RESUMO
BACKGROUND: The immune system is able to recognize substances that originate from inside or outside the body and are potentially harmful. Foreign substances that bind to immune system components exhibit antigenicity and are defined as antigens. The antigens exhibiting immunogenicity can induce innate or adaptive immune responses and give rise to humoral or cell-mediated immunity. The antigens exhibiting mitogenicity can cross-link cell membrane receptors on B and T lymphocytes leading to cell proliferation. All antigens vary greatly in physicochemical features such as biochemical nature, structural complexity, molecular size, foreignness, solubility, and so on. OBJECTIVE: Thus, this review aims to describe the molecular bases of protein-antigenicity and those molecular bases that lead to an immune response, lymphocyte proliferation, or unresponsiveness. CONCLUSION: The epitopes of an antigen are located in surface areas; they are about 880-3,300 Da in size. They are protein, carbohydrate, or lipid in nature. Soluble antigens are smaller than 1 nm and are endocytosed less efficiently than particulate antigens. The more the structural complexity of an antigen increases, the more the antigenicity increases due to the number and variety of epitopes. The smallest immunogens are about 4,000-10,000 Da in size. The more phylogenetically distant immunogens are from the immunogen-recipient, the more immunogenicity increases. Antigens that are immunogens can trigger an innate or adaptive immune response. The innate response is induced by antigens that are pathogen-associated molecular patterns. Exogenous antigens, T Dependent or T Independent, induce humoral immunogenicity. TD protein-antigens require two epitopes, one sequential and one conformational to induce antibodies, whereas, TI non-protein-antigens require only one conformational epitope to induce low-affinity antibodies. Endogenous protein antigens require only one sequential epitope to induce cell-mediated immunogenicity.
Assuntos
Proteínas de Transporte , Linfócitos T , Epitopos , Membrana CelularRESUMO
Over the last two decades, the variable domains from heavy chain-only antibodies in camelids (nanobodies) have emerged as valuable immunoreagents for analytical and diagnostic applications. One prominent use of nanobodies is for the detection of small molecules due to their ease of production, resistance to solvents used in sample extraction, facile genetic manipulation, and small size. These last two properties make it possible to produce biotinylated nanobodies in vivo, which can be loaded in an orientated manner on magnetic beads covered with avidin, creating high-density immunoadsorbenpi twbch ""ts. The method described here details the use of nanobody-based adsorbents to concentrate small molecular weight analytes for subsequent quantitative analysis by MALDI-TOF mass spectrometry. Quantitation requires the inclusion of an internal standard (IS), a compound with properties similar to those of the analyte, enabling compensation for uneven distribution during crystallization of the MALDI-TOF matrix. Since nanobody generation against small compounds requires conjugation to carrier proteins, the same conjugation chemistry can be used to synthesize the IS. By design the IS cross reacts with the capture nanobody and can be preloaded in the immunoadsorbent, facilitating quantitative detection of the target compound.
Assuntos
Anticorpos de Domínio Único , Cadeias Pesadas de Imunoglobulinas , Separação Imunomagnética , Fenômenos Magnéticos , Anticorpos de Domínio Único/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
BACKGROUND: Allergic contact dermatitis (CD) is a chronic inflammatory skin disease caused by type 1 biased adaptive immunity for which there is an unmet need for antigen (Ag)-specific immunotherapies. Exposure to skin sensitizers stimulates secretion of the proinflammatory neuropeptides substance P and hemokinin 1, which signal via the neurokinin-1 receptor (NK1R) to promote the innate and adaptive immune responses of CD. Accordingly, mice lacking the NK1R develop impaired CD. Nonetheless, the role and therapeutic opportunities of targeting the NK1R in CD remain to be elucidated. OBJECTIVE: We sought to develop an Ag-specific immunosuppressive approach to treat CD by skin codelivery of hapten and NK1R antagonists integrated in dissolvable microneedle arrays (MNA). METHODS: In vivo mouse models of contact hypersensitivity and ex vivo models of human skin were used to delineate the effects and mechanisms of NK1R signaling and the immunosuppressive effects of the contact sensitizer NK1R antagonist MNA in CD. RESULTS: We demonstrated in mice that CD requires NK1R signaling by substance P and hemokinin 1. Specific deletion of the NK1R in keratinocytes and dendritic cells, but not in mast cells, prevented CD. Skin codelivery of hapten or Ag MNA inhibited neuropeptide-mediated skin inflammation in mouse and human skin, promoted deletion of Ag-specific effector T cells, and increased regulatory T cells, which prevented CD onset and relapses locally and systemically in an Ag-specific manner. CONCLUSIONS: Immunoregulation by engineering localized skin neuroimmune networks can be used to treat cutaneous diseases that like CD are caused by type 1 immunity.
Assuntos
Dermatite Alérgica de Contato , Antagonistas dos Receptores de Neurocinina-1 , Animais , Dermatite Alérgica de Contato/tratamento farmacológico , Haptenos , Camundongos , Antagonistas dos Receptores de Neurocinina-1/farmacologia , Receptores da Neurocinina-1 , Substância PRESUMO
Visceral leishmaniasis (VL) is epidemic in Brazil with an increasing incidence of human cases and canine reservoirs, with host hypergammaglobulinemia. Conventional enzyme-linked immunosorbent assay (cELISA) based on several parasitic antigens is the main method for diagnosis and indication of treatment. Dissociative ELISA (dELISA) uses acidic treatment to free immunoglobulin G (IgG) from immune complexes, and its use revealed a significant positive fraction of suspected cases with negative serology. Looking for small molecules or haptens that block IgG antibodies, we purified by molecular exclusion chromatography, 1000-3000 MW molecules from promastigote soluble extract, mostly oligosaccharides comprising 6-13 sugar residues using MALDI-TOF analysis. Glycan-BSA complex (GBC) was constructed by conjugating promastigote glycans to BSA molecules, allowing their use in the solid support in cELISA or dELISA. Sera from experimentally infected hamsters showed higher levels of blocked monomeric IgG during infection, mostly against GBC, which was also present in lower concentrations in the promastigote soluble extract dELISA. Those data show that most of the specific monomeric IgG in serum are blocked by haptens composed by glycans produced by the parasite, better detected in the high dilution of sera in the dELISA assays. dELISA is a useful technique for detecting blocked monomeric antibodies that could have difficult clearance from blood, which could result in hypergammaglobulinemia.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Leishmaniose Visceral/imunologia , Polissacarídeos/sangue , Animais , Brasil/epidemiologia , Cricetinae , Modelos Animais de Doenças , Epidemias , Humanos , Concentração de Íons de Hidrogênio , Hipergamaglobulinemia , Leishmaniose Visceral/epidemiologia , Polissacarídeos/metabolismo , Soroalbumina Bovina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The variable VHH domains of camelid single chain antibodies have been useful in numerous biotechnology applications due to their simplicity, biophysical properties, and abilities to bind to their cognate antigens with high affinities and specificity. Their interactions with proteins have been well-studied, but considerably less work has been done to characterize their ability to bind haptens. A high-resolution structural study of three nanobodies (T4, T9, and T10) which have been shown to bind triclocarban (TCC, 3-(4-chlorophenyl)-1-(3,4-dichlorophenyl)urea) with near-nanomolar affinity shows that binding occurs in a tunnel largely formed by CDR1 rather than a surface or lateral binding mode seen in other nanobody-hapten interactions. Additional significant interactions are formed with a non-hypervariable loop, sometimes dubbed "CDR4". A comparison of apo and holo forms of T9 and T10 shows that the binding site undergoes little conformational change upon binding of TCC. Structures of three nanobody-TCC complexes demonstrated there was not a standard binding mode. T4 and T9 have a high degree of sequence identity and bind the hapten in a nearly identical manner, while the more divergent T10 binds TCC in a slightly displaced orientation with the urea moiety rotated approximately 180° along the long axis of the molecule. In addition to methotrexate, this is the second report of haptens binding in a tunnel formed by CDR1, suggesting that compounds with similar hydrophobicity and shape could be recognized by nanobodies in analogous fashion. Structure-guided mutations failed to improve binding affinity for T4 and T9 underscoring the high degree of natural optimization.