reduced the HIV-1 inhibitory activity significantly. The results suggest that hLf and SLPI are two key components responsible for HIV-1 inhibitory activity in different mucosal secretions. The variation in HIV inhibitory activity between the fluids and between individuals suggests that there may be major differences in susceptibility to HIV infection depending both on the individual and on the mucosal fluid involved.


Subject(s)
Anti-HIV Agents/analysis , HIV Infections/immunology , HIV-1/immunology , Immunity, Mucosal , Saliva/immunology , Saliva/virology , Antibodies, Blocking/analysis , Antibodies, Blocking/physiology , Cytotoxicity, Immunologic , HIV Infections/metabolism , HIV Infections/virology , Humans , Lactoferrin/analysis , Lactoferrin/physiology , Saliva/chemistry , Secretory Leukocyte Peptidase Inhibitor/analysis , Secretory Leukocyte Peptidase Inhibitor/physiology
16.
Vaccine ; 22(23-24): 3144-53, 2004 Aug 13.
Article in English | MEDLINE | ID: mdl-15297067

ABSTRACT

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.


Subject(s)
Antibodies, Blocking/biosynthesis , Tumor Necrosis Factor-alpha/immunology , Amino Acid Sequence , Animals , Antibodies, Blocking/analysis , Antibody Formation/immunology , Antibody Specificity , Cross Reactions , Drug Design , Female , Galactosamine/toxicity , Lipopolysaccharides/pharmacology , Mice , Models, Molecular , Molecular Sequence Data , Neutralization Tests , Peptide Fragments/immunology , Shock/chemically induced , Shock/prevention & control , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/biosynthesis
17.
Int Immunopharmacol ; 4(2): 289-98, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14996420

ABSTRACT

The present study was conducted to evaluate the efficacy of paternal lymphocyte (PL) immunotherapy and its relation with the development of mixed lymphocyte reaction blocking antibodies (MLR-Bf) and the success of pregnancy outcome in women with recurrent spontaneous abortion (RSA). A total of 124 women with unknown causes of abortions was registered for immunotherapy under double blind randomized trial by using the list of computer-generated numbers. Each 5 x 10(6) autologous lymphocyte (AL), third party lymphocyte (TPL) and PL was dissolved separately in 1 ml of sterile normal saline (NS). Each 1 ml of cell suspension and neat NS was injected in women with RSA through intramuscular (250 microl), intradermal (250 microl), subcutaneous (250 microl) and intravenous (250 microl) routes. All women participants with RSA received six identical immunizations at the regular interval of 4 weeks, and were then screened for the development of MLR-Bf after the completion of immunization course, and also at the first, second and third trimesters (12th, 24th and 36th weeks) of pregnancy. However, nonimmunized MLR-Bf positive women with RSA did not receive any kind of therapy (NT) and were used as one of the control group in the present study. We have observed that PL-immunized women with RSA showed a significantly increased level of MLR-Bf (>30) and pregnancy success (84%) as compared to those women with RSA who received either AL (33%), TPL (31%), NS (25%) or those who did not receive any kind of treatment (NT, 44%; P<0.001). Our results indicated the importance of immunotherapy with PL in women with RSA and also showed that MLR-Bf can be considered as one of the important factors for pregnancy improvement.


Subject(s)
Abortion, Habitual/prevention & control , Abortion, Habitual/therapy , Antibodies, Blocking/biosynthesis , Fathers , Immunization, Passive , Lymphocyte Transfusion , Abortion, Habitual/immunology , Adult , Antibodies, Blocking/analysis , Double-Blind Method , Female , Humans , Immunization Schedule , Lymphocyte Culture Test, Mixed , Male , Pregnancy , Pregnancy Outcome/epidemiology , Prospective Studies
18.
Neurodegener Dis ; 1(4-5): 160-7, 2004.
Article in English | MEDLINE | ID: mdl-16908985

ABSTRACT

The accumulation of the beta-amyloid peptide (Abeta) is a central event in the pathogenesis of Alzheimer's disease (AD). Abeta removal from the brain by immune therapy shows promising potential for the treatment of patients with AD, although the mechanisms of the antibody action are incompletely understood. In this study we compared the biological activities of antibodies raised against various Abeta fragments for Abeta reduction in vitro and in vivo. Antibodies against Abeta enhanced the uptake of Abeta42 aggregates up to 6-fold by primary microglial cells in vitro. The kinetics of Abeta42 uptake varied considerably among antibodies. Based on the activity to mediate Abeta42 uptake by microglial cells, we identified a bioactive antibody that significantly reduced Abeta42 levels in the brains of transgenic mice with neuronal expression of an AD-related mutated amyloid precursor protein. This effect depended on the epitopes recognized by the antibody. Our data suggest that the ability to facilitate Abeta42 uptake by primary microglia cells in vitro can be used to predict the biological activity of the antibody by passive immunization in vivo. This protocol may prove useful for the rapid validation of the activity of antibodies designed to be used in immune therapy of AD.


Subject(s)
Amyloid beta-Peptides/antagonists & inhibitors , Antibodies, Blocking/pharmacology , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/immunology , Amyloid beta-Peptides/metabolism , Animals , Antibodies, Blocking/analysis , Blotting, Western , Brain Chemistry/drug effects , Cells, Cultured , Epitopes/immunology , Humans , Hybridomas , Immunization, Passive , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microglia/immunology , Microglia/metabolism , Peptide Fragments/immunology , Peptide Fragments/metabolism
19.
J Immunol ; 166(2): 1292-9, 2001 Jan 15.
Article in English | MEDLINE | ID: mdl-11145712

ABSTRACT

Evidence suggests that Pseudomonas aeruginosa stromal keratitis and corneal perforation (susceptibility) is a CD4(+) T cell-regulated inflammatory response following experimental P. aeruginosa infection. This study examined the role of Langerhans cells (LC) and the B7/CD28 costimulatory pathway in P. aeruginosa-infected cornea and the contribution of costimulatory signaling by this pathway to disease pathology. After bacterial challenge, the number of LC infiltrating the central cornea was compared in susceptible C57BL/6 (B6) vs resistant (cornea heals) BALB/c mice. LC were more numerous at 1 and 6 days postinfection (p.i.), but were similar at 4 days p.i., in susceptible vs resistant mice. Mature, B7 positive-stained LC in the cornea and pseudomonas Ag-associated LC in draining cervical lymph nodes also were increased significantly p.i. in susceptible mice. To test the relevance of these data, B6 mice were treated systemically and subconjunctivally with neutralizing B7 (B7-1/B7-2) mAbs. Treatment decreased corneal disease severity and reduced significantly the number of B7-positive cells as well as the recruitment and activation of CD4(+) T cells in the cornea. IFN-gamma mRNA levels also were decreased significantly in the cornea and in draining cervical lymph nodes of mAb-treated mice. When CD28(-/-) animals were tested, they exhibited a less severe disease response (no corneal perforation) than wild-type B6 mice and had a significantly lower delayed-type hypersensitivity response to heat-killed pseudomonas Ag. These results support a critical role for B7/CD28 costimulation in susceptibility to P. aeruginosa ocular infection.


Subject(s)
Antibodies, Blocking/administration & dosage , Antibodies, Monoclonal/administration & dosage , B7-1 Antigen/physiology , CD28 Antigens/physiology , Corneal Diseases/immunology , Pseudomonas Infections/immunology , Animals , Antibodies, Blocking/analysis , Antibodies, Monoclonal/analysis , Antigens, CD/immunology , Antigens, CD/metabolism , Antigens, CD/physiology , Antigens, Differentiation, T-Lymphocyte/analysis , Antigens, Differentiation, T-Lymphocyte/immunology , Apyrase/analysis , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , B7-2 Antigen , CD28 Antigens/genetics , CD28 Antigens/immunology , CD28 Antigens/metabolism , Cell Movement/immunology , Corneal Diseases/enzymology , Corneal Diseases/pathology , Female , Genetic Predisposition to Disease , Injections, Intraperitoneal , Langerhans Cells/enzymology , Langerhans Cells/immunology , Langerhans Cells/pathology , Lymph Nodes/immunology , Lymph Nodes/pathology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Membrane Glycoproteins/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Pseudomonas Infections/enzymology , Pseudomonas Infections/pathology , Reverse Transcriptase Polymerase Chain Reaction , Species Specificity , Staining and Labeling
20.
Br J Pharmacol ; 129(6): 1178-82, 2000 Mar.
Article in English | MEDLINE | ID: mdl-10725266

ABSTRACT

1. Male, Sprague-Dawley rats were actively immunized with novel angiotensin vaccines, and their pressor responses to exogenous angiotensin I (AI) and angiotensin II (AII) were assessed in vivo. Serum antibody titres were also measured. 2. The most effective vaccine consisted of an AI analogue conjugated with a tetanus toxoid carrier protein and adjuvanted with aluminium hydroxide. When this vaccine was injected on days 0, 21 and 42, pressor responses to AI on day 63 were significantly inhibited (maximum, 8.9 fold shift), but responses to AII were unaffected. The anti-angiotensin antibody titre was increased 32,100 fold, and, uniquely, these antibodies also cross-reacted with angiotensinogen. 3. These findings indicate that active immunization against AI may be a useful approach for treating cardiovascular disorders involving the renin-angiotensin system.


Subject(s)
Angiotensin I/immunology , Angiotensin I/pharmacology , Blood Pressure/drug effects , Blood Pressure/immunology , Vaccines/immunology , Algorithms , Angiotensin I/analogs & derivatives , Angiotensin II/analogs & derivatives , Angiotensin II/immunology , Angiotensin II/pharmacology , Angiotensinogen/immunology , Angiotensinogen/pharmacology , Animals , Antibodies, Blocking/analysis , Antibodies, Blocking/immunology , Blotting, Western , Carrier Proteins/immunology , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Male , Rats , Rats, Sprague-Dawley , Serine Proteinase Inhibitors/pharmacology , Vasoconstrictor Agents/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...