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1.
J Pharm Sci ; 107(7): 1820-1830, 2018 07.
Article in English | MEDLINE | ID: mdl-29571738

ABSTRACT

Monoclonal antibodies (mAbs) represent a major class of biotherapeutics and are the fastest growing category of biologic drugs on the market. However, mAb development and formulation are often impeded by reversible self-association (RSA), defined as the dynamic exchange of monomers with native-state oligomers. Here, we present a comparative analysis of the self-association properties for 5 IgG mAbs, under matched conditions and using orthogonal methods. Concentration-dependent dynamic light scattering and sedimentation velocity studies revealed that the majority of mAbs examined exhibited weak to moderate RSA. However, because these studies were carried out at mAb concentrations in the mg/mL range, we also observed significant nonideality. Noting that nonideality frequently masks RSA and vice versa, we conducted direct boundary fitting of the sedimentation velocity data to determine stoichiometric binding models, interaction affinities, and nonideality terms for each mAb. These analyses revealed equilibrium constants from micromolar to millimolar and stoichiometric models from monomer-dimer to isodesmic. Moreover, even for those mAbs described by identical models, we observed distinct kinetics of self-association. The accuracy of the models and their corresponding equilibrium constants were addressed using sedimentation equilibrium and simulations. Overall, these results serve as the starting point for the comparative dissection of RSA mechanisms in therapeutic mAbs.


Subject(s)
Antibodies, Monoclonal/chemistry , Immunoglobulin G/chemistry , Algorithms , Dynamic Light Scattering , Humans , Protein Multimerization , Thermodynamics , Ultracentrifugation
2.
J Infect Dis ; 203(9): 1240-8, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21357942

ABSTRACT

BACKGROUND: Cigarette smoke (CS) exposure is an epidemiological risk factor for tuberculosis, although the biological basis has not been elucidated. METHODS: We exposed C57BL/6 mice to CS for 14 weeks and examined their ability to control an aerosol infection of Mycobacterium tuberculosis Erdman. RESULTS: CS-exposed mice had more M. tuberculosis isolated from the lungs and spleens after 14 and 30 d, compared with control mice. The CS-exposed mice had worse lung lesions and less lung and splenic macrophages and dendritic cells (DCs) producing interleukin12 and tumor necrosis factor α (TNF-α). There were significantly more interleukin 10-producing macrophages and DCs in the spleens of infected CS-exposed mice than in non-CS-exposed controls. CS-exposed mice also showed a diminished influx of interferon γ-producing and TNF-α-producing CD4(+) and CD8(+) effector and memory T cells into the lungs and spleens. There was a trend toward an increased number of viable intracellular M. tuberculosis in macrophages isolated from humans who smoke compared with nonsmokers. THP-1 human macrophages and primary human alveolar macrophages exposed to CS extract, nicotine, or acrolein showed an increased burden of intracellular M. tuberculosis. CONCLUSION: CS suppresses the protective immune response to M. tuberculosis in mice, human THP-1 cells, and primary human alveolar macrophages.


Subject(s)
Disease Susceptibility , Mycobacterium tuberculosis/immunology , Smoking/adverse effects , Tuberculosis/immunology , Animals , Disease Models, Animal , Female , Mice , Mice, Inbred C57BL
3.
Gastroenterology ; 139(5): 1753-61, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20659472

ABSTRACT

BACKGROUND & AIMS: Biliary atresia (BA) is a neonatal cholangiopathy of unknown etiology. The bile duct injury that occurs in patients with BA might result from a hepatobiliary viral infection followed by an autoimmune response against the bile duct epithelia. We aimed to identify autoantigens recognized by serum antibodies in the Rhesus rotavirus (RRV)-induced mouse model of BA; findings were correlated with BA in humans. METHODS: Bile duct epithelial proteins were screened for their reactivity with serum antibodies from the mouse model of BA using immunoblot assays. Unique proteins that reacted with sera antibodies were identified by mass spectrometry and verified using enzyme-linked immunosorbent assay (ELISA) and immunoblot analyses. Candidate autoantibodies in BA patient sera were analyzed by ELISA. RESULTS: A bile duct epithelial antigen that reacted strongly with serum immunoglobulin (Ig) G from the mouse model of BA was identified as α-enolase. α-Enolase autoantibody specificity was confirmed by ELISA and immunoblot analyses. Anti-RRV and anti-enolase antibodies cross-reacted with enolase and RRV proteins; we identified regions of sequence homology between RRV and enolase. Serum samples from patients with BA had increased levels of anti-enolase IgM and IgG. CONCLUSIONS: We have identified autoantibodies against α-enolase in a mouse model of BA (infected with RRV) and in serum samples from patients, indicating a role of humoral autoimmunity in disease pathogenesis. The cross-reactivity between an anti-enolase antibody and RRV proteins indicates that molecular mimicry might activate humoral autoimmunity in BA patients; further studies are required.


Subject(s)
Autoantibodies/immunology , Biliary Atresia/immunology , Immunity, Humoral , Phosphopyruvate Hydratase/immunology , Animals , Animals, Newborn , Biliary Atresia/enzymology , Biliary Atresia/pathology , Cell Line , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Humans , Immunoblotting , Mice , Mice, Inbred BALB C , Phosphopyruvate Hydratase/metabolism , Risk Factors
4.
Am J Respir Crit Care Med ; 179(11): 1011-21, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19264974

ABSTRACT

RATIONALE: Cigarette smoke (CS) is the primary cause of chronic obstructive pulmonary disease (COPD), an effect that is, in part, due to intense oxidant stress. Clearance of apoptotic cells (efferocytosis) is a critical regulator of lung homeostasis, which is defective in smokers and in patients with COPD, suggesting a role in disease pathogenesis. OBJECTIVES: We hypothesized that CS would impair efferocytosis through oxidant-dependent activation of RhoA, a known inhibitor of this process. METHODS: We investigated the effect of CS on efferocytosis in vivo and ex vivo, using acute, subacute, and long-term mouse exposure models. MEASUREMENTS AND MAIN RESULTS: Acute and subacute CS exposure suppressed efferocytosis by alveolar macrophages in a dose-dependent, reversible, and cell type-independent manner, whereas more intense CS exposure had an irreversible effect. In contrast, CS did not alter ingestion through the Fc gamma receptor. The inhibitory effect of CS on apoptotic cell clearance depended on oxidants, because the effect was blunted in oxidant-resistant ICR mice, and was prevented by either genetic or pharmacologic antioxidant strategies in vivo and ex vivo. CS inhibited efferocytosis through oxidant-dependent activation of the RhoA-Rho kinase pathway because (1) CS activated RhoA, (2) antioxidants prevented RhoA activation by CS, and (3) inhibitors of the RhoA-Rho kinase pathway reversed the suppressive effect of CS on apoptotic cell clearance in vivo and ex vivo. CONCLUSIONS: These findings advance the hypothesis that impaired efferocytosis may contribute to the pathogenesis of COPD and suggest the therapeutic potential of drugs targeting the RhoA-Rho kinase pathway.


Subject(s)
Apoptosis , Macrophages, Alveolar/drug effects , Phagocytosis/drug effects , Smoking/physiopathology , rho GTP-Binding Proteins/drug effects , Animals , Cell Line, Tumor , Humans , Macrophages, Alveolar/immunology , Male , Mice , Mice, Inbred ICR , Mice, Knockout , Neutrophils , Oxidative Stress/drug effects , Oxidative Stress/immunology , Pulmonary Disease, Chronic Obstructive/physiopathology , Signal Transduction/drug effects , Smoking/immunology , Tumor Necrosis Factor-alpha , rho GTP-Binding Proteins/metabolism , rho-Associated Kinases/drug effects , rhoA GTP-Binding Protein
5.
J Biol Chem ; 282(27): 19666-75, 2007 Jul 06.
Article in English | MEDLINE | ID: mdl-17491020

ABSTRACT

Cigarette smoke is a potent inhibitor of pulmonary T cell responses, resulting in decreased immune surveillance and an increased incidence of respiratory tract infections. The alpha,beta-unsaturated aldehydes in cigarette smoke (acrolein and crotonaldehyde) inhibited production of interleukin-2 (IL-2), IL-10, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha by human T cells but did not inhibit production of IL-8. The saturated aldehydes (acetaldehyde, propionaldehyde, and butyraldehyde) in cigarette smoke were inactive. Acrolein inhibited induction of NF-kappaB DNA binding activity after mitogenic stimulation of T cells but had no effect on induction of NFAT or AP-1. Acrolein inhibited NF-kappaB1 (p50) binding to the IL-2 promoter in a chromatin immunoprecipitation assay by >99%. Using purified recombinant p50 in an electrophoretic mobility shift assay, we demonstrated that acrolein was 2000-fold more potent than crotonaldehyde in blocking DNA binding to an NF-kappaB consensus sequence. Matrix-assisted laser desorption/ionization time-of-flight and tandem mass spectrometry demonstrated that acrolein alkylated two amino acids (Cys-61 and Arg-307) in the DNA binding domain. Crotonaldehyde reacted with Cys-61, but not Arg-307, whereas the saturated aldehydes in cigarette smoke did not react with p50. These experiments demonstrate that aldehydes in cigarette smoke can regulate gene expression by direct modification of a transcription factor.


Subject(s)
Acrolein/pharmacology , DNA/metabolism , Gene Expression Regulation/drug effects , NF-kappa B p50 Subunit/metabolism , T-Lymphocytes/metabolism , Aldehydes/pharmacology , Arginine/immunology , Arginine/metabolism , Cells, Cultured , Cysteine/immunology , Cysteine/metabolism , Cytokines/biosynthesis , Cytokines/immunology , DNA/immunology , Humans , Lung/immunology , Lung/metabolism , NF-kappa B p50 Subunit/immunology , NFATC Transcription Factors/immunology , NFATC Transcription Factors/metabolism , Protein Binding , Respiratory Tract Infections/immunology , Respiratory Tract Infections/metabolism , Smoke/adverse effects , T-Lymphocytes/immunology , Nicotiana/adverse effects , Transcription Factor AP-1/immunology , Transcription Factor AP-1/metabolism
6.
Mol Immunol ; 44(4): 488-93, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16584776

ABSTRACT

Cigarette smoking causes profound suppression of pulmonary T cell responses, which is associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. We previously demonstrated that the phenolic compounds in cigarette tar inhibit blastogenesis and interfere with human T cell cycle progression. To identify the mechanism by which cell cycle arrest occurs, we examined the effects of these compounds on cyclin-dependent kinases (Cdk) that control the G0/G1 transition. We found that hydroquinone inhibited induction of Cdk4 and Cdk6 kinase activities by >80%, while catechol and phenol were markedly less potent. HQ did not affect mitogenic induction of the Cdk6 protein, but inhibited expression of cyclin D3 by >90% resulting in a dramatic reduction in proper Cdk6/Cyclin D3 complex formation.


Subject(s)
Cyclin-Dependent Kinase 4/antagonists & inhibitors , Cyclin-Dependent Kinase 6/antagonists & inhibitors , Smoking/adverse effects , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tars/toxicity , Cell Cycle/drug effects , Cell Cycle/immunology , Cell Proliferation/drug effects , Cells, Cultured , Cyclin-Dependent Kinase 4/immunology , Cyclin-Dependent Kinase 6/immunology , Enzyme Induction/drug effects , G1 Phase , Humans , Lymphocyte Activation/drug effects , Phenols/toxicity , Resting Phase, Cell Cycle , T-Lymphocytes/cytology
7.
J Allergy Clin Immunol ; 116(4): 916-22, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16210070

ABSTRACT

BACKGROUND: Cigarette smoking inhibits T-cell responses in the lungs, but the immunosuppressive compounds have not been fully identified. Cigarette smoke extracts inhibit IL-2, IFN-gamma, and TNF-alpha production in stimulated lymphocytes obtained from peripheral blood, even when the extracts were diluted 100-fold to 1000-fold. OBJECTIVE: The objective of these studies was to identify the immunosuppressive compounds found in cigarette smoke. METHODS: Gas chromatography/mass spectroscopy and HPLC were used to identify and quantitate volatile compounds found in cigarette smoke extracts. Bioactivity was measured by viability and production of cytokine mRNA and protein levels in treated human lymphocytes. RESULTS: The vapor phase of the cigarette smoke extract inhibited cytokine production, indicating that the immunosuppressive compounds were volatile. Among the volatile compounds identified in cigarette smoke extracts, only the alpha,beta-unsaturated aldehydes, acrolein (inhibitory concentration of 50% [IC50] = 3 micromol/L) and crotonaldehyde (IC50 = 6 micromol/L), exhibited significant inhibition of cytokine production. Although the levels of aldehydes varied 10-fold between high-tar (Camel) and ultralow-tar (Carlton) extracts, even ultralow-tar cigarettes produced sufficient levels of acrolein (34 micromol/L) to suppress cytokine production by >95%. We determined that the cigarette smoke extract inhibited transcription of cytokine genes. The inhibitory effects of acrolein could be blocked with the thiol compound N-acetylcysteine. CONCLUSION: The vapor phase from cigarette smoke extracts potently suppresses cytokine production. The compound responsible for this inhibition appears to be acrolein.


Subject(s)
Acrolein/toxicity , Nicotiana/adverse effects , Smoke/adverse effects , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Acetylcysteine/pharmacology , Acrolein/antagonists & inhibitors , Acrolein/isolation & purification , Adult , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Gene Expression/drug effects , Humans , Immunosuppressive Agents/antagonists & inhibitors , Immunosuppressive Agents/toxicity , In Vitro Techniques , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin-2/biosynthesis , Interleukin-2/genetics , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Smoke/analysis , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
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