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1.
Mol Cancer ; 22(1): 22, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36721153

RESUMO

Malignant brain tumors rank among the most challenging type of malignancies to manage. The current treatment protocol commonly entails surgery followed by radiotherapy and/or chemotherapy, however, the median patient survival rate is poor. Recent developments in immunotherapy for a variety of tumor types spark optimism that immunological strategies may help patients with brain cancer. Chimeric antigen receptor (CAR) T cells exploit the tumor-targeting specificity of antibodies or receptor ligands to direct the cytolytic capacity of T cells. Several molecules have been discovered as potential targets for immunotherapy-based targeting, including but not limited to EGFRvIII, IL13Rα2, and HER2. The outstanding clinical responses to CAR T cell-based treatments in patients with hematological malignancies have generated interest in using this approach to treat solid tumors. Research results to date support the astounding clinical response rates of CD19-targeted CAR T cells, early clinical experiences in brain tumors demonstrating safety and evidence for disease-modifying activity, and the promise for further advances to ultimately assist patients clinically. However, several variable factors seem to slow down the progress rate regarding treating brain cancers utilizing CAR T cells. The current study offers a thorough analysis of CAR T cells' promise in treating brain cancer, including design and delivery considerations, current strides in clinical and preclinical research, issues encountered, and potential solutions.


Assuntos
Neoplasias Encefálicas , Imunoterapia Adotiva , Humanos , Proteínas Adaptadoras de Transdução de Sinal , Anticorpos , Antígenos CD19 , Neoplasias Encefálicas/terapia , Morte Celular , Receptores de Antígenos Quiméricos , Linfócitos T
3.
BMC Cardiovasc Disord ; 23(1): 57, 2023 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-36721091

RESUMO

BACKGROUND: Anti-mitochondrial antibody (AMA)-positive inflammatory myopathy, a rare type of idiopathic inflammatory myopathy which was frequently difficult to diagnose, can affect muscles and the structure and electrical conduction of the heart. Early identification and treatment of this myopathy can prevent serious cardiovascular adverse events and improve cardiac function. CASE PRESENTATION: We report a patient who experienced repeated syncope, ventricular tachycardia (VT) and heart failure accompanied by weakness and muscle atrophy. He was initially diagnosed with dilated cardiomyopathy and received implantable cardioverter-defibrillator therapy. He was subsequently misdiagnosed as muscular dystrophy due to progressive muscular atrophy. However, the patient developed repeated and refractory VT storms that were not alleviated by conventional therapy. Finally, he was diagnosed with AMA-positive inflammatory myopathy with cardiac injuries. The patient was markedly recovered by being treated with immunosuppressive and immunomodulatory therapy. CONCLUSION: AMA could be screened when discovering myopathies accompanied by unexplained cardiac symptoms. Our findings provide insights into the diagnosis and therapy of this rare and severe disease.


Assuntos
Insuficiência Cardíaca , Doenças Musculares , Miosite , Masculino , Humanos , Miosite/complicações , Miosite/diagnóstico , Miosite/terapia , Insuficiência Cardíaca/diagnóstico , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/terapia , Coração , Anti-Inflamatórios , Anticorpos
4.
J Nanobiotechnology ; 21(1): 36, 2023 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-36721182

RESUMO

BACKGROUND: Although a large amount of evidence has revealed that amyloid ß (Aß), especially Aß oligomers, protofibrils, and pyroglutamated Aßs, participate primarily in the pathophysiological processes of Alzheimer's disease, most clinical trials of anti-Aß antibody therapy have never acquired successful efficacy in human clinical trials, partly because peripheral administration of antibody medications was unable to deliver sufficient amounts of the molecules to the brain. Recently, we developed polymeric nanomicelles capable of passing through the blood-brain barrier that function as chaperones to deliver larger amounts of heavy molecules to the brain. Herein, we aimed to evaluate the efficacy of newly developed antibody 6H4 fragments specific to Aß oligomers encapsulated in polymeric nanomicelles on the development of Alzheimer's disease pathology in Alzheimer's disease model mice at the age of emergence of early Alzheimer's disease pathology. RESULTS: During the 10-week administration of 6H4 antibody fragments in polymeric nanomicelles, a significant reduction in the amounts of various toxic Aß species, such as Aß oligomers, toxic Aß conformers, and pyroglutamated Aßs in the brain was observed. In addition, immunohistochemistry indicated inhibition of diameters of Aß plaques, Aß-antibody immunoreactive areas, and also plaque core formation. Behavioral analysis of the mice model revealed that the 6H4 fragments-polymeric nanomicelle group was significantly better at maintaining long-term spatial reference memory in the probe and platform tests of the water maze, thereby indicating inhibition of the pathophysiological process of Alzheimer's disease. CONCLUSIONS: The results indicated that the strategy of reducing toxic Aß species in early dementia owing to Alzheimer's disease by providing sufficient antibodies in the brain may modify Alzheimer's disease progression.


Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Humanos , Animais , Camundongos , Doença de Alzheimer/tratamento farmacológico , Encéfalo , Barreira Hematoencefálica , Anticorpos , Placa Amiloide , Polímeros
5.
Cell Mol Life Sci ; 80(2): 45, 2023 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-36651994

RESUMO

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder resulting from a CAG expansion in the huntingtin (HTT) gene, which leads to the production and accumulation of mutant huntingtin (mHTT). While primarily considered a disorder of the central nervous system, multiple changes have been described to occur throughout the body, including activation of the immune system. In other neurodegenerative disorders, activation of the immune system has been shown to include the production of antibodies against disease-associated pathological proteins. However, the existence of mHTT-targeted antibodies has never been reported. In this study, we assessed the presence and titer of antibodies recognizing HTT/mHTT in patients with HD (n = 66) and age- and gender-matched healthy controls (n = 66) using a combination of Western blotting and ELISA. Together, these analyses revealed that antibodies capable of recognizing HTT/mHTT were detectable in the plasma samples of all participants, including healthy controls. When antibody levels were monitored at different disease stages, it was observed that antibodies against full-length mHTT were highest in patients with severe disease while antibodies against HTTExon1 were elevated in patients with mild disease. Combined, these results suggest that antibodies detecting different forms of mHTT peak at different disease stages.


Assuntos
Doença de Huntington , Doenças Neurodegenerativas , Humanos , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/metabolismo , Anticorpos
6.
Sci Rep ; 13(1): 567, 2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36631484

RESUMO

The majority of mammalian proteins are glycosylated, with the glycans serving to modulate a wide range of biological activities. Variations in protein glycosylation can have dramatic effects on protein stability, immunogenicity, antibody effector function, pharmacological safety and potency, as well as serum half-life. The glycosylation of therapeutic biologicals is a critical quality attribute (CQA) that must be carefully monitored to ensure batch-to-batch consistency. Notably, many factors can affect the composition of the glycans during glycoprotein production, and variations in glycosylation are among the leading causes of pharmaceutical batch rejection. Currently, the characterization of protein glycosylation relies heavily on methods that employ chromatography and/or mass spectrometry, which require a high level of expertise, are time-consuming and costly and, because they are challenging to implement during in-process biologics production or during in vitro glycan modification, are generally performed only post-production. Here we report a simplified approach to assist in monitoring glycosylation features during glycoprotein engineering, that employs flow cytometry using fluorescent microspheres chemically coupled to high-specificity glycan binding reagents. In our GlycoSense method, a range of carbohydrate-sensing microspheres with distinct optical properties may be combined into a multiplex suspension array capable of detecting multiple orthogonal glycosylation features simultaneously, using commonplace instrumentation, without the need for glycan release. The GlycoSense method is not intended to replace more detailed post-production glycan profiling, but instead, to complement them by potentially providing a cost-effective, rapid, yet robust method for use at-line as a process analytic technology (PAT) in a biopharmaceutical workflow or at the research bench. The growing interest in using in vitro glycoengineering to generate glycoproteins with well-defined glycosylation, provides motivation to demonstrate the capabilities of the GlycoSense method, which we apply here to monitor changes in the protein glycosylation pattern (GlycoPrint) during the in vitro enzymatic modification of the glycans in model glycoproteins.


Assuntos
Anticorpos , Glicoproteínas , Animais , Glicosilação , Glicoproteínas/metabolismo , Anticorpos/metabolismo , Espectrometria de Massas , Mamíferos/metabolismo , Polissacarídeos/metabolismo
7.
J Immunother Cancer ; 11(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36634919

RESUMO

BACKGROUND: Pancreatic cancer (PC) has a poor prognosis, and most patients present with either locally advanced or distant metastatic disease. Irreversible electroporation (IRE) is a non-thermal method of ablation used clinically in locally advanced PC, but most patients eventually develop distant recurrence. We have previously shown that IRE alone is capable of generating protective, neoantigen-specific immunity. Here, we aim to generate meaningful therapeutic immune effects by combining IRE with local (intratumoral) delivery of a CD40 agonistic antibody (CD40Ab). METHODS: KPC46 organoids were generated from a tumor-bearing male KrasLSL-G12D-p53LSL-R172H-Pdx-1-Cre (KPC) mouse. Orthotopic tumors were established in the pancreatic tail of B6/129 F1J mice via laparotomy. Mice were randomized to treatment with either sham laparotomy, IRE alone, CD40Ab alone, or IRE followed immediately by CD40Ab injection. Metastatic disease and immune infiltration in the liver were analyzed 14 days postprocedure using flow cytometry and multiplex immunofluorescence imaging with spatial analysis. Candidate neoantigens were identified by mutanome profiling of tumor tissue for ex vivo functional analyses. RESULTS: The combination of IRE+CD40 Ab improved median survival to greater than 35 days, significantly longer than IRE (21 days) or CD40Ab (24 days) alone (p<0.01). CD40Ab decreased metastatic disease burden, with less disease in the combination group than in the sham group or IRE alone. Immunohistochemistry of liver metastases revealed a more than twofold higher infiltration of CD8+T cells in the IRE+CD40 Ab group than in any other group (p<0.01). Multiplex immunofluorescence imaging revealed a 4-6 fold increase in the density of CD80+CD11c+ activated dendritic cells (p<0.05), which were spatially distributed throughout the tumor unlike the sham group, where they were restricted to the periphery. In contrast, CD4+FoxP3+ T-regulatory cells (p<0.05) and Ly6G+myeloid derived cells (p<0.01) were reduced and restricted to the tumor periphery in the IRE+CD40 Ab group. T-cells from the IRE+CD40 Ab group recognized significantly more peptides representing candidate neoantigens than did T-cells from the IRE or untreated control groups. CONCLUSIONS: IRE can induce local tumor regression and neoantigen-specific immune responses. Addition of CD40Ab to IRE improved dendritic cell activation and neoantigen recognition, while generating a strong systemic antitumor T-cell response that inhibited metastatic disease progression.


Assuntos
Neoplasias Hepáticas , Neoplasias Pancreáticas , Masculino , Camundongos , Animais , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Imunidade , Neoplasias Hepáticas/tratamento farmacológico , Anticorpos/uso terapêutico , Eletroporação/métodos
8.
Cell Rep Med ; 4(1): 100910, 2023 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-36603577

RESUMO

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron BA.4 and BA.5 variants caused major waves of infections. Here, we assess the sensitivity of BA.4 to binding, neutralization, and antibody-dependent cellular cytotoxicity (ADCC) potential, measured by FcγRIIIa signaling, in convalescent donors infected with four previous variants of SARS-CoV-2, as well as in post-vaccination breakthrough infections (BTIs) caused by Delta or BA.1. We confirm that BA.4 shows high-level neutralization resistance regardless of the infecting variant. However, BTIs retain activity against BA.4, albeit at reduced titers. BA.4 sensitivity to ADCC is reduced compared with other variants but with smaller fold losses compared with neutralization and similar patterns of cross-reactivity. Overall, the high neutralization resistance of BA.4, even to antibodies from BA.1 infection, provides an immunological mechanism for the rapid spread of BA.4 immediately after a BA.1-dominated wave. Furthermore, although ADCC potential against BA.4 is reduced, residual activity may contribute to observed protection from severe disease.


Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Anticorpos , Citotoxicidade Celular Dependente de Anticorpos
9.
Biosens Bioelectron ; 224: 115033, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36621082

RESUMO

Staphylococcus aureus is one of the most prevalent threats to public health. Rapid detection with high sensitivity and targeted killing is crucial to curb its spread. Herein, a metal-bearing nanocomposite, consisting of a bimetallic nanoparticle and a metal-organic framework (Au/Ir@Cu/Zn-MOF) was constructed. Upon conjugation with anti-S. aureus antibody, this nanocomposite (Ab-Au/Ir@Cu/Zn-MOF) was exploited for its dual functions, i.e. as a reporting probe in a lateral flow immunoassay and a high efficiency antibacterial reagent. Benefiting from the enrichment of Au/Ir NPs by the Cu/Zn-MOF, the Au/Ir@Cu/Zn-MOF-based lateral flow immunoassay sensor exhibited a visual limit of detection of 103 CFU/mL, which was100 times more sensitive than Au/Ir-based sensor. Moreover, the Ab-Au/Ir@Cu/Zn-MOF probe possessed synergistic photothermal-chemodynamic bactericidal effect that specifically targeted against S. aureus. Under a co-treatment by H2O2 (0.4 mM) and 808 nm near infrared irradiation (1 W/cm2, 5 min), complete sterilization of 5 × 105-106 CFU/mL S. aureus was achieved at a nanocomposite concentration as low as 6.25 µg/mL. The superior antibacterial efficiency was attributable to the three-fold properties of the Ab-Au/Ir@Cu/Zn-MOF probe: (1) enhanced multi-enzyme mimicking activities that promote reactive oxygen species generation, (2) high photothermal activity (efficiency of 53.70%), and (3) bacteria targeting ability via the antibody coating. By changing the antibody, this nanocomposite can be tailored to target a wide range of bacteria species, for detection and for precise antibacterial treatment.


Assuntos
Técnicas Biossensoriais , Imunoconjugados , Nanopartículas Metálicas , Peróxido de Hidrogênio , Bactérias , Anticorpos , Antibacterianos/farmacologia , Imunoensaio , Staphylococcus aureus , Zinco
11.
Elife ; 122023 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-36626205

RESUMO

Virus-based tumour vaccines offer many advantages compared to other antigen-delivering systems. They generate concerted innate and adaptive immune response, and robust CD8+ T cell responses. We engineered a non-replicating pseudotyped influenza virus (S-FLU) to deliver the well-known cancer testis antigen, NY-ESO-1 (NY-ESO-1 S-FLU). Intranasal or intramuscular immunization of NY-ESO-1 S-FLU virus in mice elicited a strong NY-ESO-1-specific CD8+ T cell response in lungs and spleen that resulted in the regression of NY-ESO-1-expressing lung tumour and subcutaneous tumour, respectively. Combined administration with anti-PD-1 antibody, NY-ESO-1 S-FLU virus augmented the tumour protection by reducing the tumour metastasis. We propose that the antigen delivery through S-FLU is highly efficient in inducing antigen-specific CD8+ T cell response and protection against tumour development in combination with PD-1 blockade.


Assuntos
Inibidores de Checkpoint Imunológico , Orthomyxoviridae , Masculino , Camundongos , Animais , Antígenos de Neoplasias , Proteínas de Membrana , Imunização , Anticorpos , Linfócitos T CD8-Positivos
12.
Structure ; 31(1): 2-3, 2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36608664

RESUMO

In this issue of Structure, Ge et al. report an epitope-directed strategy to select antibodies specific for Frizzled subtypes. Structural and biochemical analyses provide mechanistic insights into the target binding of the isolated antibodies that could guide the design of reagents and therapeutics targeting distinct Frizzled receptors.


Assuntos
Anticorpos , Receptores Frizzled , Epitopos/química
13.
Nat Commun ; 14(1): 454, 2023 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-36709319

RESUMO

High-affinity antibodies are often identified through directed evolution, which may require many iterations of mutagenesis and selection to find an optimal candidate. Deep learning techniques hold the potential to accelerate this process but the existing methods cannot provide the confidence interval or uncertainty needed to assess the reliability of the predictions. Here we present a pipeline called RESP for efficient identification of high affinity antibodies. We develop a learned representation trained on over 3 million human B-cell receptor sequences to encode antibody sequences. We then develop a variational Bayesian neural network to perform ordinal regression on a set of the directed evolution sequences binned by off-rate and quantify their likelihood to be tight binders against an antigen. Importantly, this model can assess sequences not present in the directed evolution library and thus greatly expand the search space to uncover the best sequences for experimental evaluation. We demonstrate the power of this pipeline by achieving a 17-fold improvement in the KD of the PD-L1 antibody Atezolizumab and this success illustrates the potential of RESP in facilitating general antibody development.


Assuntos
Anticorpos , Biblioteca de Peptídeos , Humanos , Teorema de Bayes , Reprodutibilidade dos Testes , Antígenos
14.
Transpl Int ; 36: 10840, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36713113

RESUMO

Extracorporeal photopheresis (ECP) is an immunomodulatory therapy based on the infusion of autologous cellular products exposed to ultraviolet light (UV) in the presence of a photosensitizer. The study evaluates the ECP efficacy as induction therapy in a full-mismatch kidney transplant rat model. Dark Agouti to Lewis (DA-L) kidney transplant model has been established. ECP product was obtained from Lewis rat recipients after DA kidney graft transplantation (LewDA). Leukocytes of those LewDA rats were exposed to 8-methoxy psoralen, and illuminated with UV-A. The ECP doses assessed were 10 × 106 and 100 × 106 cells/time point. Lewis recipients received seven ECP infusions. DA-L model was characterized by the appearance of donor-specific antibodies (DSA) and kidney function deterioration from day three after kidney transplant. The dysfunction progressed rapidly until graft loss (6.1 ± 0.5 days). Tacrolimus at 0.25 mg/kg prolonged rat survival until 11.4 ± 0.7 days (p = 0.0004). In this context, the application of leukocytes from LewDA sensitized rats accelerated the rejection (8.7 ± 0.45, p = 0.0012), whereas ECP product at high dose extended kidney graft survival until 26.3 ± 7.3 days, reducing class I and II DSA in surviving rats. ECP treatment increases kidney graft survival in full-mismatch rat model of acute rejection and is a suitable immunomodulatory therapy to be explored in kidney transplantation.


Assuntos
Transplante de Rim , Fotoferese , Ratos , Animais , Sobrevivência de Enxerto , Ratos Endogâmicos Lew , Rejeição de Enxerto/prevenção & controle , Anticorpos
15.
In Vivo ; 37(1): 493-497, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36593046

RESUMO

BACKGROUND/AIM: To date, no reports of interleukin (IL)-5-producing Castleman disease with nephrotic syndrome and moreover no reports of relapse after remission with rituximab treatment, have been published. CASE REPORT: A 67-year-old male presented to the Osaka Medical and Pharmaceutical University Hospital with a history of low-grade fever, papules, and nephrotic syndrome. Lymph nodes were palpated in the inguinal region. The patient showed anemia, eosinophilia, polyclonal hypergammaglobulinemia, and elevated interleukin (IL)-6 levels. Patient's serum IL-5 and IL-6 levels were measured using ELISA and immunohistochemical staining of lymph nodes was performed with antibodies specific to CD134. Histological examination confirmed diagnosis of a plasma cell variant of Castleman disease. After a total of four weekly doses of rituximab, urinary protein disappeared, and skin symptoms improved. However, one month after rituximab treatment, the skin rash worsened again, and eosinophils and IL-5 were elevated significantly. CONCLUSION: This is the first report of recurrent Castleman disease with direct evidence of increased serum IL-5. It may be reasonable to use rituximab, an anti-CD20 antibody for treating the disease, however, for IL-5-producing cases the effect of rituximab may be partial.


Assuntos
Hiperplasia do Linfonodo Gigante , Síndrome Nefrótica , Masculino , Humanos , Idoso , Hiperplasia do Linfonodo Gigante/complicações , Hiperplasia do Linfonodo Gigante/diagnóstico , Hiperplasia do Linfonodo Gigante/tratamento farmacológico , Rituximab/uso terapêutico , Síndrome Nefrótica/diagnóstico , Síndrome Nefrótica/tratamento farmacológico , Síndrome Nefrótica/etiologia , Interleucina-5/uso terapêutico , Anticorpos
16.
Alzheimers Res Ther ; 15(1): 2, 2023 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-36604729

RESUMO

BACKGROUND: Accessible and cost-effective diagnostic tools are urgently needed to accurately quantify blood biomarkers to support early diagnosis of Alzheimer's disease (AD). In this study, we investigated the ability of plasma amyloid-beta (Aß)42/Aß40 ratio measured by an antibody-free mass-spectrometric (MS) method, ABtest-MS, to detect early pathological changes of AD. METHODS: This cohort study included data from the baseline and 2-year follow-up visits from the Fundació ACE Healthy Brain Initiative (FACEHBI) study. Plasma Aß42/Aß40 was measured with ABtest-MS and compared to 18F-Florbetaben PET as the reference standard (cutoff for early amyloid deposition of 13.5 centiloids). Cross-validation was performed in an independent DPUK-Korean cohort. Additionally, associations of plasma Aß42/Aß40 with episodic memory performance and brain atrophy were assessed. RESULTS: The FACEHBI cohort at baseline included 200 healthy individuals with subjective cognitive decline (SCD), of which 36 (18%) were Aß-PET positive. Plasma Aß42/Aß40 levels were significantly lower in Aß-PET positive individuals (median [interquartile range, IQR], 0.215 [0.203-0.236]) versus Aß-PET negative subjects (median [IQR], 0.261 [0.244-0.279]) (P < .001). Plasma Aß42/Aß40 was significantly correlated with Aß-PET levels (rho = -0.390; P < .001) and identified Aß-PET status with an area under the receiver operating characteristic curve (AUC) of 0.87 (95% confidence interval [CI], 0.80-0.93). A cutoff for the Aß42/Aß40 ratio of 0.241 (maximum Youden index) yielded a sensitivity of 86.1% and a specificity of 80.5%. These findings were cross-validated in an independent DPUK-Korean cohort (AUC 0.86 [95% CI 0.77-0.95]). Lower plasma Aß42/Aß40 ratio was associated with worse episodic memory performance and increased brain atrophy. Plasma Aß42/Aß40 at baseline predicted clinical conversion to mild cognitive impairment and longitudinal changes in amyloid deposition and brain atrophy at 2-year follow-up. CONCLUSIONS: This study suggests that plasma Aß42/Aß40, as determined by this MS-based assay, has potential value as an accurate and cost-effective tool to identify individuals in the earliest stages of AD, supporting its implementation in clinical trials, preventative strategies and clinical practice.


Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Humanos , Doença de Alzheimer/diagnóstico por imagem , Estudos de Coortes , Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Disfunção Cognitiva/diagnóstico por imagem , Biomarcadores , Anticorpos , Tomografia por Emissão de Pósitrons
17.
Acta otorrinolaringol. esp ; 74(1): 50-58, enero 2023. tab, graf
Artigo em Espanhol | IBECS | ID: ibc-213930

RESUMO

Antecedentes y objetivo: Hay muchas causas de hipoacusia neurosensorial, entre las que tiene especial interés la patología autoinmune del oído interno por su posible reversibilidad con tratamiento esteroideo, a veces asociada a enfermedades sistémicas autoinmunes. La tiroiditis crónica autoinmune o de Hashimoto (TA) presenta anticuerpos que podrían afectar al oído interno independientemente del hipotiroidismo, efecto que no ha sido suficientemente estudiado y nunca mediante audiometría con extensión en altas frecuencias.El objetivo de este trabajo es estudiar si existe afectación de los umbrales auditivos en pacientes de TA, sin hipotiroidismo, en la totalidad del espectro auditivo humano (128 Hz – 20 kHz) distribuido por grupos de edad.Materiales y métodosSe han seleccionado 128 pacientes divididos en dos grupos. El primer grupo de pacientes presenta anticuerpos antitiroideos elevados sin necesitar tratamiento sustitutivo con tiroxina. El segundo grupo con tratamiento sustitutivo con tiroxina, bien controlados. Se comparan con el grupo control (GC) de 209 pacientes. En todos se realizó historia clínica, exploración otológica, estudio de niveles de anticuerpos antitiroideos, TSH (thyroid-stimulating hormone), T3 y T4 libres, timpanograma, estudio audiométrico convencional y con extensión en altas frecuencias.ResultadosTodos los pacientes fueron mujeres. Ambos grupos mostraron peor audición que los controles, siendo la diferencia estadísticamente significativa en todas las frecuencias; en el rango de frecuencias de 8 – 20 kHz con una diferencia de más de 10 dB, y en los rangos de 9-16 kHz y de 20 kHz de más de 20 dB. (AU)


Background and aim: Although sensorineural hearing loss may have different aetiologies, we focused on autoimmune hearing loss since it may be reversible with corticosteroid therapy; this entity is sometimes associated with systemic autoimmune diseases. Hashimoto's thyroiditis or chronic autoimmune thyroiditis shows antibodies and may be harmful to hearing thresholds regardless of hypothyroidism effect. To date this effect has not been sufficiently studied and never with extended high frequencies. The aim of this work is to study by age groups whether hearing thresholds in the human auditory range (128 to 20.000 Hz) are affected in Hashimoto's disease.Materials and methodsTwo groups of 128 patients affected by Hashimoto's thyroiditis were included. First group: patients with pathological antithyroid antibodies who do not need L-thyroxine treatment. Second group: patients controlled with L-thyroxine substitutive treatment. Audiometric threshold study comparing between the groups of patients and a group of 209 controls was performed. All patients underwent complete otorhinolaryngological examination, antithyroid antibodies, TSH, T3 and T4 blood levels, tympanometry, conventional pure-tone audiometry, and extended-high-frequency audiometry.ResultsAll patients were women. Both groups showed worst audiometric thresholds than the control group; both study groups showed worse hearing than controls, this difference was statistically significant in all frequencies. In the 8-20 kHz frequency range this difference was more than 10 dB, and in the 9-16 kHz and 20 kHz range this difference was more than 20 dB. When separated by age groups, in younger subjects (20-29 years) these differences were found in all frequencies, except for conversational frequencies (500 - 4,000 Hz); between 30 and 49 years the difference is statistically significant in all frequencies; and from 50 to 69 years differences are found, especially in the conversational frequencies. (AU)


Assuntos
Tireoidite Autoimune , Perda Auditiva , Doença de Hashimoto , Orelha Interna , Anticorpos
18.
Curr Protoc ; 3(1): e636, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36598346

RESUMO

Immunological memory is the basis of protection against most pathogens. Long-living memory T and B cells able to respond to specific stimuli, as well as persistent antibodies in plasma and in other body fluids, are crucial for determining the efficacy of vaccination and for protecting from a second infection by a previously encountered pathogen. Antigen-specific cells are represented at a very low frequency in the blood, and indeed, they can be considered "rare events" present in the memory T-cell pool. Therefore, such events should be analyzed with careful attention. In the last 20 years, different methods, mostly based upon flow cytometry, have been developed to identify such rare antigen-specific cells, and the COVID-19 pandemic has given a dramatic impetus to characterize the immune response against the virus. In this regard, we know that the identification, enumeration, and characterization of SARS-CoV-2-specific T and B cells following infection and/or vaccination require i) the use of specific peptides and adequate co-stimuli, ii) the use of appropriate inhibitors to avoid nonspecific activation, iii) the setting of appropriate timing for stimulation, and iv) the choice of adequate markers and reagents to identify antigen-specific cells. Optimization of these procedures allows not only determination of the magnitude of SARS-CoV-2-specific responses but also a comparison of the effects of different combinations of vaccines or determination of the response provided by so-called "hybrid immunity," resulting from a combination of natural immunity and vaccine-generated immunity. Here, we present two methods that are largely used to monitor the response magnitude and phenotype of SARS-CoV-2-specific T and B cells by polychromatic flow cytometry, along with some tips that can be useful for the quantification of these rare events. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Identification of antigen-specific T cells Basic Protocol 2: Identification of antigen-specific B cells.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , COVID-19/prevenção & controle , Pandemias/prevenção & controle , Linfócitos B , Anticorpos
19.
Molecules ; 28(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36615611

RESUMO

Fluorescent antibodies have proved to be an invaluable tool for molecular biology and diagnostics. They are routinely produced by modification of lysine residues, which leads to high heterogeneity. As such, their affinity may be compromised if the antigen-binding site is affected, the probability of which increases along with the degree of labeling. In this work, we propose a methodology for the synthesis of site-specific antibody-dye conjugates with a high degree of labeling. To this end, we synthesized two oxyamine-based branched triazide linkers and coupled them with a periodate-oxidized anti-PRAME antibody 6H8; two oxyamine-based linear monoazide linkers of similar structure were used as controls. The azide-labeled antibodies were subsequently conjugated with fluorescent dyes via SPAAC, a copper-free click reaction. Compared to their counterparts made with linear linkers, the branched conjugates possessed a higher degree of labeling. The utility of the methodology was demonstrated in the detection of the PRAME protein on the surface of the cell by flow cytometry.


Assuntos
Anticorpos , Corantes Fluorescentes , Corantes Fluorescentes/química , Antígenos
20.
Anal Chim Acta ; 1239: 340699, 2023 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-36628767

RESUMO

Antibodies against small molecules with high titer and high affinity are always pursued in the field of vaccines for drugs of abuse, antidotes to toxins and immunoassays in medical, environmental, and food safety. The exposure degree of the target molecule to the immune system is critical to induce a strongly specific antibody response, thus, the spacer arm length between the target molecule and carrier protein plays an important role. However, the influence of spacer arm length on antibody titer, affinity, and assay performance is not yet clear and highly demanded to be addressed. In the present study, we proposed a model study to answer the question by using two typical small molecules, melamine and p-nitroaniline, which were introduced by varied spacer arms with increasing alkane linear length from 2 to 12 carbon atoms brick by brick. The spacer arm lengths of the haptens were obtained by computational chemistry. The titer and affinity of mouse antisera were analyzed and compared, showing that all haptens with spacer arms of 6-8 carbon atoms, i.e. 6.3-8.8 Å in length, induced strong antibodies represented by the highest titer and affinity without exception, while the haptens with spacer arms of 2-4 carbon atoms and 10-12 carbon atoms, i.e. 1.5-3.9 Å and 11.3-13.9 Å in length, failed to induce high-quality antibody response. Moreover, the titer and sensitivity of the subsequently developed immunoassays were significantly affected by using coating haptens with different spacer arm lengths, and coating haptens with a spacer arm of 6.3-8.8 Å in length delivered the optimum detection performance. The antibody recognition mechanism study further confirmed that the hapten spacer arm length had a critical effect on the recognition properties of the induced antibody, which should be interactive with the spacer arm each other. This study showed that the hapten with appropriate spacer arm length is important to antibody response and immunoassay development, providing a valuable and general clue for the rational design of hapten.


Assuntos
Formação de Anticorpos , Haptenos , Animais , Camundongos , Haptenos/química , Anticorpos , Imunoensaio , Ensaio de Imunoadsorção Enzimática
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