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1.
Food Chem ; 443: 138539, 2024 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-38320375

RESUMO

Quinoxalines are a class of veterinary drugs with antibacterial and growth-promoting functions. They are often widely used to treat and prevent animal diseases and are illegally used as animal growth promoters to increase economic benefits. Quinoxalines could be easily metabolized in animals to various residue markers and remain in animal-derived foods, which would pose a serious threat to human health. Consequently, it is necessary to detect the residues of quinoxalines and their metabolites. This article reviewed and evaluated immunoassays for quinoxalines and their metabolites in animal-derived foods, mainly including enzyme-linked immunosorbent assays, fluorescence immunosorbent assays, immunochromatography, and surface plasmon resonance biosensors. In addition, we deeply explored the design of haptens for quinoxalines and their metabolites and analyzed the effect of haptens on antibody performance. This paper aims to provide guidance and references for their accurate and sensitive detection, thereby ensuring food safety and human public health.


Assuntos
Anticorpos , Quinoxalinas , Animais , Humanos , Quinoxalinas/análise , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio , Haptenos/química
2.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 40(2): 163-167, 2024 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-38284257

RESUMO

Objective To synthesize carbendazim artificial antigens, prepare carbendazim polyclonal antibodies and identify their characteristics. Methods Active carboxyl groups were introduced to prepare the carbendazim haptens by the mixed anhydride method. The artificial antigens and coating antigens were obtained by coupling the small molecule haptens with carriers of bovine serum albumin (BSA) and ovalbumin (OVA). Sodium dodecyl sulfate polycrylamide gel electropheresis (SDS-PAGE) was used to identify carbendazim artificial antigens. Mice were immunized with the prepared artificial antigens to obtain polyclonal antibodies against carbendazim, and the antibody titers and specificity were identified by indirect ELISA. Results Carbendazim artificial antigens were successfully prepared. The titer of polyclonal antibody was above 1:12 800 and the half-maximal inhibitory concentration ( IC50) of the antibody was 0.107 µg/mL. The cross-reactivity rates with both benomyl and thiabendazole were less than 1%. Conclusion Polyclonal antibodies with high sensitivity and high specificity were successfully prepared, laying the foundation for the establishment of a rapid detection method for carbendazim residues.


Assuntos
Anticorpos , Antígenos , Benzimidazóis , Carbamatos , Animais , Camundongos , Ensaio de Imunoadsorção Enzimática , Antígenos/química , Haptenos/química , Soroalbumina Bovina/química , Vacinas Sintéticas , Especificidade de Anticorpos
3.
BMC Vet Res ; 20(1): 15, 2024 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-38184593

RESUMO

Fish live in an aquatic environment rich in various microorganisms and pathogens. Fish mucosal-associated lymphoid tissue (MALT) plays a very important role in immune defence. This study was conducted to characterize the immune response mediated by CcIgZ3 in common carp (Cyprinus carpio.) and investigate the proliferating CcIgZ3+ B lymphocytes in gill. We determined the expression of CcIgZ3 in many different tissues of common carp following stimulation by intraperitoneal injection of TNP-LPS (2,4,6-Trinitrophenyl hapten conjugated to lipopolysaccharide) or TNP-KLH (2,4,6-Trinitrophenyl hapten conjugated to Keyhole Limpet Hemocyanin). Compared with TNP-KLH, TNP-LPS can induce greater CcIgZ3 expression in the head kidney, gill and hindgut, especially in the gill. The results indicate that the gill is one of the main sites involved in the immune response mediated by CcIgZ3. To examine the distribution of CcIgZ3+ B lymphocytes, immunohistochemistry (IHC) experiments were performed using a polyclonal antibody against CcIgZ3. The results indicated that CcIgZ3 was detected in the head kidney, hindgut and gill. To further examine whether CcIgZ3+ B lymphocytes proliferate in the gills, proliferating CcIgZ3+ B cells were analysed by immunofluorescence staining using an anti-CcIgZ3 polyclonal antibody and an anti-PCNA monoclonal antibody. CcIgZ3 and PCNA (Proliferating Cell Nuclear Antigen) double-labelled cells in the gills were located within the epithelial cells of the gill filaments of common carp stimulated with TNP-LPS at 3 dps and 7 dps, and relatively more proliferating CcIgZ3+ B cells appeared in the gills of common carp at 7 dps. These data imply that CcIgZ3+ B cells in the gills might be produced by local proliferation following TNP-LPS stimulation. In summary, compared with those in TNP-KLH, CcIgZ3 preferentially affects the gills of common carp following challenge with TNP-LPS. CcIgZ3+ B cells proliferate in the gills to quickly produce the CcIgZ3 antibody. In addition, CcIgZ3+ B cells can be activated to induce a strong immune response very early locally in the gill and produce the antibody CcIgZ3, which helps exert an immune-protective effect. These results suggest that an effective vaccine can be designed to promote production of the mucosal antibody CcIgZ3.


Assuntos
Carpas , Animais , Antígeno Nuclear de Célula em Proliferação , Brânquias , Lipopolissacarídeos/farmacologia , Anticorpos , Haptenos , Imunidade
4.
Proc Natl Acad Sci U S A ; 121(4): e2315592121, 2024 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-38227652

RESUMO

γδ T cells are essential for immune defense and modulating physiological processes. While they have the potential to recognize large numbers of antigens through somatic gene rearrangement, the antigens which trigger most γδ T cell response remain unidentified, and the role of antigen recognition in γδ T cell function is contentious. Here, we show that some γδ T cell receptors (TCRs) exhibit polyspecificity, recognizing multiple ligands of diverse molecular nature. These ligands include haptens, metabolites, neurotransmitters, posttranslational modifications, as well as peptides and proteins of microbial and host origin. Polyspecific γδ T cells are enriched among activated cells in naive mice and the responding population in infection. They express diverse TCR sequences, have different functional potentials, and include the innate-like γδ T cells, such as the major IL-17 responders in various pathological/physiological conditions. We demonstrate that encountering their antigenic microbiome metabolite maintains their homeostasis and functional response, indicating that their ability to recognize multiple ligands is essential for their function. Human γδ T cells with similar polyspecificity also respond to various immune challenges. This study demonstrates that polyspecificity is a prevalent feature of γδ T cell antigen recognition, which enables rapid and robust T cell responses to a wide range of challenges, highlighting a unique function of γδ T cells.


Assuntos
Antígenos de Grupos Sanguíneos , Receptores de Antígenos de Linfócitos T gama-delta , Humanos , Camundongos , Animais , Antígenos , Haptenos
5.
Contact Dermatitis ; 90(2): 110-115, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-37840032

RESUMO

BACKGROUND: Preservatives are a frequent cause of allergic contact dermatitis (ACD) and have caused numerous epidemics. OBJECTIVES: The objective of this study is to determine the prevalence of preservative sensitivity, assess the change in the frequency of sensitivity, identify new preservatives with increased sensitivity rates, and evaluate the situation in Turkey by comparing our findings with current literature. METHODS: A total of 201 patients diagnosed with ACD between 2018 and 2020, were patch tested with the European baseline series and additional seven preservative haptens. The change in the prevalence of sensitivity to each preservative hapten was investigated by comparing the data from the study conducted in our department between 2000 and 2004. RESULTS: Results showed that 17.4% (n = 35) of the patients were positive to preservatives. Comparison with previous data from 2000 to 2004 revealed an increase in the frequency of sensitization. The most prevalent allergen was methyldibromo glutaronitrile (9.5%), followed by methylchloroisothiazolinone/methylisothiazolinone (6.5%), and methylisothiazolinone (5%). CONCLUSION: The increase in preservative sensitivity in Turkey is the most remarkable finding. Although MDBGN was prohibited in cosmetic products, MCI/MI and MI are still widely used. Our findings suggest that awareness of preservative sensitivity should be increased and additional precautions should be taken, also in Turkey, regarding the use of preservatives.


Assuntos
Dermatite Alérgica de Contato , Conservantes Farmacêuticos , Humanos , Alérgenos/efeitos adversos , Cosméticos , Dermatite Alérgica de Contato/epidemiologia , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/diagnóstico , Haptenos , Nitrilas , Testes do Emplastro/métodos , Conservantes Farmacêuticos/efeitos adversos , Tiazóis , Turquia/epidemiologia
6.
Contact Dermatitis ; 90(3): 211-234, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37852624

RESUMO

BACKGROUND: Chemical-induced allergies at workplace represent a significant occupational health issue. These substances must be properly identified as sensitizers. In previous studies, an original model using mouse bone marrow-derived dendritic cells (BMDC) was developed for this purpose. OBJECTIVES: The aim of this study was to evaluate the predictive capacity of the BMDC model with a large panel of sensitizers (including pre- and pro-haptens) and non-sensitizers. METHODS: The readout from the BMDC model is based on expression levels of six phenotypic markers measured by flow cytometry. RESULTS: The results indicate that 29 of the 37 non-sensitizers, and 81 of the 86 sensitizers were correctly classified compared to the Local Lymph Node Assay (LLNA). Statistical analysis revealed the BMDC model to have a sensitivity of 94%, a specificity of 78%, and an accuracy of 89%. The EC2 (Effective Concentration) values calculated with this model allow sensitizers to be categorized into four classes: extreme, strong, moderate and weak. CONCLUSIONS: These excellent predictive performances show that the BMDC model discriminates between sensitizers and non-sensitizers with outstanding precision equal to or better than existing validated alternative models. Moreover, this model allows to predict sensitization potency of chemicals. The BMDC test could therefore be proposed as an additional tool to assess the sensitizing potential and potency of chemicals.


Assuntos
Dermatite Alérgica de Contato , Camundongos , Animais , Dermatite Alérgica de Contato/diagnóstico , Dermatite Alérgica de Contato/etiologia , Haptenos , Ensaio Local de Linfonodo , Citometria de Fluxo , Alérgenos/efeitos adversos
7.
Allergy ; 79(1): 52-64, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37539746

RESUMO

BACKGROUND: Tissue-resident memory T (TRM ) cells are detrimental in allergic contact dermatitis (ACD), in which they contribute to the chronicity and severity of the disease. METHODS: We assessed the impact of a standard topical corticosteroid (TCS) treatment, triamcinolone acetonide (TA), on the formation, maintenance and reactivation of epidermal TRM cells in a preclinical model of ACD to 2,4-dinitrofluorobenzene. TA 0.01% was applied at different time points of ACD response and we monitored skin inflammation and tracked CD8+ CD69+ CD103+ TRM by flow cytometry and RNA sequencing. RESULTS: The impact of TA on TRM formation depended on treatment regimen: (i) in a preventive mode, that is, in sensitized mice before challenge, TA transiently inhibited the infiltration of effector T cells and the accumulation of TRM upon hapten challenge. In contrast, (ii) in a curative mode, that is, at the peak of the ACD response, TA blocked skin inflammation but failed to prevent the formation of TRM . Finally, (iii) in a proactive mode, that is, on previous eczema lesions, TA had no effect on the survival of skin TRM , but transiently inhibited their reactivation program upon allergen reexposure. Indeed, specific TRM progressively regained proliferative functions upon TA discontinuation and expanded in the tissue, leading to exaggerated iterative responses. Interestingly, TRM re-expansion correlated with the decreased clearance of hapten moieties from the skin induced by repeated TA applications. CONCLUSIONS: Our results demonstrate that TCS successfully treat ACD inflammation, but are mostly ineffective in impeding the formation and expansion of allergen-specific TRM , which certainly restricts the induction of lasting tolerance in patients with chronic dermatitis.


Assuntos
Dermatite Alérgica de Contato , Dermatite Atópica , Fármacos Dermatológicos , Humanos , Camundongos , Animais , Células T de Memória , Linfócitos T CD8-Positivos , Pele/patologia , Dermatite Alérgica de Contato/tratamento farmacológico , Dermatite Atópica/tratamento farmacológico , Dermatite Atópica/patologia , Alérgenos , Inflamação/tratamento farmacológico , Inflamação/patologia , Haptenos , Corticosteroides , Memória Imunológica
8.
J Hazard Mater ; 465: 133221, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38103295

RESUMO

Contamination in food and the environment with fluoroquinolones (FQs) has become a serious threat to the global ecological balance and public health safety. Ofloxacin (OFL) is one of the most widely utilized sterilization agents in FQs. In the process of monitoring OFL, broad-spectrum monoclonal antibodies (mAb) cannot meet the demand for monospecific detection. Here, a computational chemistry-assisted hapten screening strategy was proposed in this study. Differences in the properties of antigenic epitopes were precisely extracted through a comprehensive comparative study of 16 common FQs molecules and a monospecific and ultrasensitive mAb-3B4 for OFL was successfully prepared. The screened fleroxacin (FLE) hapten was applied in a heterologous competition strategy resulting in a 20-fold improvement in the half inhibitory concentration (IC50) of mAb-3B4 to 0.0375 µg L-1 and cross-reacted only with marbofloxacin (MAR) in regulated FQs. In addition, a single-chain variable fragment (scFv) for OFL was constructed for the first time with an IC50 of 0.378 µg L-1. Molecular recognition mechanism studies validated the reliability of this strategy and revealed the key amino acid sites responsible for OFL specificity and sensitivity. Finally, ic-ELISA and GICA were established for OFL in real samples. This work provides new ideas for the preparation of monospecific mAb and improves the monitoring system of FQs.


Assuntos
Química Computacional , Ofloxacino , Reprodutibilidade dos Testes , Fluoroquinolonas , Ensaio de Imunoadsorção Enzimática , Haptenos , Antibacterianos/química
9.
J Immunol Methods ; 525: 113604, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38142928

RESUMO

Hapten design and synthesis have been regarded as the key factor to generate high-quality antibodies. In the present study, a novel hapten of chloramphenicol was synthesized, characterized and compared with two conventional haptens. The new hapten generated mAb 4B5 showed higher sensitivity and titer than the other two haptens-based mAbs. The haptens synthesized with the structure of chloramphenicol base generated more sensitive antibodies than the hapten with chloramphenicol succinate, and the spacer arm linked to the phenyl group hapten elicited the strongest antibody response. After optimization, a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA), both based on the mAb 4B5, were developed. The dcELISA had a half maximum inhibition concentration of 0.23 ng/mL and the LFIA showed a cutoff value of 5-10 ng/mL. The LFIA was applied to detect illegally-added chloramphenicol samples in anti-acne cosmetics, five out of 19 samples were tested chloramphenicol containing within 10 min, which result was confirmed with the dcELISA and HPLC. The LFIA has an adequate sensitivity and can be used as a point of care diagnostic device for rapidly screening chloramphenicol in cosmetics.


Assuntos
Anticorpos Monoclonais , Formação de Anticorpos , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Cloranfenicol , Haptenos/química
10.
Mikrochim Acta ; 191(1): 42, 2023 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-38114730

RESUMO

To avoid false negative results due to the low cross-reactivity rate (CR) in rapid immunoassay, a group-specific antibody with homogeneous CR toward target compounds is needed for accuracy. In this study, tylosin (TYL) and tilmicosin (TM) were selected as model molecules. Firstly, two-dimensional similarity, electrostatic potential energy, spatial conformation and charge distribution of the haptens TYL-CMO, TYL-6-ACA, TYL-4-APA, TYL-CHO and DES-CMO and target compounds of TYL and TM were obtained using Gaussian 09W and Discovery Studio. The optimal hapten was DES-CMO because it is the most similar to TYL and TM. Subsequently, the mAb 14D5 cell line was obtained with IC50 values of 1.59 and 1.72 ng/mL for TYL and TM, respectively, and a CR of 92.44%. Finally, amorphous carbon nanoparticles (ACNPs) were conjugated with mAb 14D5 to develop an accurate lateral flow immunoassay (LFA) for detection of TYL and TM by the reflectance value under natural light. The recoveries of TYL and TM ranged from 77.18 to 112.04% with coefficient of variation < 13.43%. The cut-off value in milk samples was 8 ng/mL, and the limits of detection were 11.44, 15.96, 22.29 and 25.53 µg/kg for chicken muscle, bovine muscle, porcine muscle and porcine liver samples, respectively, and the results being consistent with HPLC-UV. The results suggest that the developed LFA is accurate and potentially useful for on-site screening of TYL and TM in milk and animal tissue samples.


Assuntos
Anticorpos Monoclonais , Tilosina , Animais , Bovinos , Suínos , Ensaio de Imunoadsorção Enzimática/métodos , Imunoensaio , Haptenos
11.
Biosensors (Basel) ; 13(10)2023 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-37887114

RESUMO

Macrolide antibiotics, which are effective antimicrobial agents, are intensively used in human and veterinary medicine, as well as in agriculture. Consequently, they are found all over the world as environmental pollutants, causing harm to sensitive ecological communities and provoking a selection of resistant forms. A novel azithromycin derivative, which was used as hapten conjugate, ensured the group immunorecognition of six major macrolide representatives (105-41%), namely erythromycin, erythromycin ethylsuccinate, clarithromycin, roxithromycin, azithromycin, and dirithromycin in a competitive immunoassay based on anti-clarithromycin antibodies. The heterologous hapten-based ELISA format resulted in a 5-fold increase in sensitivity, with an IC50 value of 0.04 ng/mL for erythromycin. In this study, we proposed an underexploited strategy in an immunoassay field to significantly improve the detectability of analytes in environmental samples. Unlike most approaches, it does not require special enhancers/amplifiers or additional concentration/extraction procedures; instead, it involves analyzing a larger volume of test samples. A gradual volume increase in the samples (from 0.025 to 10 mL) analyzed using a direct competitive ELISA, immunobeads, and immunofiltration assay formats based on the same reagents resulted in a significant improvement (more than 50-fold) in assay sensitivity and detection limit up to 5 and 1 pg/mL, respectively. The suitability of the test for detecting the macrolide contamination of natural water was confirmed by the recovery of macrolides from spiked blank samples (71.7-141.3%). During 2022-2023, a series of natural water samples from Lake Onega and its influents near Petrozavodsk were analyzed, using both the developed immunoassay and HPLC-MS/MS. The results revealed no contamination of macrolide antibiotic.


Assuntos
Azitromicina , Espectrometria de Massas em Tandem , Humanos , Azitromicina/análise , Antibacterianos/análise , Macrolídeos , Eritromicina/análise , Haptenos , Água
12.
Langmuir ; 39(41): 14586-14594, 2023 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-37792480

RESUMO

The biopanning of target-specific phages is one of the most critical steps in the preparation of single-domain antibodies. In the traditional biopanning of haptens, the nonspecific binding of library phages to macromolecular proteins is one of the most challenging problems in preparing single-domain antibodies. In this research, Fe3O4@ENR-functionalized core-shell magnetic nanoparticles (FMNPs) were silylated and aminated by tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane, and target enrofloxacin was coupled onto the surface by the carbodiimide method. The magnetic nanoparticles were characterized by Fourier transform infrared spectroscopy, particle size distribution, zeta potential, transmission electron microscopy observation, and indirect enzyme-linked immunosorbent assay (ELISA). A biopanning strategy based on Fe3O4@ENR FMNPs was then established to solve the problem in the traditional solid-phase biopanning process. The results showed that a considerable number of enrofloxacin (ENR)-positive phages were screened by only one round of biopanning. Finally, two ENR-specific shark-derived single-domain genes were identified and validated by monoclonal phage ELISA, gene sequencing, and biolayer interferometry technology. Our study provides a new biopanning strategy based on Fe3O4@ENR FMNPs for efficiently providing phages specific to haptens.


Assuntos
Nanopartículas de Magnetita , Anticorpos de Domínio Único , Enrofloxacina , Nanopartículas de Magnetita/química , Bioprospecção , Haptenos
13.
J Agric Food Chem ; 71(41): 14967-14978, 2023 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-37803933

RESUMO

The synthesis of a hapten and antigen for the preparation of a monoclonal antibody (mAb) for buprofezin is described. The recognition mechanism of hapten and buprofezin by monoclonal antibodies (mAb-19F2) is described. The effectiveness of the mAb-19F2 immunoassay technique was assessed, and the effective detection of buprofezin in tea samples was achieved through the establishment of indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and colloidal gold immunochromatography assay (GICA). The mAb-19F2 subtype was IgG1, with an IC50 of 1.8 ng/mL and a linear range (IC20-IC80) of 0.6-5.4 µg/L, and had a cross-reaction rate of less than 0.18% with 29 other pesticides (neonicotinoids and insect growth regulators). The study identified π-π stacking interactions between hapten and TYR-61 at the mAb-19F2 site and alkyl/phosphate interactions with TRP-105 and ARG-103. The ic-ELISA had an IC50 of 12.9 ng/mL in green tea and 5.65 ng/mL in black tea, with a recovery rate of 92.4%-101.0% and RSD of 2.1%-4.8%. The GICA had a limit of detection (LOD) was 500 ng/mL, with the complete disappearance of the test lines visible to the naked eye. The limit of quantitation (LOQ, IC20) was determined to be 16.8 ng/mL. Additionally, the developed GICA showed no cross-reactivity with neonicotinoid pesticides. The recovery rate of tea spiked recovered samples was 83.6%-92.2%, with an RSD of 5.3%-12.6%, and the results were consistent with the LC/MS method. This study is important for the real-time detection of buprofezin residues to ensure food safety and human health.


Assuntos
Anticorpos Monoclonais , Praguicidas , Humanos , Anticorpos Monoclonais/química , Imunoensaio/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Haptenos , Neonicotinoides , Chá
14.
Anal Chem ; 95(39): 14665-14674, 2023 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-37679861

RESUMO

A sandwich immunoassay theoretically exhibits higher sensitivity and specificity compared to a competitive counterpart; however, it is extremely difficult to obtain a pair of antibodies that can bind to a small molecule simultaneously, which is always thought to be a single epitope. In the present study, abamectin (ABM) was selected to prove the effect of hapten design and antibody recognition properties on the development of a sandwich immunoassay for small molecules. First, the epitopes of ABM were roughly located, and epitope distances were determined. Then, two haptens were designed by introducing spacer arms at the C4″-OH and C5-OH of ABM, respectively, aiming to provide the longest epitope distances. A total of seven rabbit polyclonal antibodies (pAbs) and 21 mouse monoclonal antibodies (mAbs) with various recognition properties were obtained. Extensive combinatorial associations of antibody pairs for simultaneously binding to ABM were performed, and only two mAb-mAb pairs were observed to achieve a sandwich immunoassay for ABM with a total success rate of 0.27%. The best mAb pair for sandwich immunoassay was confirmed by surface plasmon resonance, used to develop a sandwich immunoassay, and then evaluated by cross-reactivities and molecular docking with structurally similar analogues and abamectin. Altogether, the study provided a theoretical foundation as well as practical experience and demonstrated the importance of careful hapten design and extensive antibody screening to successfully establish the sandwich immunoassay for small molecules.


Assuntos
Anticorpos Monoclonais , Haptenos , Animais , Camundongos , Coelhos , Simulação de Acoplamento Molecular , Anticorpos Monoclonais/química , Imunoensaio , Epitopos , Ensaio de Imunoadsorção Enzimática
15.
Int J Mol Sci ; 24(17)2023 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-37686326

RESUMO

Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Interleukin 31 (IL-31), a novel cytokine in AD, causes pruritus, typically characteristic of AD patients. The transient receptor potential vanilloid type 1 (TRPV1) is a cation channel activated by diverse noxious stimuli that has been studied in a variety of pruritic skin diseases. In this study, the AD animal model was generated by administering the hapten, trinitrochlorobenzene (TNCB), to Nc/Nga mice, and the degree of expression of the IL-31 receptor alpha (IL-31RA) and TRPV1 in the skin of these atopic models was evaluated. The Nc/Nga mice were divided into 3 groups: control, TNCB 2-weeks treated, and TNCB 8-weeks treated. After inducing AD, the skin lesions in each group were scored and compared, and the histology of the skin lesions and the IL-31RA and TRPV1 expression for each group were evaluated by analyzing immunohistochemistry. The results show a significant difference in the skin lesion scores between the groups. The immunohistochemistry evaluation highlighted the remarkable expression of IL-31RA and TRPV1 in the nerve fibers of the TNCB 8-weeks-treated group. We thus confirmed that the long-term application of TNCB induced chronic atopic-like dermatitis and that IL-31RA and TRPV1 were overexpressed in the peripheral nerve fibers in this AD model.


Assuntos
Dermatite Atópica , Animais , Camundongos , Dermatite Atópica/induzido quimicamente , Cloreto de Picrila , Pele , Prurido , Haptenos , Canais de Cátion TRPV/genética
16.
Bioconjug Chem ; 34(10): 1811-1821, 2023 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-37758302

RESUMO

Currently approved pharmacotherapies for opioid use disorders (OUDs) and overdose reversal agents are insufficient to slow the spread of OUDs due to the proliferation of fentanyl. This is evident in the 31% rise in drug overdose deaths from 2019 to 2022, with rates increasing from 21.6 to 28.3 overdoses per 100,000 deaths. Vaccines are a potential alternative or adjunct therapy for the treatment of several substance use disorders (nicotine, cocaine) but have shown limited clinical success due to suboptimal antibody titers. In this study, we demonstrate that coconjugation of a Toll-like receptor 7/8 (TLR7/8) agonist (UM-3006) alongside a fentanyl-based hapten (F1) on the surface of the carrier protein cross-reactive material 197 (CRM) significantly increased generation of high-affinity fentanyl-specific antibodies. This demonstrated enhanced protection against fentanyl challenges relative to an unconjugated (admix) adjuvant control in mice. Inclusion of aluminum hydroxide (alum) adjuvant further increased titers and enhanced protection, as determined by analysis of fentanyl concentration in serum and brain tissue. Collectively, our findings present a promising approach to enhance the efficacy of antiopioid vaccines, underscoring the need for extensive exploration of TLR7/8 agonist conjugates as a compelling strategy to combat opioid use disorders.


Assuntos
Transtornos Relacionados ao Uso de Opioides , Vacinas , Animais , Camundongos , Receptor 7 Toll-Like/agonistas , Fentanila/uso terapêutico , Adjuvantes Imunológicos/uso terapêutico , Antígenos/uso terapêutico , Haptenos , Transtornos Relacionados ao Uso de Opioides/prevenção & controle , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Analgésicos Opioides/uso terapêutico
17.
J Med Chem ; 66(17): 12407-12419, 2023 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-37646732

RESUMO

Cocaine is one of the most widely used and increasingly popular illicit psychoactive drugs. Unlike other commonly used substances of abuse, cocaine has no pharmacological therapies to treat addiction or aid in rehabilitation. Immunopharmacology has long been touted as a possible avenue to develop effective anticocaine therapies; however, lack of efficacy and designs which are not consistent with simple large-scale production have hindered vaccine translation. We have designed and synthesized a peptide-based anti-cocaine immunogen which we have shown is capable of inducing physiologically relevant immune responses in mice as part of a self-adjuvanting delivery system or in combination with the human-approved commercial adjuvant MF59. We have demonstrated that immunization with the reported vaccine elicits high titers of anti-cocaine IgG and prevents cocaine-induced hyperlocomotion in an in vivo murine model. This peptide-hapten immunogen along with self-adjuvanting liposomal-based delivery system provides a platform for the development of effective anti-drug vaccines.


Assuntos
Adjuvantes Imunológicos , Cocaína , Humanos , Animais , Camundongos , Adjuvantes Imunológicos/farmacologia , Adjuvantes Farmacêuticos , Haptenos , Imunização
18.
Biochem Biophys Res Commun ; 678: 24-32, 2023 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-37611349

RESUMO

Allergic contact dermatitis (ACD) and atopic dermatitis (AD) are common inflammatory diseases. We previously reported attenuated contact hypersensitivity (CHS) responses in AD model mice using 2,4-dinitrofluorobenzene, reflecting clinical experiments. However, previous studies have not addressed the commonality of findings across haptens and mechanisms focused on dendritic cells (DCs). Thus, this study evaluated CHS responses to fluorescein isothiocyanate (FITC) and DC migration and maturation in the sensitization phase of CHS in AD. CHS responses to FITC were compared between NC/Nga mice without and with AD induction (non-AD and AD mice, respectively). T-cell responses and DC migration and maturation after FITC-induced sensitization were examined in the draining lymph nodes of non-AD and AD mice. AD mice demonstrated reduced CHS responses to FITC under decreased T-cell proliferation following sensitization and interferon-γ production by hapten-specific T cells compared with non-AD mice. In addition, the number of FITC+CD11c+MHC class IIhigh migratory DCs 24 h after FITC sensitization was comparable between non-AD and AD mice. However, FITC+CD11c+MHC class IIhigh migratory DCs in AD mice exhibited lower expression levels of CD80 and CD86 and higher expression levels of PD-L1 and mRNA of transforming growth factor beta than non-AD mice. These findings suggest that attenuated CHS responses may be hapten-independent and the induction of the tolerogenic phenotype of hapten-bearing DCs can contribute to reduced T-cell proliferation after sensitization and CHS responses in AD.


Assuntos
Dermatite Atópica , Dermatite de Contato , Camundongos , Animais , Fluoresceína-5-Isotiocianato , Fenótipo , Fluoresceína , Haptenos , Células Dendríticas
19.
Adv Mater ; 35(47): e2303909, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37572294

RESUMO

Recruiting endogenous antibodies to the surface of cancer cells using antibody-recruiting molecules has the potential to unleash innate immune effector killing mechanisms against antibody-bound cancer cells. The affinity of endogenous antibodies is relatively low, and many currently explored antibody-recruiting strategies rely on targeting over-expressed receptors, which have not yet been identified in most solid tumors. Here, both challenges are addressed by functionalizing poly(propyleneimine) (PPI) dendrimers with both multiple dinitrophenyl (DNP) motifs, as anti-hapten antibody-recruiting motifs, and myristoyl motifs, as universal phospholipid cell membrane anchoring motifs, to recruit anti-hapten antibodies to cell surfaces. By exploiting the multivalency of the ligand exposure on the dendrimer scaffold, it is demonstrated that dendrimers featuring ten myristoyl and six DNP motifs exhibit the highest antibody-recruiting capacity in vitro. Furthermore, it is shown that treating cancer cells with these dendrimers in vitro marks them for phagocytosis by macrophages in the presence of anti-hapten antibodies. As a proof-of-concept, it is shown that intratumoral injection of these dendrimers in vivo in tumor-bearing mice results in the recruitment of anti-DNP antibodies to the cell surface in the tumor microenvironment. These findings highlight the potential of dendrimers as a promising class of novel antibody-recruiting molecules for use in cancer immunotherapy.


Assuntos
Dendrímeros , Animais , Camundongos , Anticorpos , Haptenos , Fagocitose , Dinitrobenzenos , Membrana Celular
20.
Anal Chem ; 95(29): 10895-10902, 2023 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-37433088

RESUMO

Conjugate vaccines have been demonstrated to be a promising strategy for immunotherapeutic intervention in substance use disorder, wherein a hapten structurally similar to the target drug is conjugated to an immunogenic carrier protein. The antibodies generated following immunization with these species can provide long-lasting protection against overdose through sequestration of the abused drug in the periphery, which mitigates its ability to cross the blood-brain barrier. However, these antibodies exhibit a high degree of heterogeneity in structure. The resultant variations in chemical and structural compositions have not yet been clearly linked to the stability that directly affects their in vivo functional performance. In this work, we describe a rapid mass-spectrometry-based analytical workflow capable of simultaneous and comprehensive interrogation of the carrier protein-dependent heterogeneity and stability of crude polyclonal antibodies in response to conjugate vaccines. Quantitative collision-induced unfolding-ion mobility-mass spectrometry with an all-ion mode is adapted to rapidly assess the conformational heterogeneity and stability of crude serum antibodies collected from four different vaccine conditions, in an unprecedented manner. A series of bottom-up glycoproteomic experiments was performed to reveal the driving force underlying these observed heterogeneities. Overall, this study not only presents a generally applicable workflow for fast assessment of crude antibody conformational stability and heterogeneity at the intact protein level but also leverages carrier protein optimization as a simple solution to antibody quality control.


Assuntos
Anticorpos , Imunização , Haptenos , Vacinas Conjugadas/química , Proteínas de Transporte
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